Download Mucosal Immunity in Mycobacterial Infections Anna Tjärnlund

Mucosal Immunity in Mycobacterial Infections
Anna Tjärnlund
Stockholm University
All previously published papers were reproduced with permission from the publishers.
© Anna Tjärnlund, Stockholm 2007
ISBN 91-7155-388-6
Printed in Sweden by Universitetsservice AB, Stockholm 2007
Distributor: Stockholm University Library
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Integrity without knowledge is weak and useless,
and knowledge without integrity is dangerous and dreadful.
Samuel Johnson (1709-1784)
English author, critic, & lexicographer
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SUMMARY
More than a century after the identification of the tubercle bacillus and the first attempts at
vaccination, tuberculosis (TB) still remains one of the world’s most serious infectious
diseases. TB, caused by the bacterium Mycobacterium tuberculosis, is typically a disease of
the lung, which serves both as the port of entry and as the major site of disease manifestation.
The currently used vaccine, BCG, is administered parenterally and induces a systemic
immune response. However, it fails to protect against pulmonary TB, thereby raising the
question whether vaccination targeting the mucosal immunity in the lungs could be more
favourable.
The respiratory mucosal surfaces represent the first line of defense against a multitude of
pathogens. Secretory IgA in mucosal secretions has an important function by blocking the
entrance of pathogenic organisms and preventing infections. Additionally, a role for IgA in
the modulation of immune responses is currently being revealed. In this work, we investigated
the relevance of mucosal IgA in the protection against mycobacterial infections using mice
deficient for IgA and the polymeric Ig receptor, which is the receptor responsible for mucosal
secretions of IgA. Gene-targeted mice were more susceptible to mycobacterial infections in
the respiratory tract and displayed reduced production of proinflammatory, and protective,
factors such as IFN-γ and TNF-α in the lungs. The mechanisms explaining the defective
proinflammatory responses in the lungs of deficient mice might involve impaired signalling
through Fcα receptors, or homologous receptors, which could lead to an inadequate activation
of pulmonary macrophages. This could subsequently result in suboptimal induction and
production of cytokines and chemokines important for the attraction and migration of immune
cells to the site of infection.
An induction of optimal adaptive immune responses to combat mycobacterial infections
requires prompt innate immune activation. Toll-like receptors (TLRs) are vital components of
the innate branch of the immune system, ensuring early recognition of invading pathogens.
Using TLR-deficient mice we demonstrated an important role for TLR2, and partly TLR4, in
the protection against mycobacterial infection in the respiratory tract. TLR2-deficient mice
failed to induce proper proinflammatory responses at the site of infection, and macrophages
derived from the knockout mice displayed impaired anti-mycobacterial activity.
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Experimental evidence has concluded that the immune response upon an infection can
influence the outcome of succeeding infections with other pathogens. Concurrent infections
might additionally interfere with responses to vaccinations and have deleterious effects. We
developed an in vitro model to study the effect of a malaria infection on a successive M.
tuberculosis infection. Our results demonstrate that a malaria blood-stage infection enhances
the innate immune response to a subsequent M. tuberculosis infection with a Th1 prone
profile. Reduced infectivity of malaria-exposed dendritic cells implies that a malaria infection
could impose relative resistance to ensuing M. tuberculosis infection. However, a prolonged
Th1 response may interfere with malaria parasite control.
The outcome of this work emphasizes the importance of generating effective immune
responses in the local mucosal environment upon respiratory mycobacterial infections. It
furthermore puts new light on the immunological interaction between parasites and
mycobacteria, which could have implications for future vaccine research.
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ORIGINAL ARTICLES
This doctoral thesis is based on the following papers, which are referred to by their Roman
numerals in the text:
I.
Rodríguez, A*., Tjärnlund, A*., , Ivanyi, J., Singh, M., García, I., Williams, A.,
Marsh, P. D., Troye-Blomberg, M., and Fernández, C. (2005). Role of IgA in the
defense against respiratory infections. IgA deficient mice exhibited increased
susceptibility to intransal infection with Mycobacterium bovis BCG. Vaccine
23(20): 2565-2572.
II.
Tjärnlund, A*., Rodríguez, A*., Cardona, P-J., Guirado, E., Ivanyi, J., Singh, M.,
Troye-Blomberg, M., and Fernández, C. (2006). Polymeric Ig receptor knockout
mice are more susceptible to mycobacterial infections in the respiratory tract. Int.
Immunol. 18(5):807-816.
III.
Tjärnlund, A., Guirado, E., Julian, E., Cardona, P-J., and Fernández, C. (2006).
Determinant role for TLR signalling in acute mycobacterial infection in the
respiratory tract. Microbes Infect. 8(7):1790-1800.
IV.
Tjärnlund, A., Troye-Blomberg, M., Pawlowski, A. (2007). The effect of malaria
parasite-derived materials on dendritic cell susceptibility and response to
subsequent Mycobacterium tuberculosis infection. Submitted.
* These authors contributed equally
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CONTENTS
SUMMARY
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ORIGINAL ARTICLES
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ABBREVIATIONS
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INTRODUCTION
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TUBERCULOSIS
MYCOBACTERIAL INFECTIONS
Immune evasion
IMMUNE RESPONSE TO MYCOBACTERIAL INFECTIONS
Innate immunity
Macrophages
Dendritic cells
Neutrophils
Toll-like receptors
γδT cells
Adaptive immunity
Cellular immunity
CD4+ T cells
CD8+ T cells
Humoral immunity
Granuloma formation
TB: THE DISEASE
Diagnosis
Tuberculin skin test
Microscopy
Cultivation
Molecular methods
Treatment
The current BCG vaccine
New vaccine candidates
ANIMAL MODELS OF TB
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MUCOSAL IMMUNITY IN THE RESPIRATORY TRACT
SPECIFIC IMMUNE RESPONSES
SECRETORY IgA
FUNCTIONS OF IgA
IgA RECEPTORS
IgA DEFICIENCY
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MALARIA
PARASITE LIFE CYCLE
IMMUNITY TO BLOOD-STAGE MALARIA
Adaptive immunity
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Humoral immunity
Cellular immunity
DCs in malaria
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CO-INFECTION BETWEEN TB AND MALARIA
ANIMAL STUDIES
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PRESENT STUDY
AIMS
MATERIALS AND METHODS
RESULTS AND DISCUSSION
PAPER I
PAPER II
PAPER III
PAPER IV
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CONCLUDING REMARKS
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ACKNOWLEDGEMENTS
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REFERENCES
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ABBREVIATIONS
APC
Antigen-presenting cell
BAL
Broncho-alveolar lavage
BCG
Mycobacterium bovis Bacillus Calmette-Guérin
CFU
Colony forming unit
CR
Complement receptor
CT
Cholera toxin
DC
Dendritic cell
DC-SIGN
DC-specific intercellular adhesion molecule-3 grabbing
nonintegrin
dIgA
Dimeric immunoglobulin A
DOTS
Directly observed treatment short-course
FcαR
Fc receptor for immunoglobulin A
FcR
Fc receptor
GPI
Glycosylphosphatidylinositol
HIV
Human immunodeficiency virus
Hz
Hemozoin
Ig
Immunoglobulin
i.n.
Intranasal
iNOS
Inducible nitric oxide synthase
IFN-γ
Interferon-gamma
IL
Interleukin
IRAK
Interleukin-1 receptor associated kinases
i.v.
Intravenous
M cell
Microfold cell
MALT
Mucosa-associated lymphoid tissue
MDR
Multidrug-resistant
MHC
Major histocompatibility complex
MR
Mannose receptor
MyD88
Myeloid differentiation factor 88
NO
Nitric oxide
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pIgA
Polymeric immunoglobulin A
pIgR
Polymeric immunoglobulin receptor
PPD
Purified protein derivative
PfEMP1
Plasmodium falciparum erythrocyte membrane protein 1
PfRBC
Plasmodium falciparum-infected red blood cell
RANTES
Regulated upon activation normal T-cell sequence
RBC
Red blood cell
RNI
Reactive nitrogen intermediate
SC
Secretory component
SIgA
Secretory immunoglobulin A
SIgAD
Selective immunoglobulin A deficiency
TB
Tuberculosis
TCR
T-cell receptor
TGF-β
Transforming growth factor-beta
Th
Helper-T cell
TLR
Toll-like receptor
TNF-α
Tumor necrosis factor-alpha
TST
Tuberculin skin test
XDR
Extensively drug-resistant
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INTRODUCTION
TUBERCULOSIS
Tuberculosis (TB) remains one of the leading infectious diseases and causes high mortality in
humans, resulting in almost 2 million deaths annually (WHO, 2007). The increasing global
health burden of TB is due both to the synergistic pathogenesis of co-infection with the
human immunodeficiency virus (HIV), as well as to the continued dissemination of
multidrug-resistant (MDR) Mycobacterium tuberculosis strains (Toossi, 2003; Coker, 2004).
Despite this alarming health challenge, the capacity for treating and preventing TB remains
limited, and a uniformly effective vaccine is lacking.
The M. tuberculosis complex, the cause of TB, is comprised of M. tuberculosis, M. bovis, M.
africanum, M. microti and M. canetti. Although all members can cause TB, M. tuberculosis is
the most prevalent. The natural reservoir of M. tuberculosis and M. canetti is limited to
humans and that of M. microti is mainly limited to small rodents (Kremer et al., 1998). In
contrast, the host range of M. bovis is very broad and this species can cause disease among a
wide range of wild and domestic animals, as well as in humans (Ayele et al., 2004). M.
africanum has been isolated from humans and various animal species (Thorel, 1980;
Alfredsen and Saxegaard, 1992). All members in the complex are slow-growing organisms,
with generation times ranging from 12 to 24 hours depending on environmental and microbial
variables.
MYCOBACTERIAL INFECTIONS
The causative agent of infectious TB, M. tuberculosis, is a rod-shaped, obligate aerobic
bacillus, which is shielded by a unique wax-rich cell wall composed of long-chain fatty acids,
glycolipids and other components (reviewed in Kaufmann, 2001). This robust cell wall of the
bacteria contributes to intracellular survival in host phagocytes.
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TB can manifest itself at any tissue site, but the lungs represent both the main port of entry
and the most important site of disease manifestation. Droplets containing bacilli are expelled
from individuals with active pulmonary TB, and subsequently inhaled into the respiratory
tract (Riley et al., 1995) and phagocytosed by alveolar macrophages. Only particles less than
5 μm in diameter can gain access to the alveoli (Hatch, 1942), where macrophages, resident
within the alveolar space, can phagocytose the bacillus. Most of the bacteria engulfed by
alveolar macrophages will be eliminated from the body through mucocilliary movements, and
a few bacteria will be transported by macrophages into the lung interstitium.
Once M. tuberculosis has entered the lungs, one of four potential fates is possible
(Dannenberg, 1994):
1. The initial host response can effectively kill and eliminate the bacilli. These
individuals will not develop TB at any time point in the future.
2. The bacilli can grow and multiply immediately after infection, thereby causing clinical
disease known as primary TB.
3. The bacilli may become dormant and never cause disease, resulting in a latent
infection that is manifested only as positive tuberculin skin test (TST).
4. The dormant bacilli can eventually begin to grow with resultant clinical disease known
as reactivation TB.
Immune evasion
In spite of the targeting macrophages, whose function is the elimination of microbes, M.
tuberculosis can remain viable after phagocytosis due to different strategies evolved to evade
host immune responses. The use of non-activating complement receptors (CR) may be
advantageous for the bacterium, since engagement of these receptors does not induce the
release of cytotoxic reactive oxygen intermediates (Wright and Silverstein, 1983). Moreover,
it is well known that mycobacteria can prevent the normal phagosome-lysosome fusion
resulting in persistence of the bacteria within the host cell (Armstrong and Hart, 1975). In this
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way the bacteria not only remain viable, but bacterial antigens are prevented from being
presented to T cells. Mycobacteria appear to increase the retention of the tryptophan-aspartate
containing coat (TACO) protein on the surface of the mycobacterial phagosome, thereby
preventing phagosome-lysosome fusion (Ferrari et al., 1999). Moreover, characterization of
mycobacterial phagosomes has revealed the presence of the transferrin receptor (CD71), rab5,
and early phagosome Ag 1, all markers of early endosomes, while late endosomal protonATPases are blocked from recruitment to mycobacteria-harbouring phagosomes (SturgillKoszycki et al., 1994; Clemens and Horwitz, 1995; Sturgill-Koszycki et al., 1996). In
addition, the expression of major histocompatibility complex (MHC) class II molecules is
decreased in M. tuberculosis-infected macrophages (Noss et al., 2000). Other immune evasion
mechanisms are the secretion of proteins such as superoxide dismutase and catalases by M.
tuberculosis, which are antagonistic to reactive oxygen intermediates (Andersen et al., 1991),
and the inhibition of macrophage apoptosis (Fratazzi et al., 1999). Macrophages infected with
M. tuberculosis produce inhibitory cytokines, such as transforming growth factor (TGF)-β
and interleukin (IL)-10, which reduce macrophage activation, thereby leading to decreased
clearance of bacteria (Barnes et al., 1992; Toossi et al., 1995).
IMMUNE RESPONSE TO MYCOBACTERIAL INFECTIONS
Innate immunity
The initial response to an infection is mediated by components of the innate immunity that
serves primarily to restrict the multiplication and dissemination of the pathogens, as well as to
initiate the ensuing adaptive response. In addition to macrophages, M. tuberculosis also
interacts with epithelial cells in the alveolar space of the lung and is able to invade and
replicate in this cell type (Bermudez and Goodman, 1996; Garcia-Perez et al., 2003).
However, the role of alveolar epithelium in mycobacterial infections has not been fully
elucidated. In addition to forming a physical barrier, alveolar epithelial cells can express
adhesion molecules and release cytokines and chemokines, such as IL-8 and monocyte
chemotactic protein-1, and thereby modulate the local immune response (Lin et al., 1998). M.
tuberculosis infection has also been shown to induce the expression of inducible nitric oxide
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synthase (iNOS) mRNA by epithelial cells and the production of nitric oxide (NO) (Roy et
al., 2004), and, more recently, interferon (IFN)-γ (Sharma et al., 2007).
Through the presentation of mycobacterial antigens, and the expression of costimulatory
molecules and cytokines, phagocytic cells play an important role in the initiation and direction
of the adaptive immunity.
Macrophages
Macrophages are regarded as phagocytic cells that initially ingest M. tuberculosis. Thus, they
provide an important cellular niche during infection. The macrophages are considered to be
the main cellular host for mycobacteria, and their major role is the rapid killing of the
invading organism through the release of toxic reactive oxygen and nitrogen intermediates, or
killing by lysosomal enzymes following fusion with the bacterial phagosome. The receptors
that have been implicated in the uptake of mycobacteria include, the mannose receptor (MR),
that recognizes mannose residues on mycobacteria (Schlesinger, 1993; Schlesinger et al.,
1996), Fc receptors (FcRs) binding antibody-coated bacteria, CR1, CR3, and CR4, which bind
complement factor C3-opsonized bacilli (Schlesinger et al., 1990; Hirsch et al., 1994; Aderem
and Underhill, 1999), surfactant receptors (Downing et al., 1995), and scavenger receptors
(Zimmerli et al., 1996).
Upon infection, macrophages have been shown to secrete proinflammatory cytokines, such as
tumor necrosis factor (TNF)-α, IL-1, and IL-6, which are believed to be important for the
recruitment of cells to the site of infection (Giacomini et al., 2001). Furthermore, the secretion
of TNF-α may also aid in the activation of macrophages to produce reactive oxygen and
nitrogen intermediates, and help granuloma formation (Flynn et al., 1995; Roach et al., 2002).
The significance of these toxic nitrogen oxides in the host defense against M. tuberculosis has
been well documented, both in vitro and in vivo, particularly in the murine system
(MacMicking et al., 1997; Shiloh and Nathan, 2000). In the mouse, reactive nitrogen
intermediates (RNIs) play a protective role in both the acute and chronic persistent infection
(MacMicking et al., 1997; Flynn et al., 1998). More importantly, accumulating evidence
supports a role for these reactive molecules in the host defense against human TB (Nicholson
et al., 1996; Wang et al., 1998), although this still remains controversial.
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The mechanisms by which NO and other RNIs may affect antimicrobial activity, could be
through the modification of bacterial DNA, proteins and lipids (reviewed in Chan et al.,
2001). NO can deaminate, as well as directly damage bacterial DNA, and has been
demonstrated to induce apoptosis. RNIs also have the potential to disrupt signalling pathways,
Dendritic cells
While dendritic cells (DC) do not display effective anti-microbial activity upon mycobacterial
encounters, their secretion of cytokines and expression of costimulatory molecules help in
modulating the adaptive immune response, supporting a helper T cell (Th) 1 biased T-cell
response. The major role of DCs during mycobacterial infections appears to be that of an
antigen-presenting cell (APC). It was recently shown that DCs, but not macrophages, infected
with M. tuberculosis were capable of driving Th 1 polarization of naïve CD4+ T cells
(Hickman et al., 2002). Activation of human monocyte-derived DCs with the 19 kDa
mycobacterial lipoprotein results in the preferential secretion of IL-12, a key player in host
defense against M. tuberculosis (Thoma-Uszynski et al., 2000). An additional property of
DCs that contributes to their effectiveness in initiating immune responses is their ability to
migrate from peripheral tissues to secondary lymphoid tissues after acquiring antigens. Naïve
T cells are thereby activated via antigen-presenting-, and costimulatory-molecules in the
presence of polarizing cytokines such as IL-12.
DCs and macrophages appear to have different roles during infection with mycobacteria. For
instance, activated macrophages, but not DCs, have the ability to kill intracellular M.
tuberculosis (Bodnar et al., 2001). The different intracellular behaviour of M. tuberculosis in
macrophages and DCs may reflect differences in the receptors involved in bacterial uptake in
the two cell types. DCs have lectin-surface receptors, such as the recently identified DCspecific intercellular adhesion molecule-3 grabbing nonintegrin (DC-SIGN) (Geijtenbeek et
al., 2000), that facilitate antigen uptake (Engering et al., 2002) and phagocytosis of
mycobacteria by DCs (Geijtenbeek et al., 2003; Tailleux et al., 2003). Although CR3 and MR
are also expressed by human DCs, they seem to be less important for the uptake of the
tubercle bacillus (Tailleux et al., 2003). Ligation of DC-SIGN with the M. tuberculosis-
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derived lipoarabinomanan induces IL-10 secretion in DCs, and thereby suppresses their
function (Geijtenbeek et al., 2003).
Neutrophils
It has been suggested that neutrophils participate in the host defense against mycobacterial
infections since circulating neutrophils become activated and are recruited to the lungs early
in infection. They can be found at the infection nidus at the onset of infection, as well as
several days after the initial reponse (Pedrosa et al., 2000; Fulton et al., 2002). In vivo
depletion of neutrophils prior to mycobacterial infection enhances bacterial growth in the
lungs of infected mice, whereas local treatment with the neutrophil chemoattractant
macrophage-inflammatory protein-2 enhances neutrophil recruitment and decreases
mycobacterial growth (Appelberg et al., 1995; Fulton et al., 2002). The mechanisms by which
neutrophils exert their anti-mycobacterial function are not completely resolved, although
several hypotheses have been proposed. These include the secretion of chemokines (Riedel
and Kaufmann, 1997; Seiler et al., 2003), the induction of granuloma formation (Seiler et al.,
2003), and macrophage uptake of neutrophil-specific molecules such as myeloperoxidase
(Hanker and Giammara, 1983), and lactoferrin (Silva et al., 1989). Another mechanism
whereby neutrophils indirectly contribute to the killing of mycobacteria was recently
demonstrated by Tan et al. Mycobacteria-infected macrophages acquire the contents of
neutrophil granules and their anti-microbial molecules by the uptake of apoptotic neutrophil
debris, which is trafficked to endosomes and colocalize with the intracellular bacteria (Tan et
al., 2006). The involvement of neutrophils in direct killing of mycobacteria has been a matter
of controversy. In vitro studies have demonstrated the ability of neutrophils to kill virulent M.
tuberculosis (Brown et al., 1987; Jones et al., 1990), although this has been questioned
(Denis, 1991; Aston et al., 1998). Kisich et al. showed that neutrophils in human pulmonary
lesions contained intracellular M. tuberculosis, thereby demonstrating a phagocytic role for
neutrophils in human TB. Human neutrophils were furthermore able to kill virulent M.
tuberculosis in vitro (Kisich et al., 2002).
Neutrophils are clearly important in the early immunity to bacterial infections. They respond
rapidly to chemotactic stimuli released by the bacteria or inflammed epithelium and, thus,
arrive early at the site of infection. Besides their anti-mirobial role, neutrophils have been
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implicated in the modulation of the adaptive immune response by the release of
chemoattractants, which recruit other immune cells, such as T cells, monocytes, macrophages
and DCs to the site of infection (Kasama et al., 1993; Yang et al., 2000; Scapini et al., 2001).
It was recently demonstrated that neutrophils and DCs interact physically through DC-SIGN
expressed on DCs and Mac-1 expressed on neutrophils (van Gisbergen et al., 2005;
Megiovanni et al., 2006). This interaction enables neutrophils to induce maturation of DCs
via TNF-α secretion and a preferential production of IL-12 by the matured DCs (van
Gisbergen et al., 2005), which in turn results in an enhanced activation of T cells (Megiovanni
et al., 2006).
Toll-like receptors
Besides phagocytosis, the recognition of M. tuberculosis or mycobacterial products is also
crucial for an effective host response. Central to the immune defense against microbial
pathogens are pattern recognition receptors, such as the toll-like receptors (TLRs). There are
13 members of the TLR family known today, of which TLR1-10 are found in humans
(Ulevitch, 2004). Besides microbial products, TLRs also recognize endogenous ligands, such
as heat shock proteins (Ohashi et al., 2000; Vabulas et al., 2002), extracellular matrix
breakdown products (Termeer et al., 2002; Guillot et al., 2002), and intracellular contents
from necrotic cells (Gallucci et al., 1999; Li et al., 2001). Ligation of TLRs initiates a signal
transduction pathway that culminates in the activation of NF-κB and induction of several
immuno-related genes, including cytokines and chemokines (Hoffmann et al., 1999; Aderem
and Ulevitch, 2000). TLR activation is therefore an important link between innate cellular
responses and the subsequent activation of adaptive immune response to microbial pathogens.
DCs express the broadest repertoire of TLRs through which they can recognize a plethora of
microbial compounds. Upon TLR triggering, immature DCs, apart from cytokine secretion,
undergo the process of maturation, resulting in an augmented expression of T-cell
costimulatory molecules, such as CD80 and CD86, along with antigen-presentation
molecules, such as MHC class II (Tsuji et al., 2000; Hertz et al., 2001; Michelsen et al.,
2001).
Emerging evidence suggests that TLRs play an important role in the activation of immune
cells by pathogens, including M. tuberculosis. TLR2, TLR4, and more recently, TLR1/TLR6
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that heterodimerise with TLR2, have been implicated in the recognition of mycobacterial
antigens (Bulut et al., 2001; Hajjar et al., 2001). Predominantly, a role for TLR2 in the
immune recognition of M. tuberculosis has been demonstrated. Mycobacterial products have
been shown to induce secretion of TNF-α and NO by macrophages via interactions with
TLRs, as well as inducing apoptosis in the host cell (Aliprantis et al., 1999; Brightbill et al.,
1999).
Infection studies using TLR gene-disrupted mice have, however, provided conflicting data,
depending on the experimental settings, for instance the dose of bacteria used. TLR2-/- mice
have been demonstrated to be more susceptible to M. tuberculosis infection than wild-type
mice, while others have reported a redundant role for TLR2 in this context (Reiling, et al.,
2002; Sugawara et al., 2003; Drennan et al., 2004). In addition, outcomes of infection studies
with TLR4-deficient mice show disparity (Reiling et al., 2002; Kamath et al., 2003; Shim et
al., 2003). Signal transduction by most TLRs, with the exception of TLR3, requires the
adaptor molecule myeloid differentiation factor 88 (MyD88) (Medzhitov et al., 1998; Adachi
et al., 1998; Kawai et al., 1999). MyD88 is an intracellular adaptor protein in the IL-1
receptor/IL-1 receptor associated kinases (IRAK) pathway that links TLR recognition with the
activation of IRAK and TNF receptor associated factor (TRAF), translocation of NF-κB, and
gene transcription (Akira et al., 2003). Mice deficient in MyD88 fail to generate
proinflammatory responses when stimulated through TLRs (Adachi et al., 1998; Kawai et al.,
1999), and demonstrate high susceptibility to several infectious agents, including
mycobacteria (Muraille et al., 2003; Mun et al., 2003; Feng et al., 2003; Fremond et al.,
2004).
In this way, TLRs contribute to the innate immune system by the induction of antimicrobial
effector molecules, upon ligation. In addition, the recognition of mycobacterial products by
TLRs induces secretion of cytokines, chemokines, and upregulation of immunostimulatory
molecules, and subsequently the modulation of the adaptive immune response.
γδ T cells
T cells that express the γδ T-cell receptor (TCR) participate in immunity against M.
tuberculosis (Kaufmann, 1996), and are believed to play a role in the early immune response
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(Izzo and North, 1992). Dieli et al. demonstrated an early accumulation of γδ T cells in the
lungs of pulmonary BCG-infected mice, reaching a peak 3 weeks before αβ T cells, where
they produce IFN-γ, exert cytotoxic activity against BCG-infected macrophages, and play a
regulatory role in the induction of CD8+ T cells in the lungs (Dieli et al., 2003). In addition,
studies using γδ TCR knockout mice indicate that γδ T cells may be involved in the regulation
of granuloma formation, which is critical for the control of mycobacteria (D’Souza et al.,
1997). These results indicate that γδ T cells might be important for the control of
mycobacterial infection in the period between innate and adaptive immunity. Human γδ T
cells, predominantly the Vγ9/Vδ2 TCR subset, which represents a major peripheral blood T
cell subset, have also been demonstrated to respond to M. tuberculosis antigens (Kabelitz et
al., 1991), and monocytes infected with live M. tuberculosis were seen to be particularly
effective in expanding this subset of γδ T cells (Havlir et al., 1991).
The effector functions of γδ T cells in the immune response to M. tuberculosis appear to be
both cytokine secretion and cytotoxicity. Studies with M. tuberculosis antigen-activated γδ T
cell clones or primary cells demonstrated IFN-γ production, as well as TNF-α production, in
response to phosphate antigens (reviewed in Boom, 1999). Likewise, cytotoxicity mediated
by γδ T cells has been confirmed and was dependent upon activation through the TCR (Munk
et al., 1990; Dieli et al., 2003). M. tuberculosis-reactive γδ T cells from the peripheral blood
of TST positive subjects were cytotoxic for monocytes pulsed with mycobacterial antigens.
Adaptive immunity
M. tuberculosis is a classic example of a pathogen against which the protective immune
response relies on cell-mediated immunity. The initial interaction in the lungs is with alveolar
macrophages, but after this first encounter DCs and monocyte-derived macrophages, recruited
to the site of infection, also take part in the phagocytic process (Henderson et al., 1997;
Thurnher et al., 1997). Infected DCs mature and migrate to draining lymph nodes to prime
naïve T cells via processed antigens. Inflammation in the lungs provides the signals that direct
the effector T lymphocytes back to the site of infection where granulomas are formed. The
anatomic affinity of these cells appears to be mainly determined by site-specific integrins,
“homing receptors”, on their surface and complementary mucosal tissue-specific receptors,
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“addressins”, on vascular endothelial cells (Kunkel and Butcher, 2003). In addition,
chemokines produced in the local microenvironment promote chemotaxis toward mucosal
tissues and regulate integrin expression on mucosal lymphocytes, thereby controlling cell
migration (Champbell et al., 2003).
Cellular immunity
CD4+ T cells
It is well established that CD4+ T cells are of utmost importance for protective immunity
against M. tuberculosis. CD4+ T cells recognize peptide antigens from mycobacteria,
degraded in the phago-lysosomal compartments and complexed with MHC class II molecules
(Davis and Björkman, 1988). Murine studies with antibody depletion of CD4+ T cells (Muller
et al., 1987), adoptive transfer (Orme and Collins, 1984), or the use of gene-deficient mice
(Caruso et al., 1999), have demonstrated that CD4+ T cell subsets are required for the control
of the infection. The primary effector function of CD4+ T cells is the production of cytokines;
first and foremost IFN-γ, which is crucial for the induction of microbicidal activities by
macrophages, but also TNF-α. Production of these cytokines by CD4+ T cells is important for
the control of TB (Flynn et al., 1993; Flynn et al., 1995), and studies using IFN-γ gene
depleted mice demonstrate that these mice are highly susceptible to virulent M. tuberculosis
infection, with defective macrophage activation and uncontrolled bacillar growth (Cooper et
al., 1993). Furthermore, it is known that humans, defective in genes for IFN-γ or the IFN-γ
receptor, are prone to serious mycobacterial infections, including M. tuberculosis (Ottenhof et
al., 1998). TNF-α in turn plays a key role in the granuloma formation (Kindler et al., 1989;
Senaldi et al., 1996). It induces macrophage activation, and has immunoregulatory properties
(Orme and Cooper, 1999; Tsenova et al., 1999), and is also important for the containment of
latent infection in granulomas, both in mice (Mohan et al., 2001) and humans (Ehlers, 2003).
Although IFN-γ production by CD4+ T cells is a very important effector function, CD4+ T
cells have most likely other roles in controlling M. tuberculosis infection. In MHC class II-/-or CD4-/-mice, the levels of IFN-γ were severely diminished early in infection, but returned to
wild-type levels later on (Tascon et al., 1998; Caruso, et al., 1999). Nevertheless, the gene22
deficient mice were not rescued by this later IFN-γ production and succumbed to infection.
Both CD4+ T cell clones and mycobacterial-antigen-expanded CD4+ T cells have been shown
to exhibit cytolytic effector functions against mycobacterial-antigen pulsed or mycobacteriainfected macrophages (Orme et al., 1992).
Active TB is characterized by a profound and prolonged suppression of M. tuberculosisspecific T-cell responses, demonstrated by decreased production of IL-2 and IFN-γ (Toossi et
al., 1986; Huygen et al., 1988; Zhang et al., 1994; Torres et al., 1994; Hirsch et al., 1999).
Overproduction of immunosuppressive cytokines, such as IL-10 and TGF-β, by mononuclear
phagocytes has been implicated in decreased T cell function during TB (Hirsch et al., 1996;
Gong et al., 1996; Hirsch et al., 1997; Hirsch et al., 1999). However, while IL-10 and TGF-β
levels return to normal after anti-TB treatment, M. tuberculosis-stimulated production of IFNγ remains depressed beyond completion of the treatment (Hirsch et al., 1996; Hirsch et al.,
1999).
Regulatory-T cells constitute a key component of peripheral tolerance suppressing potentially
autoreactive T cells and preventing autoimmune diseases (Sakaguchi et al., 1995; Sakaguchi,
2005). Different subsets of regulatory-T cells have been described, such as IL-10-secreting
(Tr1) or TGF-β-secreting regulatory T cells (TH3), and CD4+CD25+ regulatory T cells
(O’Garra and Vieira, 2004; Sakaguchi, 2005; Belkaid and Rouse, 2005). The latter cells share
common markers with conventional, activated CD4+ T cells, and are now identified by the
molecular marker FoxP3 (Sakaguchi, 2005; Fontenot and Rudensky, 2005; Roncador et al.,
2005). Involvement of IL-10-secreting regulatory-T cells has been proposed in TB anergic
patients (Boussiotis et al., 2000). Recent reports indicate a role for CD4+CD25+ regulatory T
cells in the negative modulation of anti-TB immune responses. An increased frequency of
CD4+CD25+FoxP3+ regulatory-T cells in the blood and at the site of infection was associated
with M. tuberculosis infection (Chen et al., 2007), corroborating a previous study showing
enhanced frequency of CD4+CD25+ T cells during active TB and sustained enhancement after
completion of anti-TB treatment (Ribeiro-Rodrigues et al., 2006).
23
CD8+ T cells
Despite the residence of the bacteria within phagosomes, CD8+ T cells take part in the
immunity against mycobacterial infections (reviewed in Smith and Dockrell, 2000).
Mycobacterial antigen-specific CD8+ T cells are restricted by either MHC class I or CD1
molecules. CD1 molecules are nonpolymorphic molecules that present lipids or glycolipids to
T cells, as opposed to peptide epitopes presented by MHC molecules (reviewed in Porcelli
and Modlin, 1999). Mice genetically disrupted in the genes for β2-microglobulin or the
transporter of antigen processing, and therefore deficient in MHC class I and non-classical
MHC class Ib molecules and, thus, unable to activate CD8+ T cells, display an increased
susceptibility to M. tuberculosis infection compared to wild-type mice (Flynn et al., 1992;
Behar et al., 1999; Sousa et al., 1999), indicating a protective role for these cells.
The mechanism by which mycobacterial proteins gain access to the MHC class I molecules is
not clear. Mycobacteria-induced pores or breaks in the phagosomal membrane have been
proposed as mechanisms (Myrvik et al., 1984; Mazzaccaro et al., 1996; Teitelbaum et al.,
1999). The bacteria could use this as a way of gaining access to cytosolic nutrients and
introducing toxic molecules into the cytoplasm. Additionally, mycobacterial antigens would
be allowed to enter the cytoplasm of infected cells and hence the MHC class I pathway.
Two principal effector functions for CD8+ T cells have been suggested: lysis of infected cells
and production of cytokines, explicitly IFN-γ, although the relative contributions of these
functions are not yet established. CD1- and MHC class I-restricted CD8+ T cells, specific for
mycobacterial antigens, have been shown to induce lysis of infected human DCs and
macrophages, resulting in reduced intracellular bacterial numbers (Stenger et al., 1997; Cho et
al., 2000). This was dependent on perforin, which is required for pore formation (Stenger et
al., 1997), while granulysin was seen to be responsible for killing the intracellular bacteria
(Stenger, et al., 1998). Moreover, antigen-specific CD8+ T cells can target and kill infected
macrophages, and also induce growth inhibition of M. tuberculosis, through apoptotic
mechanisms (Oddo et al., 1998). The role of IFN-γ in mycobacterial infections, on the other
hand, is considered to be through the activation of macrophages. CD8+ T cells from lungs of
infected mice have been shown to be primed for IFN-γ production, although this production
appears to be limited in the lungs (Serbina and Flynn, 1999).
24
Humoral immunity
While the essential role for T cells in the control of M. tuberculosis infection is well
established, the role of B cells and antibodies is less well understood. Although neglected for
a long time, their role has recently been reappreciated. In individuals infected with M
tuberculosis, a vigorous humoral response is always present, in addition to the well-defined
cellular immune response. Infection studies performed using B cell-deficient mice have
revealed conflicting data. One study reported an increase in viable bacilli in B cell-deficient
mice compared to wild-type mice (Vordermeier et al., 1996), whereas results from another
study suggested that absence of B cells, and hence antibodies, does not affect the outcome of
infection with M. tuberculosis (Johnson et al., 1997). Yet another study demonstrated that B
cell deficiency results in a reduced recruitment of neutrophils, macrophages, and CD8+ T cells
to the lungs upon M. tuberculosis infection (Bosio et al., 2000), suggesting that B cells may
influence the cellular composition at this site by regulating chemokines and adhesion
molecules. An additional role for B cells as APCs has also been suggested (Vordermeier et
al., 1996).
Passive immunization with monoclonal antibodies has been reported to protect mice against
mycobacterial infection. It was recently shown that IgA antibodies specific for the αcrystallin antigen of M. tuberculosis significantly reduced the number of colony forming units
(CFUs) in lungs of infected mice (Williams et al., 2004). These results corroborate other
studies, reporting a protective role for monoclonal antibodies and passive immunization in
experimental mycobacterial infections (Teitelbaum et al., 1998; Pethe et al., 2001; Chambers
et al., 2004; Hamasur et al., 2004).
Granuloma formation
Granuloma formation is the hallmark of M. tuberculosis infection. Granulomas are formed in
response to chronic local antigenic stimulation, and can be observed in many different
infectious diseases, including schistosomiasis, leprosy, and leishmaniasis (Reyes-Flores,
1986; Modlin and Rea, 1988; Palma and Saravia, 1997; Rumbley and Phillips, 1999; Boros,
1999). The structure and composition of granulomas vary depending on the organism. A
tuberculous granuloma is observed concomitantly with a highly activated cell-mediated
25
immune response, which generally mediates the control of mycobacterial numbers in the
lungs. The granuloma is composed of many different cells, including macrophages, CD4+-,
and CD8+-T cells, and B cells (Gonzales-Juarrero et al., 2001). These cells control the
infection by providing a local environment for cellular interactions, leading to an effective
immune response where cytokine production, macrophage activation and CD8+ T cell-effector
functions lead to killing of the mycobacteria. Granulomas also provide a way of containing
the bacilli by walling off and preventing the spread of infection. Altogether, these actions lead
to an inhibition of growth, or to the death of M. tuberculosis. However, they also result in
inflammatory pathology that contributes to the damage of the host tissue.
TB: THE DISEASE
TB is primarily a pulmonary infectious disease. Following infection with M. tuberculosis
there is an early transient influx of granulocytes, but the hallmark of mycobacterial infections
is the development of granulomatous lesions (Rook and Bloom, 1994). Although granuloma
formation provides a mean of containing the infection, granulomas may displace and destroy
adjacent tissues. Initially well-formed granulomas may gradually develop central caseation
that may lead to extensive fibrosis or cavity formation in the lungs.
TB can involve any organ system in the body. While pulmonary TB is the most common
clinical manifestation, extrapulmonary TB is also an important clinical problem. The bacilli
can spread from the initial site of infection, in the lung, through the lymphatics or blood to
other parts of the body and cause extrapulmonary TB of the pleura, lymphatics, bone,
urogenital system, meninges, peritoneum, or skin. Before the HIV pandemic, and in studies
involving immunocompetent adults, it was observed that extrapulmonary TB constituted
about 10-20% of all TB cases (Weir and Thornton, 1985; Fanning, 1999;). In HIV-positive
patients, extrapulmonary TB accounts for more than 50% of all TB cases (Theuer et al.,
1990). The most common extrapulmonary sites in HIV-positive individuals are the lymph
nodes.
Disseminated, or miliary, TB refers to the involvement of two or more organs simultaneously,
and can occur during primary infection or after reactivation of a latent infection, as well as
after reinfection.
26
Diagnosis
An early confirmation of the diagnosis of TB is a challenging problem. The established
methods have limitations in speed, sensitivity and specificity. In general, it is more difficult to
diagnose extrapulmonary TB than pulmonary TB, since this often requires invasive
procedures to obtain diagnostic specimens for histological or bacteriological confirmation.
Tuberculin skin test
The TST is currently the only widely used method for identifying latent infection with M.
tuberculosis in asymptomatic individuals. This test is based on the fact that infection with M.
tuberculosis produces a delayed-type hypersensitivity reaction to certain mycobacterial
components in the extracts of culture filtrates called “tuberculins”. The test, also known as the
Mantoux method, is administered by intradermal injection of the tuberculin purified protein
derivative (PPD), which produces a wheal of the skin. The visible induration (in mm) is
measured 48 and 72 hours after injection. There are, however, concerns regarding the TST.
Several factors may contribute to false-negative results, such as age, poor nutrition and
general health, overwhelming acute illness, or immunosuppression, such as medications or
HIV infection (American Thoracic Society, 2000). In addition, false-positive results can occur
in individuals who have been infected with other mycobacteria, including vaccination with
BCG. Because of its low sensitivity, TST cannot be used to rule out the possibility of active
TB.
Microscopy
The microscopal detection of acid-fast bacilli in stained sputum smears is the first
bacteriological evidence of the presence of mycobacteria in clinical specimens. Acid-fast
staining procedure depends on the ability of mycobacteria to retain dye when treated with
mineral acid or an acid-alcohol solution. Smear examination is rapid, inexpensive, technically
simple, and highly specific for acid-fast bacilli, such as M. tuberculosis. Additionally, it gives
a quantitative estimation of the number of bacilli being excreted. The identification of smear
positive patients is of major importance since only smear positive pulmonary TB patients are
27
regarded as highly infectious to others. However, smear microscopy cannot discriminate
between M. tuberculosis and other mycobacteria and, in addition, lacks sensitivity, since
5000-10000 bacteria/ml in the sample are needed for a positive result (American Thoracic
Society, 2000).
Cultivation
Mycobacterial growth in cultures is the ultimate proof of mycobacterial infection and is often
used as the reference method due to its high sensitivity and specificity (Schirm et al., 1995;
Walker, 2001). With this method, as few as 10 bacteria/ml within a sample are necessary for
bacterial detection (American Thoracic Society, 2000). Also, the cultivation of the etiological
agent has been essential for species identification, drug susceptibility testing and monitoring
the response to therapy. Nevertheless, the slow growth rate of M. tuberculosis and most other
mycobacterial pathogens complicates the use of cultivation as a diagnostic technique.
Molecular methods
The use of nucleic acid amplification for the diagnosis of TB is rapidly evolving. These
technologies allow for the amplification of specific target sequences of nucleic acids that can
be detected through the use of a nucleic acid probe, and both RNA and DNA amplification
systems are commercially available (Cohen et al., 1998).
Treatment
Although TB can be cured; current treatment is complex and long lasting, involving four
drugs for 2 months and two drugs for at least another 4 months. The combination of different
drugs is neccessary in order to avoid development of resistant and MDR TB. Isoniazid,
rifampicin, pyrazinamide and streptomycin constitute the first line of TB drugs that are used
and predominantely target actively growing bacteria through the inhibition of cell wall
synthesis, DNA replication and protein synthesis. The long duration for chemotherapy is due
28
to the fact that M. tuberculosis is a slow growing organism and, following the initial killing of
the majority of bacteria, persistent bacteria can revert to the state of latency.
Through the WHO sponsored, directly observed treatment short-course (DOTS) program,
effective TB therapy is now available to around 70% of the world’s population. Yet, the
likelihood of DOTS therapy resulting in the eradiction of TB is limited by the large reservoir
of latently infected individuals, as well as by delays in diagnosis. The treatment regime is
additionally demanding for the patient, labour intensive for health staff and is compromised in
settings where health services are poorly accessible.
In 1993 the WHO declared TB a global health emergency. One reason for this extraordinary
declaration was the increase in MDR TB, which was reaching epidemic proportions. The
management of MDR TB is a challenging problem, given that treatment is less effective,
more toxic and much more expensive compared to the treatment of patients with drug
susceptible TB. In the last few years the fluoroquinolone group of drugs has been added to the
chemotherapy of resistant forms of TB, and have been used as a part of regimens to treat
patients with MDR TB.
During the last year, alarming findings of what has been named extensively drug-resistant
(XDR) TB has been reported. XDR TB is caused by strains of M. tuberculosis resistant to
virtually all second-line drugs. Inappropriate treatment regimens have probably contributed to
the development of XDR TB, which raises the concern for a future epidemic of untreatable
TB.
The current BCG vaccine
The current vaccine against TB, the attenuated M. bovis bacillus Calmette-Guérin (BCG), was
developed by the French scientists Calmette and Guérin in the first decade of the 20th
century. It was first given to humans in 1921 and has now been given to more people than any
other vaccine (Fine, 1995a). Although it can prevent disseminated and meningeal TB in
young children, its efficacy against the most prevalent form of disease, pulmonary TB in
adults, has been strongly questioned. Data concerning the protective efficacy of BCG in adults
range from 0% in South India to 80% in the UK (Fine, 1995b). The reason for this variation in
29
efficacy might depend on several factors, including variation in the BCG strains used,
vaccination dose, vaccination protocols, or inappropriate handling of the vaccine (Hess and
Kaufmann, 1999). Additionally, studies from animal models suggest that prior exposure to
live environmental mycobacteria primes the host immune system against mycobacterial
antigens shared with BCG, and recall of this immune response upon vaccination results in an
accelerated clearance of BCG and therefore decreased protection against M. tuberculosis
(Kamala et al., 1996; Brandt et al., 2002). Helminth infections have, furthermore, been shown
to have an impact on the immune response against mycobacterial infections, leading to a Th2
shift in the immune profile, resulting in a reduced protective efficacy of BCG vaccination
(Elias et al., 2005a). Moreover, the hypothesis that the protection by BCG vaccination wanes
over time has also been brought up (Sterne et al., 1998).
Another factor underlying the failure of BCG-aquired protection could be the route of
vaccination. BCG is currently administered intradermally, which might not be optimal for
inducing protective immunity in the respiratory tract. Vaccination at the mucosal site has been
believed to be superior to vaccination at other sites for eliciting protective immune responses
against mucosal infectious diseases (Davis, 2001). Falero-Diaz and colleagues reported that
intranasal (i.n.) vaccination with BCG conferred potent protection against airway M.
tuberculosis challenge (Falero-Diaz et al., 2000). Another study demonstrated that a single
i.n. BCG vaccination is superior to the subcutaneous route for the protection against
pulmonary TB in mice (Chen et al., 2004). In addition, i.n. vaccination offers desirable
advantages, such as ease in administration, feasibility, and the ability to trigger both mucosal
and systemic immune activation (Davis, 2001).
Lastly, BCG vaccination is also known to stimulate cell-mediated immunity, and BCG
immunotherapy has been used in the treatment of bladder cancer resulting in improved
survival (Alexandroff et al., 1999).
New vaccine candidates
Over the past several years there has been an intensive effort to develop a new vaccine against
TB. The TB vaccines under development can be divided into two categories: prophylactic
(preexposure) or therapeutic (postexposure) vaccines. Prophylactic vaccines prevent infection
30
and subsequent disease and should be given to uninfected persons. Therapeutic vaccines aim
to prevent or reduce progression to disease, and would be given to individuals already
infected with M. tuberculosis. To date, a number of vaccine candidates have been tested in
animal models. The increasing knowledge of the tubercle proteins and the development of
techniques to help identify the most immunogenic antigens, have generated numerous subunit
vaccine candidates. Another area in which there has been substantial interest and progress is
DNA vaccines, and several mycobacterial antigens have been targeted in this manner
(Huygen, 1998). Whole bacterial vaccines have the advantage of a built-in adjuvanticity, as
well as containing both protein- and non-protein antigens. This strategy includes live
attenuated bacteria, as well as engineering and overexpression of distinct antigens to improve
the immunogenicity.
ANIMAL MODELS OF TB
Experimental animal models of TB are central to vaccine development. As for many
infectious diseases, there is no ideal animal model for TB. The most common models used for
M. tuberculosis infection are the mouse and the guinea pig. In neither of these species does
the disease perfectly match that seen in humans, however, many aspects of immunity are the
same. Nevertheless, caution must be advocated when extrapolating results from animal
infection experiments to human TB.
The mouse is the most widely used species and provides many advantages (reviewed in
Kaufmann, 2003). The mouse genome has been completely sequenced, mice are relatively
inexpensive, there is a wealth of information on their immune system, and techniques and
reagents for mechanistic studies are abundant. Additionally, the availability of genetically
targeted mice makes it possible for in vivo studies to elucidate the relevance of particular cells
and molecules. A large number of gene knockout and knockin mice, both constitutive and
conditional, has been generated. However, the mouse is relatively resistant to M. tuberculosis,
and does not develop the severe pathology seen in some human patients.
The guinea pig is generally considered even more susceptible to M. tuberculosis than humans
and therefore provides a very sensitive model for testing the efficacy of novel vaccine
candidates. Moreover, granulomatous lesions in guinea pigs are very similar to those seen in
31
human TB patients. Finally, the group 1 CD1 molecules, responsible for the presentation of
mycobacterial glycolipids to CD1-restricted T cells, are present in humans and guinea pigs
but absent in mice (Schaible and Kaufmann, 2000; Ulrichs and Kaufmann, 2002).
The non-human primate model is considered the closest match for human disease in terms of
pathology. The immune response in this model is very similar to that seen in humans, and
most reagents for human cells and molecules can be applied to primate studies. Due to ethical
reasons, experiments in non-human primates should be limited to critical experiments used for
final validations directly preceding clinical trials of vaccines and therapeutic agents in
humans.
32
MUCOSAL IMMUNITY IN THE RESPIRATORY TRACT
Mucosal surfaces lining the respiratory-, gastrointestinal-, and urogenital tracts are the major
sites of entry for pathogens. These mucosal surfaces thereby provide the first line of defense
against entrance of various bacteria and viruses. Protection of mucosal membranes against
colonization, possible entry and invasion by microbes is provided by a combination of nonspecific and specific mechanisms. Production of mucus is part of the non-specific
mechanisms that acts as a physical barrier containing substances such as lysozyme,
lactoferrin, collectin and defensins. Ciliary action can force microbes out of the respiratory
tract. The movement of microbes by the ciliae decreases the time available for the adherence
by pathogens to the epithelium. Tight junctions between neighbouring epithelial cells lining
the mucosal membranes also act as a physical barrier against penetration.
SPECIFIC IMMUNE RESPONSES
Mucosal surfaces contain specialized mucosa-associated lymphoid tissues (MALT) necessary
for antigen sampling and induction of mucosal immune responses. The immune system in the
upper and lower respiratory tract can be divided into three parts (Davis, 2001):
1. an epithelial compartment at the surface of the epithelium and the underlying
connective tissue that contains immunocompetent cells
2. the MALT, subdivided according to anatomical location: the nasal-associated
lymphoid tissue (NALT), larynx-associated lymphoid tissue (LALT), and the
bronchus-associated lymphoid tissue (BALT)
3. lymph nodes draining the respiratory system
Mucosal antigen-specific immune responses are elicited in the MALT, where foreign material
from epithelial surfaces can be sampled and transported by microfold (M) cells, and
subsequently be taken up by underlying DCs and macrophages (reviewed in Kiyono and
Fukuyama, 2004). M cells are specialized epithelial cells that transcytose particles across
33
epithelial barriers to an intraepithelial lymphoid pocket created by the basolateral surface of
the M cell (Neutra et al., 1996). The follicles of the MALT contain all immunocompetent
cells, i. e. T cells, B cells and APCs, that are required for the initiation of an immune
response. The MALT, as well as local and regional draining lymph nodes, thereby constitute
the inductive site for the generation of mucosal immunity. The common mucosal immune
system connects these inductive sites with effector sites where antigen-specific lymphocytes
perform their effector functions after extravasation from peripheral blood, directed by the
local profile of vascular adhesion molecules and chemokines (Fig. 1).
Figure 1. The common mucosal immune system. (Modified from Nat. Rev. Immunol. (2004) 4:699-710).
DCs in the lymphoid tissue capture antigens, process and then present them to lymphocytes in
the context of MHC molecules. After antigen-induced priming, proliferation, and partial
differentiation in the MALT, lymphoid memory and effector cells migrate to regional lymph
nodes where further differentiation can take place (Brandtzaeg et al., 1999). The lymphocytes
34
thereafter pass into the peripheral blood circulation whereby extravasation at mucosal effector
sites occurs. These primed cells express adhesion molecules, or “homing receptors”, specific
for corresponding determinants on endothelial cells in mucosal and exocrine glandular tissues
(Butcher and Picker, 1996).
M cells moreover express MHC class II molecules (Allan et al., 1993) and ICAM-1 on their
cell surface (Ueki et al., 1995), indicating that they can initiate an immune response. There
are reports showing that M cells can be found in the lungs as well as in the gut, and that
particulate antigens can be transported through pulmonary M cells (Tenner-Racz et al., 1979).
Teitelbaum et al. demonstrated that transcytosis by pulmonary M cells facilitates delivery of
M. tuberculosis to the broncho-tracheal lymph nodes early after infection, suggesting a role of
M cells in the early development of the local immune response (Teitelbaum et al., 1999).
SECRETORY IgA
Immunoglobulin (Ig) A is the predominant Ig isotype induced at mucosal sites (Brandtzaeg,
1989), where it is believed to mediate defense mechanisms (Mazanec et al., 1993; Lamm,
1997). Monomers of IgA are polymerized through the J chain, which is added just before the
secretion of IgA by plasma cells (Johansen et al, 2000). IgA-producing plasma cells migrate
to the basolateral surface of mucosal epithelial cells, where secreted IgA is transported to the
luminal side by the polymeric Ig receptor (pIgR), expressed at the basolateral side of
epithelial cells lining the mucosal surfaces (Mostov, 1994) (Fig. 2).
Translocation of IgA involves enzymatic cleavage of the pIgR, whereby the extracellular part
of the molecule, the secretory component (SC), in complex with dimeric IgA (dIgA), forms
the secretory IgA (SIgA), that is released into the luminal secretions (Norderhaug et al.,
1999). pIgR-mediated transcytosis does not require the presence of a ligand, resulting in a
continuous release of the SC into external secretions. In human exocrine fluids, 30 to 60% of
SC is normally in a free form (Brandtzaeg, 1973). The constitutive expression of pIgR in
epithelial cells can be further upregulated by certain cytokines, such as IFN-γ (Sollid et al.,
1987; Youngman et al., 1994; Loman et al., 1997), and TNF-α (Kvale et al., 1988).
35
Figure 2. Transport of mucosal IgA. (A) IgA translocation through the epithelium. (B) Structure of SIgA.
FUNCTIONS OF IgA
IgA is thought to be the most important Ig class for lung defense by protecting the mucosal
surfaces from penetration by microorganisms and foreign antigens, as well as by neutralizing
bacterial products such as enzymes and toxins (Mazanec et al., 1993; Lamm, 1997). Other
mechanisms include the ability of IgA to agglutinate microbes and interfere with bacterial
motility by interacting with their flagella. Immune complexes of IgA and encountered
antigens can be transported across epithelial cells from basal to apical surfaces in vitro
(Kaetzel et al., 1991), and in vivo (Robinson et al., 2001). Foreign substances that have
interfered with the mucosal surface can thereby be eliminated from the body by an IgAmediated transport back through the epithelium. Additionally, IgA appears to be able to
interact with viral antigens during transcytosis and interfere with viral synthesis and/or
assembly, thereby neutralizing viruses intracellularly (Mazanec et al., 1992). In vitro studies
have demonstrated evidence for such intraepithelial cell interactions with Sendai virus
(Fujioka et al., 1998), measles virus (Yan et al., 2002), influenza virus (Mazanec et al., 1995),
rotavirus (Burns et al., 1996; Feng et al., 2002), and recently against HIV (Huang et al.,
2005).
36
In humans, two subclasses of IgA, termed IgA1 and IgA2, exist, each being the product of a
separate gene, whereas in mice there is only one class of IgA. The major difference between
IgA1 and IgA2 resides in the hinge region, the short region between the two Fab arms and the
Fc region. IgA1 features an extended hinge due to the insertion of a duplicated stretch of
amino acids, which is lacking in IgA2. The longer hinge in IgA1 may have evolved to offer
advantages in antigen recognition by allowing higher avidity bivalent interactions with
distantly spaced antigens (Boehm et al., 1999; Furtado et al., 2004). In secretions, most of the
locally produced IgA is, as described previously, polymeric with a relative increase in the
proportion of IgA2 in human. The reason for the predominance of this subclass might be due
to the increased vulnerability to proteolytic attack that the extended stretch of amino acids
found in IgA1 bring about. Highly specific IgA-cleaving proteases are produced by a number
of important bacterial pathogens that are able to colonize mucosal surfaces and invade
mucosal tissues (Kilian et al., 1996). For instance, Neisseria meningitides, Haemophilus
influenzae, and Streptococcus pneumoniae secrete proteases that specifically cleave the IgA1
hinge region (Senior et al., 1991). IgA2 remains resistant to cleavage since the susceptile
hinge region is missing. Another strategy evolved by bacteria to circumvent effector functions
by host IgA antibodies is the expression of IgA-binding proteins. These are proteins that bind
specifically to IgA and are produced by many strains of group A Streptococcus (Stretococcus
pyogenes) and group B Streptococcus, which are major human pathogens.
IgA is the second most prevalent antibody in serum after IgG, and serum IgA is
predominantely (90%) monomeric IgA1 in humans but mainly dIgA in other animals.
IgA RECEPTORS
IgA has traditionally been viewed as a non-inflammatory antibody. It is a poor activator of
complement and does not activate the classical pathway, although its role in activation of the
alternative pathway remains controversial. Only recently, it has become apparent that dIgA or
polymeric IgA (pIgA), upon binding to mannan-binding lectin, can induce the activation of
the lectin pathway (Roos et al., 2001). The significance of complement activation by IgA in
vivo remains unclear. It can be speculated that in situations where antigen is limited, IgA
could in fact inhibit complement activation by blocking binding of IgG and IgM, which are
more potent activators of complement. The inability of SIgA to fix complement efficiently or
37
to act as an opsonin is an advantage at mucosal sites, where the induction of an inflammatory
reaction would likely affect the integrity of the mucosal surface (Kerr, 1990).
Whereas the role of SIgA in mucosal immunity is being elucidated, the function of serum IgA
antibodies is mostly unknown. Serum IgA is considered to be a “discrete housekeeper”
because IgA-immune complexes can be removed by the phagocytic system with little or no
resulting inflammation. However, characterization of FcRs for IgA (FcαRs) has challenged
the paradigm of IgA as a non-inflammatory or even anti-inflammatory Ig. The human FcαR
(FcαRI, CD89) is expressed on eosinophils (Monteiro, et al., 1990), neutrophils (HonorioFranca et al., 2001), monocytes (Patry et al., 1996), macrophage subsets, Kupffer cells, and
DCs (Geissmann et al., 2001). Whereas the interaction of IgA with the pIgR is
noninflammatory, antigen-complexed binding to CD89 mediates a broad spectrum of
proinflammatory and immunomodulatory effects depending on the cell type involved (Morton
et al., 1996; Monteiro and van de Winkel, 2003). These effects include antibody-dependent
cell-mediated cytotoxicity, phagocytosis, release of cytokines, superoxide generation, calcium
mobilization, degranulation and antigen presentation. Therefore, a second line of defense at
the interface of mucosal and systemic immunity, provided by FcαR-serum IgA interactions
on Kupffer cells, has been proposed (van Egmond et al., 2000). Under physiological
conditions, SIgA inhibits the invasion of pathogens into the mucosal surface, as a first line of
defense, without activating inflammatory responses. Under pathological conditions, the
pathogens can invade the portal circulation due to a disruption of the mucosal barrier, where
they will subsequently be exposed to serum IgA. Concomitantly produced inflammatory
cytokines induce FcαRI expression on Kupffer cells, and FcαRI-positive Kupffer cells can
thereby phagocytose the pathogens that have entered the circulation.
Despite extensive studies on FcαRs in man, there is scarce knowledge about the structure and
function of FcαRs in mice. A CD89 homologue in rats was recently identified (Maruoka et
al., 2004), but no mouse homologue has yet been found. A common Fcα/μR was newly
characterized on human and mouse B cells and macrophages, and in different tissues like
liver, spleen, and intestine (Shibuya et al., 2000). Using the human FcαR probe, transcripts of
two cDNAs, PIR-A and PIR-B (paired Ig receptors A and B), isolated from a mouse splenic
library, were detected (Kubagawa et al., 1997). The PIR-A and PIR-B genes were fund to be
expressed on B cells and cells from the myeloid lineage. IgA has furthermore been shown to
38
bind to the intracellular lectin Gal-3 (Mac-2) expressed on several cell types including
macrophages (Reljic, et al., 2004a). Gal-3 has been implicated in allergic reactions due to its
ability to activate mast cells by crosslinking receptor-bound IgE (Liu et al., 1993).
IgA DEFICIENCY
Selective IgA deficiency (SIgAD), using 0.05 g/l as the upper limit for diagnosis in adults, is
the most common form of primary immunodeficiency in the western world and affects
approximately 1/600 individuals (reviewed in Hammarström et al., 2000). The variability in
the prevalence among different ethnic groups is striking (1/18000 in Japanese and 1/4000 in
Chinese), which suggests a genetic implication for the disorder. Although SIgA has a fairly
clear biological role, SIgAD is a heterogeneous condition with symptoms ranging from none
at all to recurrent respiratory or gastrointestinal diseases, atopy, asthma, and inflammatory or
autoimmune disorders such as systemic lupus erythematosus, rheumatoid arthritis, and
pernicious anaemia (Burks and Steele, 1986; Burrows and Cooper, 1997; CunninghamRundles, 2001). However, most individuals remain asymptomatic. IgA deficiency can be
associated with IgG subclass deficiency (Oxelius et al., 1981), making it, in some cases,
difficult to distinguish between the cause and the symptoms. The fact that most IgA-deficient
individuals are healthy may be partly explained by a compensatory increase in IgM-bearing B
cells and increased secretory IgM in mucosal fluids (Norhagen et al., 1989). However,
secretory IgM does not completely replace SIgA functionally, particularly not in the upper
respiratory tract (Brandtzaeg et al., 1987).
39
MALARIA
Malaria is caused by a protozoan parasite of the genus Plasmodium. There are four species
affecting humans, Plasmodium falciparum, P. vivax, P. ovale and P. malariae. The disease is
associated with a variety of clinical syndromes ranging from asymptomatic to lethal infections
involving anaemia, organ failure, pulmonary and cerebral disease. P. ovale and P. malariae
are relatively infrequent causes of morbidity, while P. vivax is a common cause of severe
illness, especially in Asia and South America, but is rarely fatal. The vast majority of severe
malaria cases and deaths are caused by P. falciparum, which is endemic in most of subSaharan Africa, where the WHO estimates that 90% of malaria cases occur. There are over
500 million clinical malaria cases every year, with 1-3 million malaria-associated deaths per
year, of which the majority are of children under the age of five (Snow et al., 2005). In
addition to these human malaria parasites, there are other Plasmodium species that infect
various animals.
PARASITE LIFE CYCLE
The life cycle of the malaria parasite is complex and involves several developmental stages
with asexual reproduction in the human host and sexual reproduction in the Anopheles
mosquito vector (reviewed by Stevenson and Riley, 2004) (Fig. 3). An infected female
mosquito injects 10-30 sporozoites into the host during a blood meal. These sporozoites are
carried by the blood to the liver, where they invade hepatocytes within 30 minutes of a bite.
Inside the hepatocytes they undergo a process of asexual replication, which gives rise to
schizonts. Up to this point the infection is non-pathogenic and clinically silent. The liver
schizonts eventually rupture and thousands of merozoites are released into the blood, where
they invade red blood cells (RBC). Each merozoite can divide inside the RBC and develop
through different stages, such as ring and trophozoite, into schizontes. As the parasite
develops, adherent ligands are expressed on the cell membrane of the infected RBC, one
being P. falciparum erythrocyte membrane protein 1 (PfEMP1), which enables the parasitized
cell to bind to receptors expressed by endothelial cells lining the blood vessels inside organs,
such as the brain, lungs and the placenta. Upon rupture of the infected RBC, more than 30
merozoites are released that can then reinfect new RBCs. This gives rise to a cyclic blood-
40
stage infection, which takes 48-72 hours to complete, depending on the Plasmodium species.
The RBC stage of the parasite’s life cycle is responsible for the symptoms and pathology of
malaria. A small subset of merozoites differentiates into male and female gametocytes that
can be taken up by an Anopheles mosquito feeding on the host. Inside the midgut of the
mosquito these mature into gametes. This is the sexual stage of the parasite’s life cycle and
upon fertilization a motile zygote is formed, which penetrates the epithelial cell layer of the
midgut and forms an oocyst. After 10-24 days, thousands of sporozoites are released from the
oocyst, which invade the salivary glands of the mosquito and can be injected into a host upon
the mosquito’s blood meal.
Figure 3. Schematic view of the malaria parasite life cycle. (Nat. Rev. Immunol. (2001) 1:117-125.)
IMMUNITY TO BLOOD-STAGE MALARIA
Both innate and adaptive immune responses are critical for establishing clinical immunity and
control of parasitemia during an infection with P. falciparum. Innate immune responses are
important for the control of initial parasitemia, and play a role not only for non-immune
individuals infected for the first time, but also for semi-immune individuals who may be
infected with a parasite variant they have never encountered before (Stevenson and Riley,
2004). The initial response to infection usually involves splenic removal of parasitized RBCs.
41
Upon rupture of schizonts, parasite products are released into the circulation and trigger the
activation of phagocytes, resulting in a release of pro-inflammatory cytokines that cause fever
and mediate other pathological effects. One of these bioactive parasite products is
glycosylphosphatidylinosiol (GPI), which acts as a malaria pathogen-associated molecular
pattern and toxin. GPI induces the production of several factors implicated in malaria
pathogenesis, for example pro-inflammatory cytokines, such as TNF-α, IL-1 and IL-12, as
well as iNOS and various adhesion molecules that are expressed on vascular endothelium and
recognized by PfEMP1 (Carlson et al., 1992; Schofield and Hackett, 1993; Tachado et al.,
1997; Naik et al., 2000).
Adaptive immunity
In endemic areas where there is a continous exposure to the parasite, adaptive immunity is
gradually built up and the severity and incidence of malarial illness decrease with increasing
age. Since the parasites have the capacity to vary their antigens, which are major targets for
protective antibodies, repeated exposure to the parasite is required for a long-lasting
immunity. During the first months of life, infants are protected from malaria, most likely by
antibodies transferred from the immune mother. Experimental evidence for species- and
stage-specific immunity in malaria demonstrates that adaptive immunity is crucial for the
protection against malaria (reviewed in Troye-Blomberg, 1994).
Humoral immunity
Ig levels in individuals, living in highly endemic areas are strongly elevated and the level of
total anti-malarial antibodies increases with age. Antibody-mediated protection against bloodstage malaria is primarily mediated by cytophilic IgG antibodies and can involve different
mechanisms (Good and Doolan, 1999). Antibodies can inhibit merozoite invasion of RBCs,
prevent sequestration of infected RBC by inhibiting binding to adhesion molecules on the
vascular endothelium, neutralize parasite GPI, and thereby inhibit the induction of the
inflammatory cascade, prevent parasite binding to the placenta, and lastly, enhance the
clearance of infected RBCs by binding to their surface and thus promote phagocytosis and
removal of the immune complex in the spleen.
42
It has been reported that levels of total IgE, as well as anti-malarial IgE antibodies, are
elevated in malaria patients, in parallel to an elevation of TNF-α (Perlmann et al., 1994). A
pathogenic role for IgE has been suggested, whereby crosslinking of IgE receptors on
monocytes by IgE-containing immune complexes leads to a local overproduction of TNF-α.
This has recently been questioned by studies indicating a protective role for IgE (Bereczky et
al., 2004; Dolo et al., 2005).
Cellular immunity
T cells are essential for both generating, as well as regulating, immunity against blood-stage
malaria. The major T cells controlling blood stage infections are CD4+ T cells, of both the Thl
and Th2 subsets. Evidence from numerous studies, such as selective depletion of CD4+ T cells
in vivo (Weinbaum et al., 1978; Suss et al., 1988; Kumar et al., 1989), and adoptive transfer
of CD4+ T cells to immunocompromised mice (Brake et al., 1988; Meding and Langhorne,
1991; Taylor-Robinson et al., 1993; Taylor-Robinson and Phillips, 1993), have revealed the
critical role for CD4+ T cells in protective immunity against blood-stage malaria infection.
Important for the generation of protective immunity is IL-12 production, which induces a
polarized Th1 response with a production of IFN-γ and TNF-α. These cytokines activate
phagocytic cells, which are important for controlling the level of parasitaemia.
The levels of T cells expressing the γδ TCR are elevated in acute malaria infection, and they
are believed to have a protective role by exerting cytotoxicity, as well as by secretion of
cytokines, such as IFN-γ (reviewed in Dieli et al., 2001). Human NK cells, which are also
activated early during malaria infection (Orago and Facer, 1991), can be activated rapidly by
parasites in vitro (Theander et al., 1987), resulting in IFN-γ production (Artavanis-Tsakonas
et al., 2003). Since RBCs are devoid of MHC molecules, antigen-specific cytotoxic CD8+ T
cells are generally not believed to act against the asexual blood stage.
DCs in malaria
Early interactions between blood-stage malaria parasites and innate cells are thought to be
43
important for shaping the adaptive immune response to blood-stage malaria. However, the
role of DCs in the induction of protection against the blood-stage malaria parasite is not well
defined. Studies have shown that P. falciparum-infected red blood cells (PfRBC) can bind
myeloid DCs and modulate the maturation and function of DCs. Adhesion of PfRBCs has
been shown to inhibit the upregulation of maturation makers, costimulatory molecules and
adhesion molecules on DCs upon stimulation with LPS. Moreover, the ability of such DCs to
induce T cell responses was significantly reduced (Urban et al., 1999). The receptors on DCs
mediating this inhibitory effect, CD36 and CD51, are not only receptors for PfRBCs but are
also involved in recognition of apoptotic cells by phagocytes. Ligation of CD36 and/or CD51
with PfRBC or apoptotic cells results in a reduced production of IL-12 and an increased
production of IL-10 (Urban et al., 2001). The malarial pigment, hemozoin (Hz), has
furthermore been shown to have an immunomodulatory effect on phagocytic cells. Hz is the
coordinated polymerized form of heme subunits generated by the parasite during hemoglobin
catabolism (Slater et al., 1991). Malaria parasites degrade host hemoglobin for its use as
source of amino acids, which is accompanied by the release of free heme. Free heme is
oxidatively active and toxic to both the host cell and the parasite, causing parasite death. Since
the malaria parasite is unable to catabolyze heme, it protects itself through the crystallization
of heme into Hz (Sullivan et al., 1996a). During a Plasmodium infection, Hz is released into
the blood through the rupture of PfRBC and is readily taken up, together with PfRBC, by
macrophages and monocytes (Schwarzer et al., 1992; Schwarzer et al., 1998).
Investigations on DC function in murine Plasmodium infections have yielded contradictory
results. In vitro and in vivo studies in murine models have shown that, in contrast to the effect
on human DCs, P. chabaudi-infected RBC induced the maturation and activation of murine
DCs (Seixas et al., 2001; Perry et al., 2004; Leisewitz et al., 2004). In contrast, other studies
have reported a suppressive effect on DCs (Ocana-Morgner et al., 2003; Pouniotis et al.,
2004). However, a general trend can be noted where there is an early phase of DC activation
during a blood-stage infection, with a condition of low parasitemia, and a late phase of
inhibition, when the condition is characterised by high parasitemia. Interestingly, a recent
study demonstrated that overstimulation of DCs via TLRs renders them refractory to further
activation (Perry et al., 2005). Since malaria is associated with an exponential increase in
parasite load, DCs are expected to develop TLR tolerance as infection progresses (Perry et al.,
2005). The consequence would be subsequent dysfunction of DCs at later stages of infection.
However, whether DC function is impaired during malaria infection still remains to be
44
established.
CO-INFECTION BETWEEN TB AND MALARIA
The WHO estimates that 90% of the malaria cases occur in sub-Saharan Africa where the
highest incidence of TB per capita also exists. Considering the overlapping geographical
endemic areas for malaria and TB, co-infection is expected to occur very frequently. Given
the chronic character of infections caused by M. tuberculosis and P. falciparum, it is highly
plausible that an interaction between one of these microorganisms and the host’s immune
system would have an impact on the course of infection and disease caused by the other
microorganism. Investigations have established that the immune response induced upon
infection with a pathogen may influence the outcome of subsequent infections or vaccinations
(Curry et al., 1995; Helmby et al., 1998). For instance, helminth infections have been shown
to have an impact on the immune response against mycobacterial infections owing to a Th2
shift in the immune profile (Elias et al., 2005a; Elias et al., 2005b). However, the mutual
modulation of host immune responses by M. tuberculosis and P. falciparum in concurrent
infections has not been extensively studied.
Malaria infection affects the host’s T cells, both qualitatively and quantitatively. Since the
containment of a mycobacterial infection and protective immunity against TB depend on an
intact cellular immunity, malaria-induced impairment of the immune system could influence
susceptibility to, or progression of a M. tuberculosis infection. Clinical evidence on the
outcome of infection or disease in individuals with double Plasmodium/M. tuberculosis
infection, as compared to those infected with a single pathogen, is scarce in the literature. One
indirect piece of evidence comes from a study in Guinea-Bissau that demonstrated a lower
risk of death due to malaria in children with a BCG scar (Roth et al., 2005). This supports
earlier suggestions that the presence of a BCG vaccination scar may be associated with a
general non-specific enhancement of the immune responses to some unrelated infections, and
reduced mortality in children (Velema et al., 1991; Kristensen et al., 2000; Roth et al., 2004),
although revaccination with BCG did not reduce morbidity from malaria in African children
(Rodrigues et al., 2007). A statistically significant association between the presence of
antimalarial antibodies and the diagnosis of TB was reported by Adebajo et al., indicating that
malaria could affect the progression of the mycobacterial disease (Adebajo et al., 1994).
45
ANIMAL STUDIES
The effect of malaria on chronic TB has been investigated in a study utilizing a murine model,
showing increased M. tuberculosis loads in the lungs, liver and spleen of co-infected mice
(Scott et al., 2004). Another study examined the effect of TB on malaria in M. tuberculosisinfected C57BL/6 mice subsequently infected with Plasmodium yoelii. A protective effect of
prior M. tuberculosis infection against P. yoelii was detected and was associated with an
enhanced Th1 type of immune response (Page et al., 2005). In contrast, Th2-prone BALB/C
mice were not protected against malaria infection by prior M. tuberculosis infection. The
results obtained with the C57BL/6, but not BALB/c mice, corroborate previous studies
demonstrating protection against malaria in animals infected with mycobacteria (BazazMalik, 1973; Clark et al., 1976; Murphy et al., 1981; Stevenson et al., 1984; Matsumoto et
al., 2001).
Although animal models serve as a valuable tool for studying immune responses and
dissecting underlying mechanisms, caution is advised when extrapolating these results to
humans. Animal models of malaria infection suffer from the lack of specificity caused by host
restriction of malaria species. P. falciparum, the clinically most important human malaria
parasite, does not produce disease in mice.
46
PRESENT STUDY
AIMS
The upper respiratory tract is the port of entrance for the mycobacterium, and the first
encounter between the host and the pathogen takes place in the lung. This raises the
hypothesis that protective immunity against mycobacterial infections is highly dependent on a
local respiratory mucosal immunity in the lungs, in addition to cell-mediated immune
responses. In paper I-II we therefore aimed to investigate the importance of IgA, the major Ig
isotype present at mucosal sites, in the respiratory mucosal immunity against infection with
intracellular mycobacteria.
An efficient immune response against a pathogen is critically dependent on rapid detection of
the invading organism by the innate immunity and the activation of the subseqent adaptive
immune response. Emerging evidence supports the concept that innate immune responses are
vital for the host defense against M. tuberculosis. TLRs represent critical pathogenrecognition receptors whose signals lead to the generation of important effector responses,
including Th1 responses. We therefore studied the role of TLR signalling in early and late
stages of mycobacterial infection in paper III.
Considering the overlapping geographical endemic areas for two of the leading infectious
causes of morbidity and mortality worldwide, malaria and TB, a high frequency of coinfection can be presumed. Since DCs have a unique capacity to prime naïve T cells, and
subsequently direct the downstream immunological events that occur after the initial
encounter with the pathogens, they provide a critical link between the innate and adaptive
immune responses. In paper IV we developed a model for studying concurrent
Plasmodium/M. tuberculosis infection using DCs. We assessed the consequences of a malaria
blood-stage infection on a subsequent M. tuberculosis infection of DCs.
47
The specific objectives were:
ƒ
To evaluate the relevance of IgA in the respiratory tract in the protection against
mycobacterial infection using IgA-deficient mice (paper I).
ƒ
To study the role of actively secreted IgA in the protection against mycobacterial
infection in the respiratory tract using pIgR-deficient mice (paper II).
ƒ
To assess the role of different TLRs in the defense against primary mycobacterial
infection using TLR-deficient mice (paper III).
ƒ
To analyse the effect of malaria antigens on DC susceptibility and response to a
subsequent M. tuberculosis infection (paper IV).
MATERIALS AND METHODS
The materials and methods used in this work are described in the individual papers (I-IV).
RESULTS AND DISCUSSION
PAPER I
In paper I we investigated the role of IgA in the protection against i.n. challenge with BCG
using IgA-deficient mice (Harriman et al., 1999). The most characteristic component of
mucosal immunity is SIgA. An induction of IgA responses in the respiratory tract could
provide a protective function against diseases caused by respiratory infections, among them
TB. We therefore wanted to investigate the relevance of IgA in the respiratory mucosal
immunity against mycobacterial infection, after i.n. immunization with a mycobacterial
antigen.
The antigen used for immunization was the endotoxin-free recombinant PstS-1 protein. This
38 kDa lipoprotein is a putative phosphate-transport receptor, and a known B-, and T-cell
48
stimulant that has been suggested as a potential immunodiagnostic reagent (Wilkinson et al.,
1997). As an adjuvant, for targeting the mucosal immune system, the cholera toxin (CT) was
used. CT is a strong mucosal adjuvant that markedly increases antigen presentation by DCs,
macrophages and B cells (reviewed in Holmgren et al., 2005). It acts by an upregulation of
MHC- and costimulatory molecules as well as chemokine receptors on APCs, and by
increasing the permeability of the epithelium leading to enhanced uptake of co-administered
antigens.
Although protective immunity against the intracellular pathogen M. tuberculosis relies on
efficient cell-mediated immune responses, the function of antibodies is being reappreciated.
(Winslow et al., 2000; Edelson and Unanue, 2001; Hellwig et al., 2001; Glatman-Freedman,
2003). In concordance with this, our results demonstrated that IgA-deficient mice, immunized
with our formulation, were more susceptible to BCG infection compared to immunized wildtype mice, as shown by a higher bacterial load in the lungs and broncho-alveolar lavage
(BAL). This result suggests involvement of IgA in the protection against infection with
mycobacteria in the respiratory tract.
To address the mechanisms underlying the increased susceptibility to BCG infection
displayed by the IgA-deficient mice, we analyzed the induced local immune responses, after
i.n. immunization with PstS-1 formulated with CT. No IgA was detected in either saliva or
BAL from IgA-/- mice. However, they displayed higher levels of both antigen-specific and
total IgM compared to IgA+/+ mice. Moreover, IgA-deficient mice displayed an impaired T
cell response, as revealed by significantly reduced TNF-α- and IFN-γ-production in the lungs,
when compared to wild-type mice.
Previous studies have demonstrated impaired T cell responses in both IgA-deficient and Bcell-deficient mice (Vordermeier et al., 1996; Arulanandam et al., 2001; Zhang et al., 2002).
However, the mechanistic details behind have not been clarified. One possibility is
involvement of signalling through FcαRs. The human FcαR (CD89) has been well
characterized, whereas no murine homologue has yet been identified, although a common
Fcα/μR, both human and murine, has been identified, which is expressed on B cells and
macrophages (Shibuya et al., 2000; Sakamoto et al., 2001). Moreover, activated mouse T
cells express FcαRs (Sandor et al., 1992). In vitro studies have demonstrated that monomeric
49
and polymeric IgA, but not IgG or IgM, stimulate TNF-α and NO production, as well as
induction of apoptosis in mouse macrophage cell lines (Reljic et al., 2004b). It is therefore
possible that absence of IgA could lead to a reduced or inadequate activation of macrophages
at mucosal sites, and thereby reduced bacterial clearance. This would furthermore affect
subsequent immunological events, since activated macrophages secrete TNF-α and other
molecules with chemoattractant properties for immune cells.
Reljic et al. (2004a) demonstrated that IgA binds to the intracellular lectin Gal-3, expressed
on activated macrophages, indicating a further role for IgA-mediated inhibition of
intracellular mycobacteria. Gal-3 has been shown to accumulate only in those phagosomes
containing live M. tuberculosis through binding to phosphatidylinositol mannosides, and
appears to influence the clearance of infection (Beatty et al., 2002), since bacterial interaction
with the phagosomal membrane is required for mycobacterial inhibition of phago-lysosome
fusion (de Chastellier and Thilo, 1997). It is possible that the interaction between Gal-3 and
its mycobacterial ligand could be further amplified by IgA, which provides additional
crosslinking of Gal-3. This may keep the pathogen tightly fixed to Gal-3 and away from other
membrane components. In other words, IgA antibodies bound to mycobacteria, or
mycobacterial components, may interfere with interactions between opsonised bacteria and
the membrane of phagosomes, and thereby prevent the survival of mycobacteria within
macrophages.
Important in this context is the study by Geissmann et al. showing that human DCs located
beneath the epithelium express the IgA Fc receptor CD89, and cosslinking by IgA
immunocomplexes results in internalization of the ligand, enhanced expression of the
costimulatory molecule CD86, as well as MHC class II molecules, and an increase in the
allostimulatory activity by the cells (Geissmann et al., 2001).
Another indication of an impaired activation of lung macrophages in the absence of IgA
antibodies, comes from studies demonstrating that only lung-derived macrophages from IgAdeficient mice, and not bone marrow-derived macrophages, display an impaired activation
phenotype and reduced cytokine production upon stimulation, when compared to wild-type
controls (Rodríguez A., personal communication).
50
All together, our results suggest a role for IgA in the protection against mycobacterial
infection in the respiratory tract, most likely by modulating the locally induced
proinflammatory immune responses through macrophage activation. Another possible
mechanism is the opsonisation of bacteria and blocking the entrance of the bacilli into the
lungs.
PAPER II
In addition to investigating a possible role of IgA in the protection against mycobacterial
infection in the respiratory tract, we also addressed the role of actively secreted antibodies,
specifically SIgA. For this purpose we used pIgR-deficient mice (Shimada et al., 1999).
Although B-cell deficient mice have previously been used in infection studies with
mycobacteria (Vordermeier et al., 1996; Boiso et al., 2000; Turner et al., 2001), mice lacking
actively secreted antibodies have never been used. Generation of pIgR-knockout mice
presents a unique opportunity to explore the relative contribution of local secretory antibodies
versus systemic immunity in the protection against mycobacteria, as has been done previously
in virus-infection studies (Asashi et al., 2002).
Initially, the immunological status regarding antibodies, as well as the induced immune
responses upon i.n. immunization with the same formulation as in paper I, namely PstS-1 in
combination with CT, was assessed in the pIgR-/- mice. Interestingly, although no antigenspecific IgA was detected in saliva from the knockout mice, IgA levels in BAL were
comparable between the two groups. This finding indicates differences between the upper and
lower respiratory tract concerning the mechanisms of IgA transport to mucosal secretions.
The possibility of transport mechanisms other than through the pIgR cannot be ruled out, nor
can leakage through the epithelial membrane, which has been previously reported (Johansen
et al., 1999). Also, their respective contributions may be different in the upper and lower
respiratory tract. Our finding indicates that the transport of IgA in the upper respiratory tract
is to a larger extent pIgR-mediated than in the lower respiratory tract, where contributions
from other transport mechanisms or leakage may have a greater impact.
Protection studies against i.n. infection with BCG revealed that pIgR-/- mice were more
susceptible to mycobacterial infection than wild-type mice, with significantly higher bacterial
51
burden in the lungs. Although these mice had similar levels of IgA antibodies in the BAL
after i.n. immunization as wild-type mice, their higher bacterial load in the lungs after BCG
infection could indicate that the SC plays a role for the protective capacity of SIgA. The SC
has been associated with regulation of innate, non-specific, responses to pathogens, mainly
due to binding of N-glycans of the SC to bacterial and host factors. One such function is the
stabilization and protection of dIgA and pIgA, and although proteolytic degradation is less
extensive in the respiratory tract than in the gut lumen, the absence of pIgR, and hence the
SC, could imply that the IgA antibodies found in the BAL of pIgR-/- mice are more rapidly
degraded and therefore not able to perform their protective function. A further function
attributed to the SC is its binding to bacterial components, such as Clostridium difficile toxin
A (Dallas and Rolfe, 1998) and fimbriae of enterotoxigenic Escherichia coli (de Oliviera et
al., 2001), thereby limiting infection and morbidity. Although no interactions between the SC
and mycobacteria, or mycobacterial components, have been reported, the possibility cannot be
ruled out. Additonally, it was recently demonstrated, in a mouse model, that SIgA was more
protective than pIgA against respiratory infection with Shigella flexneri, due to carbohydratedependent adherence of the SIgA to the mucus lining of the epithelial cell layer (Phalipon et
al., 2002). SIgA autoantibodies found in saliva from normal subjects were moreover shown to
be polyreactive against pathogenic elements, suggesting an innate function of these antibodies
(Quan et al., 1997). This property was recently confirmed for gastrointestinal SIgA in an
infection study using Salmonella typhimurium (Wijburg et al., 2006). In conclusion, these
properties of the SC suggest that dIgA/pIgA antibodies without the SC, which would be found
in pIgR-/- mice, are less protective than SIgA antibodies that are produced in wild-type mice.
In this study, we also saw that BCG-infected pIgR-/- mice displayed a considerable reduction
in TNF-α and IFN-γ production in lung mononuclear cells. Since these cytokines are central
for protective immunity against mycobacterial infections (Cooper et al., 1993; Flynn et al.,
1995), this impaired mycobacterium-induced proinflammatory response most likely
contributed to the higher bacterial load in the lungs of these mice. We demonstrated in paper I
that IgA-deficient mice have a defective proinflammatory immune response, probably due to
an impaired activation of macrophages or other IgA receptor-bearing cells, and thereby
reduced secretion of cytokines and chemokines. Although the pIgR-knockout mice have IgA
antibodies, the lack of pIgR and SC might render these IgA antibodies less functionable, even
in the sense of their immunomodulary effect. Alveolar macrophages and DCs extending out in
the alveoli might therefore not be optimally activated. Interestingly, a recent study by Favre et
52
al. demonstrated that SIgA works as a weak immunopotentiator in the mucosal environment
in the gastrointestinal tract (Favre et al., 2005). SIgA transported from lumen to the
subepithelial compartment via M cells stimulated mucosal immune responses via upregulated
expression of the costimulatory molecules CD80 and CD86 on DCs.
We next addressed the role of SIgA in the protection against natural infection with virulent M.
tuberculosis. Viable bacterial counts in the lungs clearly demonstrated that pIgR-knockout
mice were more susceptible than wild-type mice at the early phase of infection. At the late
phase of infection no differences between the two groups could be seen. The higher
susceptibility to M. tuberculosis infection was associated with significantly reduced
expression of important protective factors in the lungs. Expression of the proinflammatory
cytokines IFN-γ and TNF-α was substantially reduced, which corroborates the findings in the
BCG-infected pIgR-deficient mice. Moreover, expression of iNOS was also reduced.
Production of RNIs is an important defense mechanism against microbial pathogens exerted
by macrophages. A reduced level of iNOS in the lungs of infected pIgR-/- mice is therefore in
line with the higher bacterial load in the lungs of these mice. Moreover, reduced expression of
the regulated upon activation normal T-cell sequence (RANTES) was also seen in the lungs of
infected knockout mice. This C-C chemokine is involved in attracting monocytes and
lymphocytes to sites of infection, as well as promoting Th1 type of responses (Taub et al.,
1996; Dairaghi et al., 1998; Chensue et al., 1999). These findings indicate that pIgR-/- mice
display lower expression of factors important for protective immunity against TB and proper
granuloma formation. Indeed, histological analysis of granulomas in the lungs demonstrated a
decreased infiltration of macrophages and lymphocytes, but increased numbers of neutrophils
in the pIgR-knockout mice compared to wild-type mice. In addition, a higher degree of
necrosis and karyorrhexis was evident in the granulomas in the knockout mice. This
qualitative histopathological difference was detected at the early phase of infection, whereas
no major differences in granuloma composition where seen during the late phase. In this
context, it is interesting to note that human SC has been reported to bind and inactivate IL-8
(Marshall et al., 2001), a cytokine important for the attraction of neutrophils. Although no
rodent IL-8 homologue has been identified, a murine IL-8 receptor homologue exists (Cerretti
et al., 1993), and mice deficient for this receptor display impaired neutrophil responses and
reduced leukocyte migration (Lee et al., 1995; Becker et al., 2000; Hang et al., 2000). It is
therefore tempting to speculate that the ligand for this murine receptor homologue could, in a
similar fashion to human IL-8, bind to SC, whereby mucosal secretions from pIgR-/- mice
53
would contain higher levels of the putative IL-8 homologue compared to wild-type mice,
resulting in increased chemotaxis of neutrophils in the knockout mice.
Our findings suggest that mycobacteria-infected pIgR-/- mice display a delayed immune
response, due to reduced expression of RANTES and TNF-α and consequently impaired
attraction of macrophages and lymphocytes to the site of infection, as well as a lower
proinflammatory response. As a consequence, pIgR-deficient mice allow more extensive
bacterial growth in the lungs, which in turn triggers an increased granulomatous infiltration in
order to control the bacilli. At the later phase of infection pIgR-/- mice have managed to
control the bacterial growth, displaying comparable viable bacterial counts in the lungs as the
wild-type mice.
Collectively, our results imply a role for SIgA in the modulation of mycobacteria-induced
proinflammatory immune responses and subsequently a role in the protection against
mycobacterial infections.
PAPER III
Resistance against mycobacterial infection and active TB is dependent on the host’s ability to
generate a Th1 type of immune response. Activation of TLRs is an important link between
innate cellular responses and the subsequent initiation of adaptive immune defenses against
microbial pathogens, including mycobacteria. In paper III we investigated the importance of
TLR2 and TLR4 in early and late stage of infection using TLR2-/- mice (Takeuchi et al.,
2004), and TLR4-/- mice (Hoshino et al., 1999) with a C57BL/6 background.
Studies on the interaction between mycobacterial components and TLRs are extensive as well
as numerous, and have established that the 19 kDa lipoprotein and lipid derivatives of
mycobacterium mainly interact with TLR2 to induce predominantely proinflammatory
responses (Krutzik and Modlin, 2004). Although these in vitro studies are essential for
dissecting interactions at a molecular level, the physiological role of TLRs can only be
evaluated using whole mycobacteria. The engagement of multiple TLRs, as well as other
receptors, may affect the outcome of each single interaction. We therefore initially
investigated the susceptibility of TLR-deficient mice to BCG infection. Given that TB is
54
primarily acquired through inhalation of airborne droplets containing the bacterium, we
infected the experimental animals via the respiratory tract, in order to resemble a natural
infection. Analysis of the bacterial burden in the lungs of infected mice demonstrated an
impaired control of bacterial growth in the lungs of TLR2-/- mice at the early stage of
infection, whereas no difference between wild-type and knockout mice could be detected at
the later stage. The role of TLR4 signalling appears less important in the control of
mycobacterial growth, both at early and late stages of infection. In addition, TLR6- and
TLR9-deficient mice did not differ from wild-type mice in terms of inhibition of bacillary
growth. The fact that an increased susceptibility among TLR2-/- mice was only evident during
early stages of infection, suggests that the absence of signalling through TLR2 could be
compensated for as the infection proceeded. Other danger signals, such as endogenous factors
released upon inflammation-induced tissue pathology, might contribute to evoking an immune
response through other TLRs or additional receptors. Heat shock proteins (Ohashi et al.,
2000; Vabulas et al., 2002), hyaluronan (Termeer et al., 2002) and fibronectin (Smiley et al.,
2001) released from damaged cells have been shown to trigger TLR4 signalling.
When using the intravenous (i.v.) route of infection no differences between any of the
experimental groups could be seen, indicating the importance of the chosen route of infection.
Several studies have addressed the role of TLRs in inducing protective immunity against
mycobacterial infection by infecting experimental animals i.v. Since TLR expression on
resident, as well as attracted immune cells, may differ in various parts of the body, results
from i.v. infections might not be relevant for an airborne bacterium. For instance, human
alveolar epithelial cells express both TLR2 and TLR4 (Armstrong et al., 2004). However,
considering the possibility of dissemination of a mycobacterial infection, i.v. infection can
provide supplementary information. In the light of our results, TLR-dependent effector
mechanisms appear to be especially important in the respiratory tract. Indirect support for this
comes from studies demonstrating TLR-mediated activation of human bronchial epithelial
cells and subsequent secretion of beta defensin-2 (Kagnoff and Eckmann, 1997; Hertz et al.,
2003). Pulmonary epithelial cells have additionally been shown to produce IFN-γ in response
to M. tuberculosis infection (Sharma et al., 2007). Although no receptor interaction was
assessed in that study, this finding implies a role for the alveolar epithelial cells in the innate
immunity against mycobacterial infections. Data on the TLR distribution on murine
pulmonary epithelial cells is, however, lacking.
55
In addition to BCG, we also used virulent M. tuberculosis as the infectious agent. Our results
from aerosol infection of mice were in line with the BCG findings, with the exception that
TLR4-/- mice, in addition to TLR2-/- mice, displayed an impaired control of mycobacterial
growth at the early stage of infection, which was not seen during BCG infection. We believe
the reason for this difference could be due to the different mycobacteria used. Considering
that the latter experiment involved the pathogen causing TB, these findings indicate that
TLR4 signalling, besides TLR2, is of importance for inducing protective immunity against
virulent M. tuberculosis infections. We also included TLR6-/-- and TLR9-/- mice in this
experiment, however, no differences between these mouse strains and wild-type mice were
detected.
Alveolar-resident macrophages within the lung are considered to be the main cellular host for
mycobacteria, and the major role for these cells is rapid killing of the invading pathogen. This
is due to the release of toxic reactive oxygen and nitrogen intermediates, or to the killing by
lysosomal enzymes following fusion of the lysosome with the mycobacteria-containing
phagosome. In addition, cytokine production by these cells is essential for the initiation of
cellular immune responses. Given the important function of macrophages, and the fact that
these cells express TLRs (Punturieri et al., 2004), we evaluated the intracellular growth of
mycobacteria in bone marrow-derived macrophages from TLR-deficient mice, as well as their
cyokine response. Our results suggest that the higher bacterial load in TLR2-/- mice is a result
from impaired activation of macrophages, since they display impaired capacity to control
intracellular growth of BCG, in addition to a reduced secretion of TNF-α upon infection with
mycobacteria. The absence of TLR4 signalling appears less harmful since TLR4-/macrophages were not as affected as macrophages from the TLR2-/- mice. Phagosome
maturation has been demonstrated to be regulated by TLR signalling (Blender and Medzhitov,
2004), which could explain the growth advantage of BCG in TLR2-deficient macrophages.
TNF-α produced by activated macrophages is important not only for further activation of
macrophages, but also for attracting other immune cells and granuloma formation via
induction of chemokines (Algood et al., 2004). We moreover detected completely abolished
TNF-α production by MyD88-deficient macrophages, indicating that this signalling pathway
is the principal mediator in initiating anti-mycobacterial responses in macrophages.
Additional experiments revealed a reduced iNOS mRNA expression in TLR2-deficient
56
macrophages upon BCG infection, giving further evidence for the importance of TLR2
signalling for the induction of effector functions against mycobacteria in macrophages.
TLR-deficient mice, particularly TLR2-/-mice, furthermore showed defects in the generation
of proinflammatory and Th1-associated cytokine responses when using an antigen
restimulation assay. This suggests defective T-cell priming in vivo in these mice, which is
supported by a previous study (Heldwein et al., 2003). The absence of T cell activation and
expansion following infection could be a consequence of impaired antigen presentation and
costimulation, involving macrophages as well as DCs. Although an intrinsic defect in the
previously reported TLR2-deficient T cell function could not be ascribed the impaired T cell
priming in vivo (Heldwein et al., 2003), the possibility still remains. Not only classical innate
cells, but also T cells have been shown to express certain TLRs (Kabelitz, 2007). It is
therefore possible that TLRs could act as costimulatory molecules to enhance the proliferation
and cytokine production of TCR-stimulated T cells.
In contrast to in vitro restimulation assays, ex vivo studies investigating the expression of
protective molecules, such as IFN-γ, TNF-α, RANTES and iNOS, in the lungs of M.
tuberculosis-infected mice demonstrated no differences between TLR-knockout mice and
wild-type controls. Since TLR2- and TLR4-deficient mice displayed higher bacterial burdens
in the lungs at the early stage of M. tuberculosis infection, it is possible that an impairment of
the studied molecules, that initially might have caused the increased susceptbility to infection,
had already been compensated for at the time of assessment. Histological evaluation of the
lungs revealed a higher granulomatous response in the lungs of infected TLR-knockout mice
at the later stage of infection, most likely a consequence of the initially higher bacterial load
that triggered a stronger granulomatous infiltration. This response would then sufficient to
control the bacterial growth.
Collectively, our results demonstrate a protective role for TLR2, and partly TLR4, in the host
defense against mycobacterial infection in the respiratory tract. These receptors are
particularly important during the early phase of the infection. A deficiency in mainly TLR2,
but also to some extent TLR4, results in an impaired anti-mycobacterial macrophage activity,
and a defective proinflammatory response.
57
PAPER IV
Malaria, an infectious disease causing around 2 million deaths every year, shares a common
geographical distribution with TB. The chronic character of the infections caused by the two
pahogens, M. tuberculosis and P. falciparum, implies that the interaction of one of these
pathogens with the host’s immune system could have an impact on the course of infection
caused by the other. Such mutual interactions have previously been demonstrated for other
co-infecting pathogens (Curry et al., 1995; Helmby et al., 1998). We therefore established a
model to study the effect of blood-stage malaria on human DC response and the susceptibility
of DCs to a succeeding M. tuberculosis infection. Given the unique qualities of DCs as
inducers and modulators of immune responses upon interactions with microbial substances,
we believe that this model is highly relevant for dissecting the effect of a concurrent M.
tuberculosis and P. falciparum infection. In endemic areas, malaria infection is likely to
precede a M. tuberculosis infection, since Plasmodium infections are aquired during early
childhood, whereas the risk of M. tuberculosis infection increases later in life (Chadha et al.,
2003; Balasubramanian et al., 2004; Kolappan et al., 2004). Our initial purpose was therefore
to assess the effect a malaria blood-stage infection would have on human DCs, in particular
with regard to a succeeding M. tuberculosis infection.
P. falciparum parasites (strain F32, mycoplasma free) were cultured (Trager and Jensen,
1976) in human O+ RBC, and schizont- and late trophozoite stage PfRBCs were harvested for
co-culturing with human DCs. Alternatively, the malarial pigment Hz was used to represent a
malaria blood-stage infection. Hz is the aggregated form of heme polymers, generated after
parasite degradation of host hemoglobin, and released in the blood stream upon rupture of
parasite-infected RBC. To evaluate the effect of Hz, and not contaminating proteins,
membranes or GPI molecules, we used synthetic Hz (Egan et al., 2001). Synthetic Hz (βhematin) is structurally (Slater et al., 1991; Sullivan et al., 1996b) and biologically (Sherry et
al., 1995) similar to Hz formed naturally by parasites. These two preparations, PfRBC and
Hz, were used as malaria parasite-derived material (MDM) in the study.
We initially assessed the susceptibility to M. tuberculosis infection of DCs pre-exposed to
either PfRBC or Hz. It has previously been reported that PfRBCs suppress DC function
(Urban et al., 1999; Urban et al., 2001), and that Hz inhibits the differentiation of human
58
monocytes into DCs, and reduces their responsiveness to maturation signals (Skorokhod et
al., 2004). This suggested that DCs exposed to PfRBC or Hz could be more susceptible to M.
tuberculosis infection. However, our results revealed the opposite, since MDM-exposed DCs
were infected by M. tuberculosis to a lower extent as compared to non-exposed controls.
Light microscopic analysis additionally revealed that the two pathogens, or pathogen-derived
substances, could co-exist in the same cell.
In order to elucidate the mechanism of the reduced susceptibility to M. tuberculosis infection,
we analysed the expression of M. tuberculosis uptake-mediating receptors after pre-exposure
of DCs to either PfRBC or Hz. The recently identified C-type lectin DC-SIGN is the major
receptor for M. tuberculosis on DCs (Geijtenbeek et al., 2003; Tailleux et al., 2003). When
evaluating the cell surface expression of DC-SIGN after co-culturing DCs with Hz, we
detected no change in expression as compared to untreated cells. In addition, exposing DCs to
PfRBC induced a slight increase in the expression of DC-SIGN. The same results were
obtained for MR, another M. tuberculosis receptor expressed on DCs, as well as on
macrophages (Schlesinger, 1993). These findings imply that the decreased susceptibility of
DCs to M. tuberculosis infection cannot be attributed to changed uptake of bacteria due to an
altered receptor expression.
It is possible that the reduced uptake of M. tuberculosis by DCs after pre-exposure to malaria
antigens is simply a consequence of a diminished phagocytic capacity and not a specific event
for M. tuberculosis. To address this question we assessed the phagocytosis of latex beads after
treatment of DCs with Hz. There was a clear loss in phagocytic ability by DCs exposed to Hz,
which indicates a general loss in phagocytic activity as the cause for the reduced uptake of M.
tuberculosis by these DCs. Immature DCs are efficient in antigen uptake and act as sentinels
in the periphery, where they capture antigens and then migrate to draining lymph nodes to
prime T cells. The process of migration is associated with functional and phenotypical
maturation such that, upon arrival in the lymph node the DC has acquired the capacity to
effectively stimulate naïve T cells. This is related to upregulation of MCH class II- and
costimulatory molecules (Steinman, 1991). The functional consequence of maturation is the
DCs’ loss in phagocytic ability. After co-culturing DCs with PfRBC or Hz, we detected an
upregulation of MHC class II- and the costimulatory molecules CD86 and CD40, which
indicated a maturation of the cells, in line with the finding of their reduced phagocytic ability.
59
Efficient induction of T-cell activation is dependent on the interaction between ligands on T
cells with costimulatory molecules expressed on APCs. Considering the previously reported
inhibition of PfRBC of DC responses to the maturation stimulus LPS (Urban et al., 1999), we
expected that the response of DCs to M. tuberculosis infection would be downmodulated after
prior treatment with the malaria antigens. In contrast to the findings by Urban et al., the M.
tuberculosis-induced maturation of DCs was not affected by exposure to either PfRBC or Hz
prior to M. tuberculosis infection. These discrepant findings might be due to the different
stimuli used, and their divergent signalling pathways. M. tuberculosis interacts with various
receptors, including different TLRs (Geijtenbeek et al., 2003; Quesniaux et al., 2004; Bafica
et al., 2005; Floto et al., 2006), while LPS interacts only with TLR4 (Poltorak et al., 1998). In
addition to serving as receptor for PfRBCs and apoptotic cells, CD36 was recently implicated
in mediating responses to microbial ligands through TLR2, by binding to diacylglycerides and
enhancing signalling thorugh the TLR2/TLR6 heterodimer (Hoebe et al., 2005). In addition,
TLR2 agonists are able to block TLR4-induced cytokine production upon concomitant
engagement of TLR2 and TLR4 in human DCs (Re and Strominger, 2004). Binding to CD36
by PfRBC could thereby recruit TLR2 into a signalling complex and affect the signalling
casade induced by LPS through TLR4 (Stevenson and Urban, 2006). Considering the
complexity in the interactions between mycobacteria and DCs, and subsequently the divergent
signalling pathways engaged, the inhibitory effect of PfRBC may be overcome upon M.
tuberculosis infection. Moreover, the induction of maturation by Hz has been shown to be
dependent on TLR9 (Coban et al., 2005). Since human myeloid DCs do not express this
receptor (Mazzoni and Segal, 2004), this pathway should not be of importance in our model.
Besides TCR-mediated recognition of antigens presented by MHC molecules and interaction
with costimulatory molecules expressed on the surface of APCs, activation of T cells is highly
dependent on production of cytokines by APCs. Analysis of cytokine production by DCs after
M. tuberculosis infection demonstrated an enhanced Th1 type of response after prior
treatment to malaria-derived antigens. Both PfRBC- and Hz-treated DCs displayed increased
secretion of TNF-α and reduced secretion of IL-10, following M. tuberculosis infection. In
addition, pre-exposure to PfRBC also triggered IFN-γ production by M. tuberculosis-infected
DCs, while Hz had no effect in this context. As stated earlier, a Th1-biased immune response,
with initiation of cell-mediated immunity, is crucial for resistance against mycobacterial
infections and development of active TB, with IFN-γ and TNF-α being key components. In
60
light of this, our findings allow for the speculation that malaria infection could impose relative
resistance to M. tuberculosis infection, although the ensuing T-cell reponse was not studied in
our model. Moreover, dissemination of mycobacterial infections can be achieved by migration
of infected DCs, implying that the reduced infectivity of PfRBC- or HZ-exposed DCs could
result in a decreased spread of a M. tuberculosis infection. However, although the protective
effect by TNF-α is well established, an exaggerated production can result in host pathology.
Moreover, IL-10 can reduce the extent of inflammatory-induced injury by its
immunouppressive effect (Bogdan et al., 1991; Cunha et al., 1992; Flesch et al., 1994;
Murray et al., 1997; Marshall et al., 1997; Balcewicz-Sablinska et al., 1998).
It is tempting to speculate that an enhanced innate response to M. tuberculosis infection after
prior exposure to MDM, which we found in this study, could have a harmful effect on the
control of malaria infection. The initial response to malaria infection is characterised by a Th1
type of immunity that eventually shifts to a predominantly Th2 type, in order to initiate
antibody-mediated immune mechanisms important for parasite clearance (Taylor-Robinson
and Smith, 1991; Langhorne et al., 2002). A prolonged proinflammatory response could
potentially interfere with this immunological shift.
Taken together, results of our study demonstrate that malaria-blood stage infection can
influence the function of human DCs and have an impact on the outcome and response to a
concurrent M. tuberculosis infection.
61
CONCLUDING REMARKS
TB remains a major health threat worldwide and is the largest cause of death attributable to a
single infectious agent. The causative agent of the disease, M. tuberculosis, has a particular
tropism for the lungs, making pulmonary TB the most common and dangerous form of the
disease. Mucosal surfaces in the respiratory tract are constantly challenged by massive
amounts of inhaled microorganisms and antigens, and in many cases the main protective
effector function against mucosal infections is the production of pathogen-specific local SIgA
responses, achieved solely via activation of the mucosal immune system. The importance of B
cells and antibodies in protection against the intracellular pathogen M. tuberculosis remains
controversial, although a protective capacity of antibodies has lately been reconsidered.
Based on the fact that TB is transmitted via the respiratory tract, where the infection
additionally is manifested, and that IgA in mucosal secretions is the most characteristic
component of mucosal immunity, we investigated the relevance of IgA in protection against
mycobacterial infection in the respiratory tract. We conclude from our studies that IgA
antibodies play a role in the protection against mycobacterial infection in the respiratory tract,
by modulating the locally induced proinflammatory immune responses and/or by blocking the
entrance of the bacillus into the lungs. The mechanisms explaining the defective
proinflammatory responses in the lungs of IgA- and pIgR-deficient mice might involve
impaired signalling through FcαRs, or homologous receptors, which may lead to inadequate
activation of pulmonary macrophages. Insufficient activation of macrophages could
consequently result in suboptimal production of factors important for attraction or migration
of immune cells to the lungs, as well in reduced killing of the bacteria inside the
macrophages. The SC, being extraordinary abundant at mucosal sites, provide additional
innate-like properties that could be of importance for protection of mucosal surfaces.
Induction of effective immune responses cannot be achived without prior activation of the
innate immunity. Production of cytokines and chemokines by macrophages and DCs is crucial
for initiating protective immune reponses against M. tuberculosis, making early recognition of
the invading pathogen essential for containment of infection. TLRs are key molecules in
bridging innate and adaptive immunity. Since M. tuberculosis infects professional phagocytes
that express a diverse array of TLRs, interest is focused on the function and importance of
62
these receptors in combating mycobacterial infections. Our studies demonstrate a protective
role for TLR2, and partly TLR4, in anti-mycobacterial immune responses. However,
important to consider when evaluating involvement of host factors to immunological
responses against infectious agents is the natural route of infection. This is obvious from
divergent results using different routes of infection.
There is increasing evidence that the immune response against one pathogen can influence the
outcome of subsequent infections with other pathogens. Similarly, unrelated infections can
modulate vaccine-induced protective responses to other pathogens. In the light of alarming
reports on the deleterious interaction of co-infecting HIV and M. tuberculosis with the host,
and the now generally accepted harmful conseqences of co-infection beween HIV and
malaria, studies of concurrent infections with more than one pathogen should be emphasized.
Information from such studies will be of importance for vaccine reseach and drug design.
Little is known about the outome of concurrent M. tuberculosis/P. falciparum infections,
although malaria and TB occur endemically in overlapping geographical areas. We developed
an in vitro model for studying the interaction beween the pathogens causing TB and malaria
and human DCs, and our results suggest that a malaria blood-stage infection can affect the
reponse to and outome of an ensuing M. tuberculosis infection. These findings encourage
further studies on the complex immunological events occurring in individuals simultaneously
infected with these two extremely important pathogens.
For the future perspective, it is important to consider the advantages of efficiently targeting
vaccine antigens to appropriate components within the systemic and mucosal immune
compartments. As TB is primarily an infection of the respiratory tract, a successful vaccine
against TB should stimulate appropriate immunological responses to counteract the tubercle
bacillus in the lungs. The effect of concurrent infectious agents, or potential infections, on
vaccination is moreover an essential question to address.
63
ACKNOWLEDGEMENTS
These studies were financially supported by the European Commission specific RTD programs QLK2-CT-199900367 and QLK2-CT-2002-00846, Hjärt- och Lungfonden, the Swedish Agency for Research for Research with
Developing Countries (Sida/SAREC) and EU LSHP-CT-2004 503578.
I wish to express my warm gratitude to everyone who has contributed in any way in making
this thesis a reality and given me support during these intense and fascinating years,
especially:
My supervisors Marita Troye-Blomberg and Andrzej Pawlowski. Marita, my warmest
gratitude for always finding solutions, for believing in me and giving me the trust, strength
and opportunity to carry on and finishing my studies. Thanks for your concern for me and my
situation and for always having my best interest in mind. Andrzej, my deepest gratitude for
dedicating your time and interest in me and my studies, for being a warm, caring, patient, and
helpful person, and for making me grow as a researcher and as a person, and for making me
believe in myself. Thanks for always being willing to answer questions. And for always
knowing the answers!
My previous supervisor Carmen Fernández for your commitment and dedication for my
work and the results we achieved.
Gunilla Källenius for welcoming me to SMI and the TB section and letting me continue my
PhD studies there, and for being a kind and caring person.
My dear friend, former colleague and co-author Ariane Rodríguez for introducing me into
the various techniques and strategies at the department. I am immensely grateful for your
friendship and all the support you have, and continue to give me.
My cherished friend Nancy Vivar for your help, support, patience, laughs, chats at SMI and
MTC. Tanks for being my “ventil” and for your endless patience with me and for your
willingness to teach me everything you know. You inspire me to be a better person!
64
Margareta “Maggan” Hagstedt, the foundation on which the department is built on. My
warmest gratitude to you for all your help and support these years and especially for your help
with our “friends”. I have enjoyed working with you and miss our coffee breaks.
My collaborators and co-authors in Barcelona, Pere-Joan Cardona, Evelyn Guiardo,
Esther Julian, for excellent collaboration and valuable discussions.
Former and present seniors at the department of immunology: Klavs Berzins, Manuchehr
Abedi Valugerdi, Eva Severinson, Eva Sverremark-Ekström and Alf Grandien. Thank
you for sharing your vast knowledge in the field of immunology and for your support and
help with my work.
Gelana Yadeta, for making the department run smoothly and for all your help with one or the
other thing.
Shanie Saghafian Hedengren, I treasure our friendship deeply and I am grateful for all your
support. When the going gets tough, the tough gets going!
Nora Bachmayer and Yvonne Sundström, thanks for your friendship, support and all the
fun we have together! What would I do without you?
Hedvig Perlmann, for your positive and friendly character and for being an inspiration in
science. I would like to recognize late Peter Perlmann whose generous and dedicated
personality has been, and continues to be, an inspiration for researchers in the field of
immunology.
My old roomies Karin Lindroth, Monika Hansson and Masashi Hayano, thanks for your
guidance during my first trembling time at the department, and for being caring and good
friends, even after leaving the department.
Everyone in the TB section at SMI:
Maria Andersson, Solomon Ghebremichael, Melles Haile, Beston Hamasur, Sven
Hoffner, Emma Huitric, Lech Ignatowicz, Pontus Juréen, Tuija Koivula, Lisbeth Klintz,
Jolanta Mazurek, Alexandra Pennhag, Ramona Peterson, Senia Rosales, Juan Carlos
65
Toro and Jim Werngren. Thank you for welcoming me to SMI and making me feel part of
the section. I also want to thank everyone at the Department of Bacteriology, SMI, for
providing a friendly atmosphere where I have enjoyed working. A special thanks to Kristina
Jonas for all your help.
Past and present colleagues at the department of immunology – Petra Amoudruz, John
Arko-Mensah, Nancy Awah, Halima Balogun, Ahmed Bolad, Mounira Djerbi, Salah
Eldin Farouk, Pablo Giusti, Ben Gyan, Ulrika Holmlund, Elisabet Hugosson, Nnaemeka
Iriemenam, Lisa Israelsson, Gun Jönsson, Anna-Karin Larsson, Khosro Masjedi, Jacob
Minang, Amre Nasre, Alice Nyakeriga, Jubayer Rahman, Camilla Rydström, Valentina
Screpanti, Ylva Sjögren, Ankie Söderlund, Khaleda Qazi Rahman, Manijeh Vafa, NinaMaria Vasconcelos, Stefania Varani. A special thanks to Ann Sjölund for your warm
personality and “magic hands” with cells.
Francesca Chiodi at MTC, and the students in her group for being kind and helpful whenever
I came asking for something or desperately wondered where Nancy was.
The staff at the animal house, Eva Nygren and Solveig Sundberg, for taking such good care
of my mice and always greeting me with a smile.
My wonderful and fantastic friends who keep me balanced in life, especially:
Anneli and Mats for being the most wonderful people I have ever met! You make my life
brighter and happier. Thanks for your generosity and letting me enjoy Minus, Yoda and
Snufs whenever I want.
Anna H, for being so stable and making me realize that life is more than work and science,
and for help and support whenever I needed it.
My Friskis companion and dear friend Pernilla for putting up with all my delays during the
years. Maybe a gympa-dinner-movie evening soon?
Eva, min allra käraste vän, thank you for always listening to me, being there for me, and
making my life happy.
66
Marie, you are my role model in life. Your fantastic strength inspires me and keeps me
focused on what is import in life. Little Douglas for being the most adorable boy in the world!
Finally, and most of all, I would like to express my deepest and warmest gratitude to my
beloved family, my mother Margareta, father Sigge, sister Karin and grandmother Vera.
Where in this world would I be without you? What would I be? Thank you for your love and
support throughout my life, and especially the last five years.
Simon, thanks for joining the Tjärnlund family, for your warm and generous personality and
for putting up with all the “dos talk”.
67
REFERENCES
Adachi, O., Kawai, T., Takeda, K., Matsumoto, M., Tsutsui, H., Sakagami, M., Nakanishi, K., and Akira,
S. (1998). Targeted disruption of the MyD88 gene results in loss of IL-1- and IL-18-mediated function. Immunity
9:143-150.
Adebajo, A. O., Smith, D. J., Hazleman, B. L., and Wreghilt, T. G. (1994). Seroepidemiological associations
between tuberculosis, malaria, hepatitis B, and AIDS in West Africa. J. Med. Virol. 42:366-368.
Aderem, A., and Ulevitch, R. J. (2000). Toll-like receptors in the induction of the innate immune response.
Nature 406:782-787.
Aderem, A., and Underhill, D. M. (1999). Mechanisms of phagocytosis in macrophages. Annu. Rev. Immunol.
17:593-623.
Akira, S. (2003). Mammalian Toll-like receptors. Curr. Opin. Immunol. 15:5-11.
Alexandroff, A. B., Jackson, A. M., O’Donnel, M. A., and James, K. (1999). BCG immunotherapy of bladder
cancer: 20 years on. Lancet 353:1689-1694.
Alfredsen, S., and Saxegaard, F. (1992). An outbreak of tuberculosis in pigs and cattle caused by
Mycobacterium africanum. Vet. Rec. 11:51-53.
Algood, H. M., Lin, P. L., Yankura D., Jones, A., Chan, J., and Flynn J. L. (2004). TNF influences
chemokine expression of macrophages in vitro and that of CD11+ cells in vivo during Mycobacterium
tuberculosis infection. J. Immunol. 172:6846-6857.
Aliprantis, A. O., Yang, R. B., Mark, M. R., Suggett, S., Devaux, B., Radolf, J. D., Klimpel, G. R.,
Godowski, P., and Zychlinsky, A. (1999). Cell activation and apoptosis by bacterial lipoproteins through tolllike recptor-2. Science 285:736-739.
Allan, C. H., Mendrick, D. L., and Trier, J. S. (1993). Rat intestinal M cells contain acidic endosomallysosomal compartments and express class II major histocompatibility complex determinants. Gastroenterology
104:698-708.
American Thoracic Society. (2000). Diagnosis standards and classification of tuberculosis in adults and
children. Am. J. Respir. Crit. Care Med. 161:1376-1395.
Andersen, P., Askgaard, D., Ljungvist, L., Bennedsen, J., and Heron, I. (1991). Proteins released from
Mycobacterium tuberculosis during growth. Infect. Immun. 59:1905-1910.
Appelberg, R., Castro, A. G., Gomes, S., Pedrosa, J., and Silva, M. T. (1995). Susceptibility of beige mice to
Mycobacterium avium: role of neutrophils. Infect. Immun. 63:3381-3387.
Arulanandam, B. P., Raeder, R. H., Nedrud, J. G., Bucher, D. J., Le, J., and Metzger, D. W. (2001). IgA
immunodeficiency leads to inadequate Th cell priming and increased susceptibility to influenza virus infection.
J. Immunol. 166:226-231.
Armstrong, L., Medford, A. R., Uppington, K. M., Robertson, J., Withereden, I. R., Tetley, T .D., and
Millar, A. B. (2004). Expression of functional toll-like receptor-2 and -4 on alveolar epithelial cells. Am. J.
Respir. Cell. Mol. Biol. 31:241-245.
Armstrong, J. A., and Hart, P. D. (1975). Phagosome-lysosome interactions in cultured macrophages infected
with virulent tubercle bacilli. Reversal of the usual nonfusion pattern and observations on bacterial survival. J.
Exp. Med. 142:1-16.
68
Artavanis-Tsakonas, K., Eleme, K., McQueen, K. L., Cheng, N. W., Parham, P., Davis, D. M., and Riley,
E. M. (2003). Activation of a subset of human NK cells upon contact with Plasmodium falciparum-infected
erythrocytes. J. Immunol. 171:5396-5405.
Asashi, Y., Yoshikawa, T., Watanabe, I., Iwasaki, T., Hasegawa, H., Sato, Y., Shimada, S., Nanno, M.,
Matsuoka, Y., Ohwaki, M., Iwakura, Y., Suzuki, Y., Aizawa, C., Sata, T., Kurata, T., and Tamura, Y.
(2002). Protection against influenza virus infection in polymeric Ig receptor knockout mice immunized
intranasally with adjuvant-combined vaccines. J. Immunol. 168:2930-2938.
Aston, C., Rom, W. N., Talbot, A. T., and Reibman, J. (1998). Early inhibition of mycobacterial growth by
human alveolar macrophages is not due to nitric oxide. Am. J. Respir. Crit. Care. Med. 157:1943-1950.
Ayele, W. Y., Neill, S. D., Zinsstag, J., Weiss, M. G., and Pavlik, I. (2004). Bovine tuberculosis: an old
disease but a new threat to Africa. Int. J. Tuberc. Lung Dis. 8:924-937.
Bafica, A., Scanga, C. A., Feng, C. G., Leifer, C., Cheever, A., and Sher, A. (2005). TLR9 regulates Th1
responses and cooperates with TLR2 in mediating optimal resistance to Mycobacterium tuberculosis. J. Exp.
Med. 202:1715-1724.
Balasubramanian, R., Garg, R., Santha, T., Gopi, P. G., Subramani, R., Chandrasekaran, V., Thomas, A.,
Rajeswari, R., Anandakrishnan, S., Perumal, M., Niruparani, C., Sudha, G., Jaggarajamma, K., Frieden,
T. R., and Narayanan, P. R. (2004). Gender disparities in tuberculosis: report from a rural DOTS programme
in south India. Int. J. Tuberc. Lung Dis. 8:323-332.
Balcewicz-Sablinska, M. K., Keane, J., Kornfeld, H., and Remold, H. G. (1998). Pathogenic Mycobacterium
tuberculosis evades apoptosis of host macrophages by release of TNF-R2, resulting in inactivation of TNFalpha. J. Immunol. 161:2636-2641.
Barnes, P. F., Chatterjee, D., Abrams, J. S., Lu, S., Wang, E., Yamamura, M., Brennan, P. J., and Modlin,
R. L. (1992). Cytokine production induced by Mycobacterium tuberculosis lipoarabinomannan. Relationship to
chemical structure. J. Immunol. 149:541-547.
Bazaz-Malik, G. (1973). Increased resistance to malaria after Mycobacterium tuberculosis infection. Indian. J.
Med. Res. 61:1014-1024.
Beatty, W. L., Rhoades, E. R., Hsu, D. K., Liu, F. T., and Russell, D. G. (2002). Association of a macrophage
galactoside-binding protein with Mycobacterium-containing phagosomes. Cell. Microbiol. 4:167-176.
Becker, M. D., O’Rourke, L. M., Blackman, W. S., Planck, S. R., and Rosenbaum, J. T. (2000). Reduced
leukocyte migration, but normal rolling and arrest, in interleukin-8 receptor homologue knockout mice. Invest.
Ophthalmol. Vis. Sci. 41:1812-1817.
Behar, S. M., Dascher, C. C., Grusby, D. G., Wang, C. R., and Brenner, M. B. (1999). Susceptibility of mice
deficient in CD1D or TAP1 to infection with Mycobacterium tuberculosis. J. Exp. Med. 189:1973-1980.
Belkaid, Y., and Rouse, B. T. (2005). Natural regulatory T cells in infectious disease. Nat. Immunol. 6:353-360.
Bereczky, S., Montgomery, S. M., Troye-Blomberg, M., Rooth, I., Shaw, M. A., Farnet, A. (2004). Elevated
anti-malarial IgE in asymptomatic individuals is associated with reduced risk for subsequent clinical malaria. Int.
J. Parasitol. 34:935-942.
Bermudez, L. E., and Goodmann, J. (1996). Mycobacterium tuberculosis invades and replicates within type II
alveolar cells. Infect. Immun. 64:1400-1406.
Blender, J. M., and Medzhitov, R. (2004). Regulation of phagosome maturation by signals from toll-like
receptors. Science 304:1014-1018.
Bodnar, K. A., Serbina, N. V., and Flynn, J. L. (2001). Fate of Mycobacterium tuberculosis within murine
dendritic cells. Infect. Immun. 69:800-809.
69
Boehm, M. K., Woof, J. M., Kerr, M. A., and Perkins, S. J. (1999). The Fab and Fc fragments of IgA1 exhibit
a different arrangement from that in IgG: a study by X-ray and neutron solution scattering and homology
modelling. J. Mol. Biol. 286:1421-1447.
Bogdan, C., Vodovotz, Y., and Nathan, C. (1991). Macrophage deactivation by interleukin 10. J. Exp. Med.
174:1549-1555.
Boom, H. W. (1999). γδ T cells and Mycobacterium tuberculosis. Microbes Infect. 1:187-195.
Boros, D. L. (1999). T helper cell populations, cytokine dynamics, and pathology of the schistosome egg
granuloma. Microbes Infect. 1:511-516.
Bosio, C. M., Gardner, D., and Elkins, K. L. (2000). Infection of B cell-deficient mice with CDC 1551, a
clinical isolate of Mycobacterium tuberculosis: delay in dissemination and development of lunh pathology. J.
Immunol. 164:6417-6425.
Boussiotis, V. A., Tsai, E. Y., Yunis, E. J., Thim, S., Delgado, J. C., Dascher, J. J., Berezovskaya, A.,
Rousset, D., Reynes, J M., and Goldfeld, A. E. (2000). IL-10-producing T cells suppress immune responses in
anergic tuberculosis patients. J. Clin. Invest. 105:1317-1325.
Brake, D. A., Long, C. A., and Weidanz, W. P. (1988). Adoptive protection against Plasmodium chabaudi
adami malaria in athymic nude mice by a cloned T cell line. J. Immunol. 140:1989-1993.
Brandt, L., Cunha, J. F., Olsen, A. W. Chilima, B., Hirsch, P., Appelberg, R., and Andersen, P. (2002).
Failure of the Mycobacterium bovis BCG vaccine: some species of environmental mycobacteria block
multiplication of BCG and induction of protective immunity to tuberculosis. Infect. Immun. 70:672-678.
Brandtzaeg, P. (1973). Structure, synthesis and external transfer of mucosal immunoglobulins. Ann. Immunol.
124:417-438.
Brandtzaeg, P. (1989). Overview of the mucosal immune system. Curr. Top. Microbiol. Immunol. 146:13-25.
Brandtzaeg, P., Farstad, I. N., Jahnsen, F. L., Johansen, F-E., Nilsen, E. M., and Yamanaka, T. (1999).
Integration and segregation of the mucosal immune system. 1. What happens in the microcompartments?
Immunol. Today 20:141-151.
Brandtzaeg, P., Karlsson, G., Hansson, G., Petruson, B., Björkander, J., and Hanson, L. A. (1987). The
clinical condition of IgA-deficient patients is related to the proportion of IgD- and IgM-producing cells in their
nasal mucosa. Clin. Exp. Immunol. 67:626-636.
Brightbill, H. D., Libraty, D. H., Krutzik, S. R., Yang, R. B., Belisle, J. T., Bleharski, J. R., Maitland, M.,
Norgard, M. W., Plevy, S. E., Smale, S. T., Brennan, P. J., Bloom, B. R., Godowski, P. J., and Modlin, R.
L. (1999). Host defense mechanisms triggered by microbial lipoproteins trough toll-like receptors. Science
285:732-736.
Brown, A. E., Holzer, T. J., and Andersen, B. R. (1987). Capacity of human neutrophils to kill Mycobacterium
tuberculosis. J. Infect. Dis. 156:985-989.
Bulut, Y., Faure, E., Thomas, L., Equils, O., and Arditi, M. (2001). Cooperation of Toll-like receptor 2 and 6
for cellular activation by soluble tuberculosis factor and Borrelia burgdorferi outer surface protein A lipoprotein:
role of Toll-interacting protein and IL-1 receptor signaling molecules in Toll-like receptor 2 signaling. J.
Immunol. 167:987-994.
Burks, A. W. Jr., and Steele, R. W. (1986). Selective IgA deficiency. Ann. Allergy 57:3-13.
Burns, J. W., Siadat-Pajouh, M., Krishnaney, A. A., and Greenberg, H. B. (1996). Protective effect of
rotavirus VP6-specific IgA monoclonal antibodies that lack neutralizing activity. Science 272:104-107.
Burrows, P. D., and Cooper, M. D. (1997). IgA deficiency. Adv. Immunol. 65:245-276.
70
Butcher, E. C., and Picker, L. J. (1996). Lymphocyte homing and homeostasis. Science 272:60-66.
Carlson, J., Ekre, H. P., Helmby, H., Gysin, J., Greenwood, B. M, and Wahlgren, M. (1992). Disruption of
Plasmodium falciparum erythrocyte rosettes by standard heparin and heparin devoid of anticoagulant activity.
Am. J. Trop. Med. Hyg. 46:595-560.
Caruso, A. M., Serbina, N., Klein, E., Triebold, K., Bloom, B. R., and Flynn, J. L. (1999). Mice deficient in
CD4 T cells have only transiently diminished levels of IFN-γ, yet succumb to tuberculosis. J. Immunol.
162:5407-5416.
Cerretti, D. P., Nelson, N., Kozlosky, C. J., Morrissey, P. J., Copeland, N. G., Gilbert, D. J., Jenkins, N. A.,
Dosik, J. K., and Mock, B. A. (1993). The murine homologue of the human interleukin-8 receptor type B maps
near the Ity-Lsh-Bcg disease resistance locus. Genomics 18:410-413.
Chadha, V. K., Vaidyanathan, P. S., Jagannatha, P. S., Unnikrishnan, K. P., Savanur, S. J., and Mini, P.
A. (2003). Annual risk of tuberculous infection in the western zone of India. Int. J. Tuberc. Lung Dis. 7:536-542.
Chambers, M. A., Gavier-Widen, D., and Hewinson, R. G. (2004). Antibody bound to the surface antigen
MPB83 of Mycobacterium bovis enhances survival against high dose and low dose challenge. FEMS Immunol.
Med. Microbiol. 41:93-100.
Champbell, D. J., Debes, G. F., Johnston, B., Wilson, E., and Butcher, E. C. (2003). Targeting T cell
responses by selective chemokine receptor expression. Semin. Immunol. 15:277-286.
Chan, E. D., Chan, J., and Schluger, N. W. (2001). What is the role of nitric oxide in murine and human host
defense against tuberculosis? Am. J. Respir. Cell Mol. Biol. 25:606-612.
Chen, L., Wang, J., Zganiacz, A., and Xing, Z. (2004). Single intranasal mucosal Mycobacterium bovis BCG
vaccination confers improved protection compared to subcutaneous vaccination against pulmonary tuberculosis.
Infect. Immun. 72:238-246.
Chen, X., Zhou, B., Li, M., Deng, Q., Wu, X., Le, X., Wu, C., Larmonier, N., Zhang, W., Zhang, H., Wang,
H., and Katsanis, E. (2007). CD4(+)CD25(+)FoxP3(+) regulatory T cells suppress Mycobacterium tuberculosis
immunity in patients with active disease. Clin. Immunol. 123:50-59.
Chensue, S. W., Warmington, K. S., Allenspach, E. J., Lu, B., Gerard, C., Kunkel, S. L., and Lukacs, N.
W. (1999). Differential expression and cross-regulatory function of RANTES during mycobacterial (type 1) and
schistosomal (type 2) antigen-elicited granulomatous inflammation. J. Immunol. 163:165-173.
Cho, S., Mehra, V., Thoma-Uszynski, S., Stenger, S., Serbina, N., Mazzaccaro, R. J., Flynn, J. L., Barnes,
P. F., Southwood, S., Celis, E., Bloom, B. R., Modlin, R. L., and Sette, A. (2000). Antimicrobial activity of
MHC class I-restricted CD8+ T cells in human tuberculosis. Proc. Natl. Acad. Sci. USA 97:12210-12215.
Clark, I. A., Allison, A. C., and Cox, F. E. (1976). Protection of mice against Babesia and Plasmodium with
BCG. Nature 259:309-311.
Clemens, D. L., and Horwitz, M A. (1995). Characterization of the Mycobacterium tuberculosis phagosome
and evidence that phagosomal maturation is inhibited. J. Exp. Med. 181:257-270.
Coban, C., Ishii, K. J., Kawai, T., Hemmi, H., Sato, S., Uematsu, S., Yamamoto, M., Takeuchi, O., Itagaki,
S., Kumar, N., Horii, T., and Akira, S. (2005). Toll-like receptor 9 mediates innate immune activation by the
malaria pigment hemozoin. J. Exp. Med. 201:19-25.
Cohen, R. A., Muzaffar, S., Schwartz, D., Bashir, S., Luke, S., McGartland, L. P., and Kaul, K. (1998).
Diagnosis of pulmonary tuberculosis using PCR assays on sputum collected within 24 hours of hospital
admission. Am. J. Respir. Crit. Care Med. 157:156-161.
Coker, R. J. (2004). Multidrug-resistant tuberculosis: public health challenges. Trop. Med. Int. Health 9:25-40.
71
Cooper, A. M., Dalton, D. K., Stewart, T. A., Griffin, J. P., Russell, D. G., and Orme, I. M. (1993).
Disseminated tuberculosis in interferon gamma gene-disrupted mice. J. Exp. Med. 178:2243-2247.
Cunha, F. Q., Moncada, S., and Liew, F. Y. (1992). Interleukin-10 (IL-10) inhibits the induction of nitric
oxide synthase by interferon-gamma in murine macrophages. Biochem. Biophys. Res. Commun. 182:1155-1159.
Cunningham-Rundles, C. (2001). Physiology of IgA and IgA deficiency. J. Clin. Immunol. 21:303-309.
Curry, A. J., Else, K. J., Jones, F., Bancroft, A., Grencis, R. K., and Dunne, D. W. (1995). Evidence that
cytokine-mediated immune interactions induced by Schistosoma mansoni alter disease outcome in mice
concurrently infected with Trichuris muris. J. Exp. Med. 181:769-774.
Dairaghi, D. J., Soo, K. S., Oldham, E. R., Premack, B. A., Kitamura, T., Bacon, K. B., and Schall, T. J.
(1998). RANTES-induced T cell activation correlates with CD3 expression. J. Immunol. 160:426-433.
Dallas, S. D., and Rolfe, R. D. (1998). Binding of Clostridium difficile toxin A to human milk secretory
component. J. Med. Microbiol. 47:879-888.
Dannenberg, A. M. Jr. (1994). Roles of cytotoxic delayed-type hypersensitivity and macrophage-activating
cell-mediated immunity in the pathogenesis of tuberculosis. Immunobiology 191:461-473.
Davis, S. S. (2001). Nasal vaccines. Adv. Drug Deliv. Rev. 51:21-42.
Davis, M. M., and Björkman, P. J. (1988). T-cell antigen receptor gene and T-cell recognition. Nature
334:395-402.
de Chastellier, C., and Thilo, L. (1997). Phagosome maturation and fusion with lysosomes in relation to
surface property and size of the phagocytic particle. Eur. J. Cell. Biol. 74:49-62.
Denis, M. (1991). Human neutrophils, activated with cytokines or not, do not kill virulent Mycobacterium
tuberculosis. J. Infect. Dis. 163:919-920.
de Oliviera, I. R., de Araujo, A. N., Bao, S. N., and Giugliano, L. G. (2001). Binding of lactoferrin and free
secretory component to enterotoxigenic Escherichia coli. FEMS Microbiol. Lett. 203:29-33.
Dieli, F., Ivanyi, J., Marsh, P., Williams, A., Naylor, I., Sireci, G., Caccamo, N., Di Sano, C., and Salerno,
A. (2003). Characterization of lung γδ T cells following intranasal infection with Mycobacterium bovis bacillus
Calmette-Guerin. J. Immunol. 170:463-469.
Dieli, F., Troye-Blomberg, M., Farouk, S. E., Sirecil, G., and Salerno, A. (2001). Biology of gammadelta T
cells in tuberculosis and malaria. Curr. Mol. Med. 1:437-46.
Dolo, A., Modiano, D., Maiga, B., Daou, M., Dolo, G., Guindo, H., Ba, M., Maiga, H., Coulibaly, D.,
Perlmann, H., Troye-Blomberg, M., Toure, Y. T., Coluzzi, M., and Doumbo, O. (2005). Difference in
susceptibility to malaria between two sympatric ethnic groups in Mali. Am. J. Trop. Med. Hyg. 72:243-248.
Downing, J. F., Pasula, R., Wright, J. R., Twigg III, H. L., and Martin II, W. J. (1995). Surfactant protein A
promotes attachment of Mycobacterium tuberculosis to alveolar macrophages during infection with human
immunodeficiency virus. Proc. Natl. Acad. Sci. USA 92:4848-4852.
Drennan, M. B., Nicolle, D., Quesniaux, V. J., Jacobs, M., Allie, N., Mpagi, J., Fremond, C., Wagner, H.,
Kirschning, C., and Ryffel, B. (2004). Toll-like receptor 2-deficient mice succumb to Mycobacterium
tuberculosis infection. Am. J. Pathol. 164:49-57.
D’Souza, C. D., Cooper, A. M., Frank, A. A., Mazzaccaro, R. J., Bloom, B. R., and Orme, I. M. (1997). An
anti-inflammatory role for gamma delta T lymphocytes in aquired immunity to Mycobacterium tuberculosis. J.
Immunol. 158:1217-1221.
Edelson, B. T., and Unanue, E. R. (2001). Intracellular antibody neutralizes Listeria growth. Immunity 14:503512.
72
Egan, T. J., Mavuso, W. W., and Ncokazi, K. K. (2001). The mechanism of beta-hematin formation in acetate
solution. Parallels between hemozoin formation and biomineralization processes. Biochemistry 40:204-213.
Ehlers, S. (2003). Role of tumour necrosis factor (TNF) in host defence against tuberculosis: implications for
immunotherapies targeting TNF. Ann. Rheum. Dis. 62:Suppl 2:ii37-42.
Elias, D., Akuffo, H., Pawlowski, A., Haile, M., Schon, T., and Britton, S. (2005a). Schistosoma mansoni
infection reduces the protective efficacy of BCG vaccination against virulent Mycobacterium tuberculosis.
Vaccine 23:1326-1334.
Elias, D., Akuffo, H., Thors, C., Pawlowski, A., and Britton, S. (2005b). Low dose chronic Schistosoma
mansoni infection increases susceptibility to Mycobacterium bovis BCG infection in mice. Clin. Exp. Immunol.
139:398-404.
Engering, A, Geijtenbeek, T. B. H., van Vliet, S. J., Wijers, M., van Liempt, E., Demaurex, N.,
Lanzavecchia, A., Fransen, J., Figdor, C. G., Piguet, V., and van Kooyk, Y. (2002). The dendritic cellspecific adhesion receptor DC-SIGN internalizes antigen for presentation to T cells. J. Immunol. 168:2118-2126.
Falero-Diaz, G., Challacombe, S., Banerjee, D., Douce, G., Boyd, A., and Ivanyi, J. (2000). Intranasal
vaccination of mice against infection with Mycobacterium tuberculosis. Vaccine 18:3223-3229.
Fanning, A. (1999). Tuberculosis: 6. Extrapulmonary disease. CMAJ 160:1597-1603.
Favre, L., Spertini, F., and Corthésy, B. (2005). Secretory IgA possesses intrinsic modulatory properties
stimulating mucosal and systemic immune responses. J. Immunol. 175:2793-2800.
Feng, C. G., Scanga, C. A., Collazo-Custodio, C. M., Cheever, A. W., Hieny, S., Caspar, P., and Sher, A.
(2003). Mice lacking myeloid differentiation factor 88 display profound defects in host resistance and immune
responses to Mycobacterium avium infection not exhibited by Toll-like receptor 2 (TLR2)- and TLR4-deficient
animals. J. Immunol. 171:4758-4764.
Feng, N., Lawton, J. A., Gilbert, J., Kuklin, N., Vo, P., Prasad, B. V., and Greenberg, H. B. (2002).
Inhibition of rotavirus replication by a non-neutralizing, rotavirus VP6-specific IgA mAb. J. Clin. Invest.
109:1203-1213.
Ferrari, G., Langen, H., Naito, M., and Pieters, J. (1999). A coat protein on phagosomes involved in the
intracellular survival of mycobacteria. Cell 97:435-447.
Fine, P. E. M. (1995a). Bacille Calmette-Guérin vaccines: a rough guide. Clin. Infect. Dis. 20:11-14.
Fine, P. E. M. (1995b). Variation in protection by BCG – implication of and for heterologous immunity. Lancet
346:1339-1345.
Flesch, I. E., Hess, J. H., Oswald, I. P., and Kaufmann, S. H. (1994). Growth inhibition of Mycobacterium
bovis by IFN-gamma stimulated macrophages: regulation by endogenous tumor necrosis factor-alpha and by IL10. Int. Immunol. 6:693-700.
Floto, R. A., MacAry, P. A., Boname, J. M., Mien, T. S., Kampmann, B., Hair, J. R., Huey, O. S., Houben,
E. N., Pieters, J., Day, C., Oehlmann, W., Singh, M., Smith, K. G., and Lehner, P. J. (2006). Dendritic cell
stimulation by mycobacterial Hsp70 is mediated through CCR5. Science 314:454-458.
Flynn, J. L., Chan, J., Triebold, K. J., Dalton, D. K., Stewart, T., and Bloom, B. R. (1993). An essential role
for Interferon-γ in resistance to Mycobacterium tuberculosis infection. J. Exp. Med. 178:2249-2254.
Flynn, J. L., Goldstein, M. M., Triebold, K. J., Koller, B., and Bloom, B. R. (1992). Major histocompatibility
complex class I-restricted T cells are required for resistance to Mycobacterium tuberculosis infection. Proc. Natl.
Acad. Sci. USA 89:12013-12017.
73
Flynn, J. L., Goldstein, M. M., Chan, J., Triebold, K. J., Pfeffer, K., Lowenstain, C. J., Schreiber, R., Mak,
T. W., and Bloom, B. R. (1995). Tumor necrosis factor-alpha is required in the protective immune response
against Mycobacterium tuberculosis in mice. Immunity 2:561-572.
Flynn, J. L., Scanga, C. A., Tanaka, K. E., and Chan, J. (1998). Effects of aminoguanidine on latent murine
tuberculosis. J. Immunol. 160:1796-1803.
Fontenot, J. D., and Rudensky, A. Y. (2005). A well adapted regulatory contrivance: regulatory T cell
development and the forkhead family transcription factor Foxp3. Nat. Immunol. 6:331-337.
Fratazzi, C., Arbeit, R. D., Carini, C., Balcewicz-Sablinska, M. K., Keane, J., Kornfeld, H., and Remold, H
G. (1999). Macrophage apoptosis in mycobacterial infections. J. Leukoc. Biol. 66:763-764.
Fremond, C. M., Yeremeev, V., Nicolle, D. M., Jacobs, M., Quesniaux, V. F., and Ryffel, B. (2004). Fatal
Mycobacterium tuberculosis infection despite adaptive immune response in the absence of MyD88. J. Clin.
Invest. 114:1790-1799.
Fujioka, H., Emancipator, S. N., Aikawa, M., Huang, D. S., Blatnik, F., Karban, T., DeFife, K., and
Mazanec, M. B. (1998). Immunocytochemical colocalization of specific immunoglobulin A with sendai virus
protein in infected polarized epithelium. J. Exp. Med. 188:1223-1229.
Fulton, S. A., Reba, S. M., Martin, T. D., and Boom, W. H. (2002). Neutrophil-mediated mycobacteriocidal
immunity in the lung during Mycobacterium bovis BCG infection in C57BL/6 mice. Infect. Immun. 70:53225317.
Furtado, P. B., Whitty, P. W., Robertson, A., Eaton, J. T., Almogren, A., Kerr, M. A., Woof, J. M., and
Perkins, S. J. (2004). Solution structure determination of monomeric human IgA2 by X-ray and neutron
scattering, analytical ultracentrifugation and constrained modelling: a comparison with monomeric human IgA1.
J. Mol. Biol. 338:921-941.
Gallucci, S., Lolkema, M., and Matzinger, P. (1999). Natural adjuvants: endogenous activators of dendritic
cells. Nat. Med. 5:1249-1255.
Garcia-Perez, B. E., Mondragen-Flores, R., and Luna-Herrera, J. (2003). Internalization of Mycobacterium
tuberculosis by macropinocytosis in non-phagocytic cells. Microb. Pathog. 35:49-55.
Geijtenbeek, T. B. H., Torensma, R., van Vliet, S. J., van Duijnhoven, G. C. F., Adema, G. J., van Kooyk,
Y., and Figdor, C. G. (2000). Identification of DC-SIGN, a novel dendritic cell-specific ICAM-3 receptor that
supports primary immune responses. Cell 100:575-585.
Geijtenbeek, T. B. H., van Vliet, S. J., Koppel, E. A., Sanchez-Hernandez, M., Vandenbroucke-Grauls, C.
M., Appelmelk, B., and van Kooyk Y. (2003). Mycobacteria target DC-SIGN to suppress dendritic cell
function. J. Exp. Med. 197:7-17.
Geissmann, F., Launay, P., Pasquier, B., Lepelletier, Y., Leborgne, M., Lehuen, A., Brousse, N., and
Monteiro, R. C. (2001). A subset of human dendritic cells expresses IgA Fc receptor (CD89), which mediates
internalization and activation upon cross-linking by IgA complexes. J. Immunol. 166:346-352.
Giacomini, E., Iona, E., Ferroni, L., Miettinen, M., Fattorini, L., Orefici, G., Julkunen, I., and Coccia, E.
M. (2001). Infection of human macrophages and dendritic cells with Mycobacterium tuberculosis induces a
differential cytokine gene expression that modulates T cell response. J. Immunol. 166:7033-7041.
Glatman-Freedman, A. (2003). Advances in antibody-mediated immunity against Mycobacterium tuberculosis:
implications for a novel vaccine strategy. FEMS Immunol. Med. Microbiol. 39:9-16.
Gong, J. H., Zhang, M., Modlin, R. L., Linsley, P. S., Iyer, D., Lin, Y., and Barnes, P. F. (1996). Interleukin10 downregulates Mycobacterium tuberculosis-induced Th1 responses and CTLA-4 expression. Infect. Immun.
64:913-918.
74
Gonzalez-Juarrero, M., Turner, O. C., Turner, J., Marietta, P., Brooks, J. V., and Orme, I. M. (2001).
Temporal and spatial arrangement of lymphocytes within lung granulomas induced by aerosol infection with
Mycobacterium tuberculosis. Infect. Immun. 69:1722-1728.
Good, M. F., and Doolan, D. L. (1999). Immune effector mechanisms in malaria. Curr. Opin. Immunol.
11:412-419.
Guillot, L., Balloy, V., McCormack, F. X., Golenbock, D. T., Chignard, M., and Si-Tahar, M. (2002).
Cutting edge: the immunostimulatory activity of the lung surfactant protein-A involves Toll-like receptor 4. J.
Immunol. 168:5989-5992.
Hajjar, A. M., O’Mahony, D. S., Ozinsky, A., Underhill, D. M., Aderem, A., Klebanoff, S. J., and Wilson,
C. B. (2001). Cutting edge: functional interactions between toll-like receptor (TLR) 2 and TLR1 or TLR6 in
response to phenol-soluble modulin. J. Immunol. 166:15-19.
Hamasur, B., Haile, M., Pawlowski, A., Schröder, U., Källenius, G., Svenson, S. B. (2004). A mycobacterial
lipoarabinomannan specific monoclonal antibody and its F(ab') fragment prolong survival of mice infected with
Mycobacterium tuberculosis. Clin. Exp. Immunol. 138:30-38.
Hammarström, L., Vorechovsky, I., and Webster, D. (2000). Selective IgA deficiency (SIgAD) and common
variable immunodeficiency (CVID). Clin. Exp. Immunol. 120:225-231.
Hang, L., Frendeus, B., Godaly, G., and Svanborg, C. (2000). Interleukin-8 receptor knockout mice have
subepithelial neutrophil entrapment and renal scarring following acute pyelonephritis. J. Infect. Dis. 182:17381748.
Hanker, J. S., and Giammara, B. L. (1983). Neutrophil pseudoplatelets: their discrimination by
myeloperoxidase demonstration. Science 220:415-417.
Harriman, G. R., Bogue, M., Rogers, P., Finegold, M., Pacheco, S., Bradley, A., Zhang, Y., and Mbawuike,
I. N. (1999). Targeted deletion of the IgA constant region in mice leads to IgA deficiency with alterations in
expression of other Ig isotypes. J. Immunol. 162:2521-2529.
Hatch, T. F. (1942). Behavior of microscopic particles in the air and the respiratory system. Aerobiology, Publ.
No. 17. Amer. Assn. Adv. Sci. 102-105.
Havlir, D. V., Ellner, J. J., Chervenak, K. A., and Boom, W. H. (1991). Selective expansion of human
gamma delta T cells by monocytes infected with live Mycobacterium tuberculosis. J. Clin. Invest. 87:729-733.
Heldwein, K. A., Liang, M. D., Andresen, T. K., Thomas, K. E., Marty, A. M., Cuesta, N., Vogel, S. N., and
Fenton, M. J. (2003). TLR2 and TLR4 serve disinct roles in the host immune response against Mycobacterium
bovis BCG. J. Leukoc. Biol. 74:277-286.
Hellwig, S. M., van Spriel, A. B., Schellekens, J. F., Mooi, F. R., and van de Winkel, J. G. (2001).
Immunoglobulin A-mediated potection against Bordetella pertussis infection. Infect. Immun. 69:4846-4850.
Helmby, H., Kullberg, M., and Troye-Blomberg, M. (1998). Altered immune responses in mice with
concomitant Schistosoma mansoni and Plasmodium chabaudi infections. Infect. Immun. 66:5167-5174.
Hendersen, R. A., Watkins, S. C., and Flynn, J. L. (1997). Activation of human dendritic cells following
infection with Mycobacterium tuberculosis. J. Immunol. 159:635-643.
Hertz, C. J., Kiertscher, S. M., Godowski, P. J., Bouis, D. A., Norgard, M. V., Roth, M. D., and Modlin
RL. (2001). Microbial lipopeptides stimulate dendritic cell maturation via Toll-like receptor 2. J. Immunol.
166:2444-2450.
Hertz, C. J., Wu, Q., Porter, E. M., Zhang, Y. J., Weismuller, K. H., Godowski, P. J., Ganz, T., Randell, S.
H., and Modlin, R. L. (2003). Activation of Toll-like receptor 2 on human tracheobronchial epithelial cells
induces the antimicrobial peptide human beta defensin-2. J. Immunol. 171:6820-6826.
75
Hess, J., and Kaufmann, S. H. (1999). Live antigen carriers as tools for improved antituberculosis vaccines.
FEMS Immunol. Med. Microbiol. 23:165-173.
Hickman, S. P., Chan, J., and Salgame, P. (2002). Mycobacterium tuberculosis induces differential cytokine
production from dendritic cells and macrophages with divergent effects on naïve T cell polarization. J. Immunol.
168:4636-4642.
Hirsch, C. S., Ellner, J. J., Blinkhorn, R., and Toossi, Z. (1997). In vitro restoration of T cell responses in
tuberculosis and augmentation of monocyte effector function against Mycobacterium tuberculosis by natural
inhibitors of transforming growth factor beta. Proc. Natl. Acad. Sci. USA. 94:3926-3931.
Hirsch, C. S., Ellner, J. J., Russell, D. G., and Rich, E. A. (1994). Complement receptor-mediated uptake and
tumor necrosis factor-alpha-mediated growth inhibition of Mycobacterium tuberculosis by human alveolar
macrophages. J. Immunol. 152:743-753.
Hirsch, C. S., Hussein, R., Toossi, Z., Dawood, G., Shahid, F., and Ellner, J. J. (1996). Cross-modulation by
transforming growth factor beta in human tuberculosis: suppression of antigen-driven blastogenesis and
interferon gamma production. Proc. Natl. Acad. Sci. USA. 93:3193-3198.
Hirsch, C. S., Toossi, Z., Othieno, C., Johnson, J. L., Schwander, S. K., Robertson, S., Wallis, R. S.,
Edmonds, K., Okwere, A., Mugerwa, R., Peters, P., and Ellner, J. J. (1999). Depressed T-cell interferongamma responses in pulmonary tuberculosis: analysis of underlying mechanisms and modulation with therapy. J.
Infect. Dis. 180:2069-2073.
Hoebe, K., Georgel, P., Rutschmann, S., Du, X., Mudd, S., Crozat, K., Sovath, S., Shamel, L., Hartung, T.,
Zahringer, U., and Beutler, B. (2005). CD36 is a sensor of diacylglycerides. Nature 433:523-527.
Hoffmann, J. A., Kafatos, F. C., Janeway, C. A., and Ezekowitz, R. A. (1999). Phylogenetic perspectives in
innate immunity. Science 284:1313-1318.
Holmgren, J., Adamsson, J., Anjuere, F., Clemens, J., Czerkinsky, C., Eriksson, K., Flach, C. F., GeorgeChandy, A., Harandi, A. M., Lebens, M., Lehner, T., Lindblad, M., Nygren, E,. Raghavan, S., Sanchez, J.,
Stadford, M., Sun, J. B., Svennerholm, A. M., and Tengvall, S. (2005). Mucosal adjuvants and anti-infection
and anti-immunopathology vaccines based on cholera toxin, cholera toxin B subunit and CpG DNA. Immunol.
Lett. 97:181-188.
Honorio-Franca, A. C., Launay, P., Carneiro-Sampaio, M. M., and Monteiro, R. C. (2001). Colostral
neutrophils express Fc alpha receptors (CD89) lacking gamma chain association and mediate noninflammatory
properties of secretory IgA. J. Leukoc. Biol. 69:289-296.
Hoshino, K., Takeuchi, O., Kawai, T., Sanjo, H., Ogawa, T., Takeda, Y., Takeda, K., and Akira, S. (1999).
Cutting edge: Toll-like receptor 4 (TLR4)-deficient mice are hyporesponsive to lipopolysaccaride: Evidence for
TLR4 as the Lps gene product. J. Immunol. 162:3749-3752.
Huang, Y. T., Wright, A., Gao, X., Kulick, L., Yan, H., and Lamm, M. E. (2005). Intraepithelial cell
neutralization of HIV-1 replication by IgA. J. Immunol. 174:4828-4835.
Huygen, K. (1998). DNA vaccines: application to tuberculosis. Int. J. Tuberc. Lung. Dis. 2:971-978.
Huygen, K., Van Vooren, J. P., Turneer, M., Bosmans, R., Dierchx, P., and De Bruyn, J. (1988). Specific
lymphoproliferation, gamma interferon production, and serum immunoglobulin G directed against a purified 32
kDa mycobacterial protein antigen (P32) in patients with active tuberculosis. Scand. J. Immunol. 27:187-194.
Izzo, A. A., and North, R. J. (1992). Evidence for an α/β T cell-independent mechanism of resistance to
mycobacteria. Bacillus-Calmette-Guerin causes progressive infection in severe combined immunodeficient mice,
but not in nude mice or in mice depleted o CD4+ and CD8+ T cells. J. Exp. Med. 176:581-586.
Johansen, F. E., Pekna, M., Norderhaug, I. N., Haneberg, B., Hietala, M. A., Krajci, P., Betsholtz, C., and
Brandtzaeg, P. (1999). Absence of epithelial immunoglobulin A transport, with increased mucosal leakiness, in
polymeric immunoglobulin receptor/secretory component-deficient mice. J. Exp. Med. 190:915-922.
76
Johansen, F. E., Braathen, R., and Brandtzaeg, P. (2000). Role of J chain in secretory immunoglobulin
formation. Scand. J. Immunol. 52:240-248.
Johnson, C. M., Cooper, A. M., Frank, A. A., Bonorino, C. B., Wysoki, L. J., and Orme, I. M. (1997).
Mycobacterium tuberculosis aerogenic rechallenge infections in B cell-deficient mice. Tuber. Lung Dis. 78:257261.
Jones, G. S., Amiarult, H. J., and Andersen, B. R. (1990). Killing of Mycobacterium tuberculosis by
neutrophils: a nonoxidative process. J. Infect. Dis. 162:700-704.
Kabelitz, D. (2007). Expression and function of Toll-like receptors in T lymphocytes. Curr. Opin. Immunol.
19:39-45.
Kabelitz, D., Bender, A., Prospero, T., Wesselborg, S., Janssen, O., and Pechhold, K. (1991). The primary
response of human gamma/delta T cells to Mycobacterium tuberculosis is restricted to V gamma 9-bearing cells.
J. Exp. Med. 172:1331-1338.
Kaetzel, C. S., Robinson, J. K., Chintalacharuvu, K. R., Vaerman, J-P., and Lamm, M. E. (1991). The
polymeric immunoglobulin receptor (secretory component) mediates transport of immune complexes across
epithelial cells: a local defense function of IgA. Proc. Natl. Acad. Sci. USA 88:8796-8800.
Kagnoff, M. F., and Eckmann, L. (1997). Epithelial cells as sensors for microbial infection. J. Clin. Invest.
100:6-10.
Kamala, T., Paramasivan, C. N., Herbert, D., Venkatesan, P., and Prabhakar, R. (1996). Immune response
and modulation of immune response induced in the guinea-pigs by Mycobacterium avium complex (MAC) & M.
fortuitum complex isolates from different sources in the south Indian BCG trial area. Ind. J. Med. Res. 103:201211.
Kamath, A. B., Alt, J., Debbabi, H., and Behar, S. M. (2003). Toll-like receptor 4-defective C3H/HeJ mice
are not more susceptible than other C3H substrains to infection with Mycobacterium tuberculosis. Infect. Immun.
71:4112-4118.
Kasama, T., Strieter, R. M., Standiford, T. J, Burdick, M. D., and Kunkel, S. L. (1993). Expression and
regulation of human neutrophil-derived macrophage inflammatory protein 1 alpha. J. Exp. Med. 178:63-72.
Kaufman, S. H. E. (1996). Gamma/delta and other unconventional T lymphocytes: what do they see and what
do they do? Proc. Natl. Acad. Sci. USA 93:2272-2279.
Kaufmann, S. H. E. (2001). How can immunology contribute to the control of tuberculosis? Nat. Rev. Immunol.
1:20-30.
Kaufmann, S. H. E. (2003). Immune response to tuberculosis: experimental animal models. Tuberculosis
(Edinb.) 83:107-111.
Kawai, T., Adachi, O., Ogawa, T., Takeda, K., and Akira, S. (1999). Unresponsiveness of MyD88-deficient
mice to endotoxin. Immunity 11:115-122.
Kerr, M. A. (1990). The structure and function of human IgA. Biochem. J. 271:285-296.
Kilian, M., Reinholdt, J., Lomholt, H., Poulsen, K., and Frandsen, E V. (1996). Biological significance of
IgA1 proteases in bacterial colonization and pathogenesis: critical evaluation of experimental evidence. APMIS
104:321-338.
Kindler, V., Sappino, A P., Grau, G. E., Piguet, P. F., and Vassalli, P. (1989). The inducing role of tumor
necrosis factor in the development of bactericidal granulomas during BCG infection. Cell 56:731-740.
Kisich, K. O., Higgins, M., Diamond, G, and Heifets, L. (2002). Tumor necrosis factor alpha stimulates killing
of Mycobacterium tuberculosis by human neutrophils. Infect. Immun. 70:4591-4599.
77
Kiyono, H., and Fukuyama, S. (2004). NALT-versus Peyer’s patch-mediated mucosal immunity. Nat. Rev.
Immunol. 4:699-710.
Kolappan, C., Gopi, P. G., Subramani, R., Chadha, V. K., Kumar, P., Prasad, V. V., Appegowda, B. N.,
Rao, R. S., Sashidharan, R., Ganesan, N., Santha, T., and Narayanan, P. R. (2004). Estimation of annual
risk of tuberculosis infection (ARTI) among children aged 1-9 years in the south zone of India. Int. J. Tuberc.
Lung Dis. 8:418-423.
Kremer, K., van Soolingen, D., van Embden, J., Hughes, S., Inwald, J., and Hewinson, G. (1998).
Mycobacterium microti: more widespread than previously thought. J. Clin. Microbiol. 36:2793-2794.
Kristensen, I., Aaby, P., and Jensen, H. (2000). Routine vaccinations and child survival: follow up study in
Guinea-Bissau, West Africa. B. M. J. 321:1435-1438.
Krutzik, S. R., and Modlin, R. L. (2004). The role of Toll-like receptor in combating mycobacteria. Semin.
Immunol. 16:35-41.
Kubagawa, H., Burrows, P., and Cooper, M. D. (1997). A novel pair of immunoglobulin-like receptors
expressed by B cells and myeloid cells. Proc. Natl. Acad. Sci. USA 94:5261-5266.
Kumar, S., Good, M. F., Dontfraid, F., Vinetz, J. M., and Miller, L. H. (1989). Interdependence of CD4+ T
cells and malarial spleen in immunity to Plasmodium vinckei vinckei. Relevance to vaccine development. J.
Immunol. 143:2017-2023.
Kunkel, E. J., and Butcher, E. C. (2003). Plasma-cell homing. Nat. Rev. Immunol. 3:822-829.
Kvale, D., Lovhaug, D., Sollid, L. M., and Brandtzaeg, P. (1988). Tumor necrosis factor-α up-regulates
expression of secretory component, the epithelial receptor for polymeric Ig. J. Immunol. 140:3086-3089.
Lamm, M. E. (1997). Interaction of antigens and antibodies at mucosal surfaces. Annu. Rev. Microbiol. 51:311340.
Langhorne, J., Quin, S. J., and Sanni, L. A. (2002). Mouse models of blood-stage malaria infections: immune
responses and cytokines involved in protection and pathology. Chem. Immunol. 80:204-228.
Lee, J., Cacalano, G., Camerato, T., Toy, K., Moore, M. W., and Wood, W. I. (1995). Chemokine binding
and activities mediated by the mouse IL-8 receptor. J Immunol. 155:2158-2164.
Leisewitz, A. L., Rockett, K. A., Gumede, B., Jones, M., Urban, B., and Kwiatkowski, D. P. (2004).
Response of the splenic dendritic cell population to malaria infection. Infect. Immun. 72:4233-4239.
Li, M., Carpio, D. F., Zheng, Y., Bruzzo, P., Singh, V., Ouaaz, F., Medshitov, R. M., and Beg, A. A. (2001)
An essential role of the NF-kappa B/Toll-like receptor pathway in induction of inflammatory and tissue-repair
gene expression by necrotic cells. J. Immunol. 166:7128-7135.
Lin, Y., Zhang, M., and Barnes, P. F. (1998). Chemokine production by a human alveolar epithelial cell line in
response to Mycobacterium tuberculosis. Infect. Immun. 66:1121-1126.
Liu, F. T., Frigeri, L. G., Gritzmacher, C. A., Hsu, D. K., Robertson, M. W., and Zuberi, R. I. (1993).
Expression and function of an IgE-binding animal lectin (epsilon BP) in mast cells. Immunopharmacology
26:187-195.
Loman, S., Radl, J., Jansen, H. M., Out, T. A., and Lutter, R. (1997). Vectorial transcytosis of dimeric IgA
by the Calu-3 human lung epithelial cell line: upregulation by IFN-γ. Am. J. Physiol. Lung Cell. Mol. Physiol.
272:L951-L958.
MacMicking, J., Xie, Q-W., and Nathan, C. (1997). Nitric oxide and macrophage function. Annu. Rev.
Immunol. 15:323-350.
78
Maruoka, T., Nagata, T., and Kasahara, M. (2004). Identification of the rat IgA Fc receptor encoded in the
leukocyte receptor complex. Immunogenetics 55:712-716.
Marshall, B. G., Chambers, M. A., Wangoo, A., Shaw, R. J., and Young, D. B. (1997). Production of tumor
necrosis factor and nitric oxide by macrophages infected with live and dead mycobacteria and their suppression
by an interleukin-10-secreting recombinant. Infect. Immun. 65:1931-1935.
Marshall, L. J., Perks, B., Ferkol, T., and Shute, J. K. (2001). IL-8 released constitutively by primary
bronchial epithelial cells in culture forms an inactive complex with secretory component. J. Immunol. 167:28162823.
Matsumoto, S., Yukitake, H., Kanbara, H., Yamada, H., Kitamura, A., and Yamada, T. (2001).
Mycobacterium bovis bacillus calmette-guerin induces protective immunity against infection by Plasmodium
yoelii at blood-stage depending on shifting immunity toward Th1 type and inducing protective IgG2a after the
parasite infection. Vaccine 19:779-787.
Mazanec, M. B., Kaetzel, C. S., Lamm, M. E., Fletcher D., and Nedrud, J. G. (1992). Intracellular
neutralization of virus by immunoglobulin A antibodies. Proc. Natl. Acad. Sci. USA 89:6901-6905.
Mazanec, M. B., Nedrud, J. G., Kaetzel, C. S., and Lamm, M. E. (1993). A three-tiered view of the role of
IgA in mucosal defense. Immunol. Today 14:430-435.
Mazanec, M. B., Coudret, C. L., and Fletcher D. R. (1995). Intracellular neutralization of influenza virus by
immunoglobulin A anti-hemagglutinin monoclonal antibodies. J. Virol. 69:1339-1343.
Mazzaccaro, R. J., Gedde, M., Jensen, E. R., van Santem, H. M., Ploegh, H. L., Rock, K. L., and Bloom, B.
R. (1996). Major histocompatibility class I presentation of soluble antigen facilitated by Mycobacterium
tuberculosis infection. Proc. Natl. Acad. Sci. USA 93:11786-11791.
Mazzoni, A., and Segal, D. M. (2004). Controlling the Toll road to dendritic cell polarization. J. Leukoc. Biol.
75:721-730.
Meding, S. J., and Langhorne, J. (1991). CD4+ T cells and B cells are necessary for the transfer of protective
immunity to Plasmodium chabaudi chabaudi. Eur. J. Immunol. 21:1433-1438.
Medzhitov, R., Preston-Hurlburt, P., Kopp, E., Stadlen, A., Chen, C., Ghosh, S., and Janeway, C. A. Jr.
(1998). MyD88 is an adaptor protein in the hToll/IL-1 receptor family signaling pathways. Mol. Cell 2:253-258.
Megiovanni, A. M., Sanchez, F., Robledo-Sarmiento, M., Morel, C., Gluckman, J. C., and Boudaly, S.
(2006). Polymorphonuclear neutrophils deliver activation signals and antigenic molecules to dendritic cells: a
new link between leukocytes upstream of T lymphocytes. J. Leukoc. Biol. 79:977-988.
Michelsen, K. S., Aicher, A., Mohaupt, M., Hartung, T., Dimmeler, S., Kirschning, C. J., and Schumann,
R. R. (2001). The role of Toll-like receptors (TLRs) in bacteria-induced maturation of murine dendritic cells
(DCs). Peptidoglycan and lipoteichoic acid are inducers of DC maturation and require TLR2. J. Biol. Chem.
276:25680-25686.
Modlin, R. L., and Rea, T. H. (1988). Immunopathology of leprosy granulomas. Springer Semin.
Immunopathol. 10:359-374.
Mohan, V. P., Scanga, C. A., Yu, K., Scott, H. M., Tanaka, K. E., Tsang, E., Tsai, M. M., Flynn, J. L., and
Chan, J. (2001). Effects of tumor necrosis factor alpha on host immune response in chronic persistent
tuberculosis: possible role for limiting pathology. Infect. Immun. 69:1847-1855.
Monteiro, R. C., Kubagawa, H., and Copper, M. D. (1990). Cellular distribution, regulation, and biochemical
nature of an Fc alpha receptor in humans. J. Exp. Med. 171:597-613.
Monteiro, R. C., and van de Winkel, J. G. (2003). IgA Fc receptors. Annu. Rev. Immunol. 21:177-204.
79
Morton, H. C., van Egmond, M., and van de Winkel, J. G. (1996). Structure and function of human IgA Fc
receptors (FcαR). Crit. Rev. Immunol. 16:423-440.
Mostov, K. E. (1994). Transepithelial transport of immunoglobulins. Annu. Rev. Immunol. 12:63-84.
Muller, I., Cobbold, S. P., Waldmann, H., and Kaufmann, S. H. (1987). Impaired resistance to
Mycobacterium tuberculosis infection after selective in vivo depletion of L3T4+ and Lyt-2+ T cells. Infect.
Immun. 55:2037-2041.
Mun, H. S., Aosai, F., Norose, K., Chen, M., Piao, L. X., Takeuchi, O., Akira, S., Ishikura, H., and Yano,
A. (2003). TLR2 as an essential molecule for protective immunity against Toxoplasma gondii infection. Int.
Immunol. 15:1081-1087.
Munk, M. E., Gatrill, A. J., and Kaufmann, S. H. (1990). Target cell lysis and IL-2 secretion by gamma/delta
T lymphocytes after activation with bacteria. J. Immunol. 145:2434-2439.
Muraille, E., De Trez, C., Brait, M., De Baetselier, P., Leo, O., and Carlier, Y. (2003). Genetically resistant
mice lacking MyD88-adapter protein display a high susceptibility to Leishmania major infection associated with
a polarized Th2 response. J. Immunol. 170:4237-4241.
Murphy, J. R. (1981). Host defenses in murine malaria: nonspecific resistance to Plasmodium berghei
generated in response to Mycobacterium bovis infection or Corynebacterium parvum stimulation. Infect. Immun.
33:199-211.
Murray, P. J., Wang, L., Onufryk, C., Tepper, R. I., and Young, R. A. (1997). T cell-derived IL-10
antagonizes macrophage function in mycobacterial infection. J. Immunol. 158:315-321.
Myrvik, Q. N., Leake, E. S., and Wright, M. J. (1984). Disruption of phagosomal membranes of normal
alveolar macrophages by the H37Rv strain of Mycobacterium tuberculosis. A correlate of virulence. Am. Rev.
Respir. Dis. 129:322-328.
Naik, R. S., Branch, O. H, Woods, A. S., Vijaykumar, M., Perkins, D. J., Nahlen, B. L., Lal, A. A., Cotter,
R. J., Costello, C. E., Ockenhouse, C. F., Davidson, E. A., and Gowda, D. C. (2000).
Glycosylphosphatidylinositol anchors of Plasmodium falciparum: molecular characterization and naturally
elicited antibody response that may provide immunity to malaria pathogenesis. J. Exp. Med. 192:1563-1576.
Neutra, M. R., Frey, A., and Kraehenbuhl, J. P. (1996). Epithelial M cells: gateways of mucosal infection and
immunization. Cell 86:345-348.
Nicholson, S., Bonecini-Almeida, M., Silva, J. R. L., Nathan, C., Xie, Q-W., Mumford, R., Weidner, J. R.,
Calaycay, J., Geng, J., Boechat, N., Linhares, C., Rom, W., and Ho, J. L. (1996). Inducible nitric oxide
synthase in pulmonary alveolar macrophages from patients with tuberculosis. J. Exp. Med. 184:2293-2302.
Norderhaug, I. N., Johansen, F. E., Chjerven, H., and Brandtzaeg, P. (1999). Regulation of the formation
and external transport of secretory immunoglobulins. Crit. Rev. Immunol. 19:481-508.
Norhagen, G., Engström, P. E., Hammarström, L., Söder, P. O., and Smith, C. I. (1989). Immunoglobulin
levels in saliva in individuals with selective IgA deficiency: compensatory IgM secretion and its correlation with
HLA and susceptibility to infections. J. Clin. Immunol. 9:279-286.
Noss, E. H., Harding, C. V., and Boom, W. H. (2000). Mycobacterium tuberculosis inhibits MHC class II
antigen processing in murine bone marrow macrophages. Cell. Immunol. 201:63-74.
Ocana-Morgner, C., Mota, M. M., and Rodriguez, A. (2003). Malaria blood stage suppression of liver stage
immunity by dendritic cells. J. Exp Med. 197:143-151.
Oddo, M., Renno, T., Attinger, A., Bakker, T., MacDonald, H. R., and Meylan, P. R. (1998). Fas ligandinduced apoptosis of infected human macrophages reduces the viability of intracellular Mycobacterium
tuberculosis. J. Immunol. 160:5448-5454.
80
O’Garra, A., and Vieira, P. (2004). Regulatory T cells and mechanisms of immune system control Nat. Med.
10:801-805.
Ohashi, K., Burkart, V., Flohe, S., and Kolb, H. (2000). Cutting edge: heat shock protein 60 is a putative
endogenous ligand of the toll-like receptor-4 complex. J. Immunol. 164:558-561.
Orago, A., and Facer, C. A. (1991). Cytotoxicity of human natural killer (NK) cell subsets for Plasmodium
falciparum erythrocytic schizonts: stimulation by cytokines and inhibition by neomycin. Clin. Exp. Immunol.
86:22-29.
Orme, I. M., and Collins, F. M. (1984). Adoptive protection of the Mycobacterium tuberculosis-infected lung.
Cell. Immunol. 84:113-120.
Orme, I. M., and Cooper, A. M. (1999). Cytokine/chemokine cascades in immunity to tuberculosis. Immunol.
Today 20:307-312.
Orme, I. M., Miler, E. S., Roberts, A. D., Furney, S. K., Griffin, J. P., Dobos, K. M., Chi, D., Rivoire, B.,
and Brennan, P. J. (1992). T lymphocytes mediating protection and cellular cytolysis during the course of
Mycobacterium tuberculosis infection, Evidence for different kinetics and recognition of a wide spectrum of
protein antigens. J. Immunol. 148:189-196.
Ottenhof, T. H., Kumararatne, D, and Casanova, J. L. (1998). Novel human immunodeficiencies reveal the
essential role of type-1 cytokines in immunity to intracellular bacteria. Immunol. Today 19:491-494.
Oxelius, V. A., Laurell, A. B., Lindquist, B., Golebiowska, H., Axelsson, U., Bjorkander, J., and Hanson,
L. A. (1981). IgG subclasses in selective IgA deficiency: importance of IgG2-IgA deficiency. N. Engl. J. Med.
304:1476-1477.
Page, K. R., Jedlicka, A. E., Fakheri, B., Noland, G. S., Kesavan, A. K., Scott, A. L., Kumar, N., and
Manabe, Y. C. (2005). Mycobacterium-induced potentiation of type I immune responses and protection against
malaria are host specific. Infect. Immun. 73:8369-8380.
Palma, G. I., and Saravia, N. G. (1997). In situ characterization of the human host response to Leishmania
panamensis. Am. J. Dermatopathol. 19:585-590.
Patry, C., Sibille, Y., Lehuen, A., and Monteiro, R. C. (1996). Identification of Fc alpha receptor (CD89)
isoforms generated by alternative splicing that are differentially expressed between blood monocytes and
alveolar macrophages. J. Immunol. 156:4442-4448.
Pedrosa, J., Saunders, B. M., Appelberg, R., Orme, I. M., Silva, M. T., and Cooper, A. M. (2000).
Neutrophils play a protective nonphagocytic role in systemic Mycobacterium tuberculosis infection of mice.
Infect. Immun. 68:577-583.
Perlmann, H., Helmby, H., Hagstedt, M., Carlson, J., Larsson, P., Troye-Blomberg, M., and Perlmann, P.
(1994). IgE elevation and IgE anti-malarial antibodies in Plasmodium falciparum malaria: association of high
IgE levels with cerebral malaria. Clin. Exp. Immunol. 97:284-292.
Perry, J. A., Rush, A., Wilson, R. J., Olvers, C. S., and Avery, A. C. (2004). Dendritic cells from malariainfected mice are fully functional APC. J. Immunol. 172:475-482.
Perry, J. A., Olver, C. S., Burnett, R. C., and Avery, A.C. (2005). Cutting edge: the acquisition of TLR
tolerance during malaria infection impacts T cell activation. J. Immunol. 174:5921–5925.
Pethe, K., Alonso, S., Biet, F., Delogu, G., Brennan, M. J., Locht, C., and Menozzi, F D. (2001). The
heparin-binding haemagglutinin of M. tuberculosis is required for extrapulmonary dissemination. Nature
412:190-194.
Phalipon, A., Cardona, A., Kraehenbuhl, J. P., Edelman, L., Sansonetti, P. J., and Corthesy, B. (2002).
Secretory component: a new role in secretory IgA-mediated immune exclusion in vivo. Immunity 17:107-115.
81
Poltorak, A., He, X., Sminova, I., Liu, M. Y., Van Huffel, C., Du, X., Birdwell, D., Alejos, E., Silva, M.,
Galanos, C., Freudenberg, M., Ricciardi-Castagnoli, P., Layton, B., and Beutler, B. (1998). Defective LPS
signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 282:2085-2088.
Porcelli, S. A., and Modlin, R. L. (1999). The CD1 system: antigen-presenting molecules for T cell recognition
of lipids and glycolipids. Annu. Rev. Immunol. 17:297-329.
Pouniotis, D. S., Proudfoot, O., Bogdanka, V., Apostolopoulos, V., Fifis, T., and Plebanski, M. (2004).
Dendritic cells induce immunity and long-lasting protection against blood-stage malaria despite an in vitro
parasite-induced maturation defect. Infect. Immun. 72:5331-5339.
Punturieri, A., Alviani, R. S., Polak, T., Copper, P., Sonstein, J., and Curtis, J. L. (2004). Specific
engagement of TLR4 or TLR3 does not lead to IFN-beta-mediated innate signal amplification and STAT1
phosphorylation in resident murine alveolar macrophages. J. Immunol. 173:1033-1042.
Quan, C. P., Berneman, A., Pires, R., Avrameas, S., and Bouvet, J. P. (1997). Natural polyreactive secretory
immunoglobulin A autoantibodies as a possible barrier to infection in humans. Infect. Immun. 65:3997-4004.
Quesniaux, V., Fremond, C., Jacobs, M., Parida, S., Nicolle, D., Yeremeev, V., Bihl, F., Erard, F., Botha,
T., Drennan, M., Soler, M. N., Le Bert, M., Schnyder, B., and Ryffel, B. (2004). Toll-like receptor pathways
in the immune responses to mycobacteria. Microbes Infect. 6:946-959.
Re, F., and Strominger, J. L. (2004). IL-10 released by concomitant TLR2 stimulation blocks the induction of
a subset of Th1 cytokines that are specifically induced by TLR4 or TLR3 in human dendritic cells. J. Immunol.
173:7548-7555.
Reiling, N., Holscher, C., Fehrenbach, A., Kroger, S., Kirschning, C. J., Goyert, S., and Ehlers, S. (2002).
Cutting edge: Toll-like receptor (TLR)2- and TLR4-mediated pathogen recognition in resistance to airborne
infection with Mycobacterium tuberculosis. J. Immunol. 169:3480-3484.
Reljic, R., Crawford, C., Challacombe, S., and Ivanyi, J. (2004a). Mouse monoclonal IgA binds to the
galectin-3/Mac-2 lectin from mouse macrophage cell lines. Immunol. Lett. 93:51-56.
Reljic, R., Crawford, C., Challacombe, S., and Ivanyi, J. (2004b). Mouse IgA inhibits cell growth by
stimulating tumor necrosis factor-alpha production and apoptosis of macrophage cell lines. Int. Immunol.
16:607-614.
Reyes-Flores, O. (1986). Granulomas induced by living agents. Int. J. Dermatol. 25:158-165.
Ribeiro-Rodrigues, R., Resende Co, T., Rojas, R., Toossi, Z., Dietze, R., Boom, W. H., Maciel, E., and
Hirsch, C. H. (2006). A role for CD4+CD25+ T cells in regulation of the immune response during human
tuberculosis. Clin. Exp. Immunol. 144:25-34.
Riedel, D. D., and Kaufmann, S. H. (1997). Chemokine secretion by human polymorphonuclear granulocytes
after stimulation with Mycobacterium tuberculosis and lipoarabinomannan. Infect. Immun. 65:4620-4623.
Riley, R. L., Mills, C. C., Nyka, W., Weinstock, N., Storey, P. B., Sultan, L. U., Riley, M. C., and Wells, W.
F. (1995). Aerial dissemination of pulmonary tuberculosis. A two-year study of contagion in a tuberculosis ward.
1959. Am. J. Epidemiol. 142:3-14.
Roach, D. R., Bean, A. G., Demangel, C., France, M. P., Briscoe, H., and Britton, W. J. (2002). TNF
regulates chemokine induction essential for cell recruitment, granuloma formation, and clearance of
mycobacterial infection. J. Immunol. 168:4620-4627.
Robinson, J. K., Blanchard, T. G., Levine, A. D., Emancipator, S. N., and Lamm M. E. (2001). A mucosal
IgA-mediated excretory immune system in vivo. J. Immunol. 166:3688-3692.
Rodrigues, A., Schellenberg, J. A., Roth, A., Benn, C. S., Aaby, P., and Greenwood, B. (2007).
Revaccination with Bacillus Calmette-Guerin (BCG) vaccine does not reduce morbidity from malaria in African
children. Trop. Med. Int. Health 12:224-229.
82
Roncador, G., Brown, P. J, Maestre, L., Hue, S., Torrecuadrada, J. L., Ling, K. L., Pratap, S., Toms, C.,
Fox, B. C., Cerundolo, V., Powrie, F., and Banham, A. H. (2005). Analysis of FOXP3 protein expression in
human CD4+CD25+ regulatory T cells at the single-cell level. Eur. J. Immunol. 35:1681-1691.
Rook, G. A. W., and Bloom, B. R. (1994). Mechanisms of pathogenesis in tuberculosis. In: Tuberculosis:
Pathogenesis, Protection and Control (Bloom, B. R., Ed.), pp. 485-502. ASM Press, Washington, DC.
Roos, A., Bouwman, L. H., van Gijlswijk-Janssen, D. J., Faber-Krol, M. C., Stah, G. L., and Daha, M. R.
(2001). Human IgA activates the complement system via the mannan-binding lectin pathway. J. Immunol.
167:2861-2868.
Roth, A., Gustafson, P., Nhaga, A., Djana, O., Poulsen, A., Garly, M. L., Jensen, H., Sodemann, M.,
Rodriguez, A., and Aaby, P. (2005). BCG vaccination scar associated with better childhood survival in GuineaBissau. Int. J. Epidemiol. 34:540-547.
Roth, A., Jensen, H., Garly, M. L., Djana, Q., Martins, C. L., Sodemann, M., Rodriguez, A., and Aaby, P.
(2004). Low birth weight infants and Calmette-Guerin bacillus vaccination at birth: community study from
Guinea-Bissau. Pediatr. Infect. Dis. J. 23:544-550.
Roy, S., Sharma, S., Sharma, M., Aggarwal, R., and Bose, M. (2004). Induction of nitric oxide release from
the human alveolar epithelial cell line A549: an in vitro correlate of innate immune response to Mycobacterium
tuberculosis. Immunology 112:471-480.
Rumbley, C. A., and Phillips, S. M. (1999). The schistosome granuloma: an immunoregulatory organelle.
Microbes Infect. 1:499-504.
Sakaguchi, S. (2005). Naturally arising Foxp3-expressing CD25+CD4+ regulatory T cells in immunological
tolerance to self and non-self. Nat. Immunol. 6:345-352.
Sakaguchi, S., Sakaguchi, N., Asano, M., Itoh, N., and Toda, M. (1995). Immunologic self-tolerance
maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single
mechanism of self-tolerance causes various autoimmune diseases. J. Immunol. 155:1151-1164.
Sakamoto, N., Shibuya, K., Shimizu, Y., Yotsumoto, K., Miyabayashi, T., Sakano, S., Tsuji, T., Nakayama,
E., Nakauchi, H., and Shibuya, A. (2001). A novel Fc receptor for IgA and IgM is expressed on both
hematopoietic and non-hematopoietic tissues. Eur. J. Immunol. 31:1310-1316.
Sandor, M., Ibraghimov, A., Rosenberg, M. G., Teeraratkul, P., and Lynch, R. G. (1992). Expression of
IgA and IgM Fc receptors on murine T lymphocytes. Immunol. Res. 11:169-180.
Scapini, P., Laudanna, C., Pinardi, C., Allavena, P., Mantovani, A., Sozzani, S., and Cassatella, M. A.
(2001). Neutrophils produce biologically active macrophage inflammatory protein-3alpha (MIP-3alpha)/CCL20
and MIP-3beta/CCL19. Eur. J. Immunol. 31:1981-1988.
Schaible, U. E., and Kaufmann, S. H. (2000). CD1 and CD1-restricted T cells in infections with intracellular
bacteria. Trends. Microbiol. 8:419-425.
Schirm, J., Oostendorp, L. A., and Mulder, J. G. (1995). Comparison of amplicor, in-house PCR, and
conventional culture for detection of Mycobacterium tuberculosis in clinical samples. J. Clin. Microbiol.
33:3221-3224.
Schlesinger, L. S. (1993). Macrophage phagocytosis of virulent but not attenuated strains of Mycobacterium
tuberculosis is mediated by mannose receptors in addition to complement receptors. J. Immunol. 150:2920-2930.
Schlesinger, L. S., Bellinger-Kawahara, C. G., Payne, N. R., and Horwitz, M. A. (1990). Phagocytosis of
Mycobacterium tuberculosis is mediated by human monocyte complement receptors and complement component
C3. J. Immunol. 144:2771-2780.
83
Schlesinger, L. S., Kaufmann, T. M., Iyer, S., Hull, S. R., and Marchiando, L. K. (1996). Differences in
mannose receptor-mediated uptake of lipoarabinomannan from virulent and attenuated strains of Mycobacterium
tuberculosis by human macrophages. J. Immunol. 157:4568-4575.
Schofield, L., and Hackett, F. (1993). Signal transduction in host cells by a glycosylphosphatidylinositol toxin
of malaria parasites. J. Exp. Med. 177:145-153.
Schwarzer, E., Turrini, F., Ulliers, D., Giribaldi, G., Ginsburg, G., and Arese, P. (1992). Impairment of
macrophage functions after ingestion of Plasmodium falciparum-infected erythrocytes or isolated malarial
pigment. J. Exp. Med. 176:1033-1041.
Schwarzer, E., Alessio, M., Ulliers, D., and Arese, P. (1998). Phagocytosis of the malarial pigment, hemozoin,
impairs expression of major histocompatibility complex class II antigen, CD54, and CD11c in human
monocytes. Infect. Immun. 66:1601-1606.
Scott, C. P., Kumar, N., Bishai, W. R., and Manabe, Y. (2004). Short report: Modulation of Mycobacterium
tuberculosis infection by Plasmodium in the murine model. Am. J. Trop. Med. Hyg. 70:144-148.
Seiler, P., Aichele, P., Bandermann, S., Hauser, A. E., Lu, B., Gerard, N. .P., Gerard, C., Ehlers, S.,
Mollenkopf, H. J., and Kaufmann, S. H. (2003). Early granuloma formation after aerosol Mycobacterium
tuberculosis infection is regulated by neutrophils via CXCR3-signaling chemokines. Eur. J. Immunol. 33:26762686.
Seixas, E., Cross, C., Quin, S., and Langhorne, J. (2001). Direct activation of dendritic cells by the malaria
parasite, Plasmodium chabaudi chabaudi. Eur. J. Immunol. 31:2970-2978.
Senaldi, G., Yin, S., Shaklee, C. L., Piguet, P. F., Mak, T. W., and Ulich, T. R. (1996). Corynebacterium
parvum- and Mycobacterium bovis bacillus Calmette-Guerin-induced granuloma formation is inhibited in TNF
receptor I (TNF-RI) knockout mice and by treatment with soluble TNF-RI. J. Immunol. 157:5022-5026.
Senior, B. W., Loomes, L. M., and Kerr, M. A. (1991). Microbial IgA proteases and virulence. Rev. Med.
Microbiol. 2:200-207.
Serbina, N. V., and Flynn, J. L. (1999). Early emergence of CD8(+) T cells primed for production of type 1
cytokines in the lungs of Mycobacterium tuberculosis-infected mice. Infect. Immun. 67:3980-3988.
Sharma, M., Sharma, S., Roy, S., Varma, S., and Bose, M. (2007). Pulmonary epithelial cells are a source of
interferon-gamma in response to Mycobacterium tuberculosis infection. Immunol. Cell Biol. In press.
Sherry, B. A., Alava, G., Tracey, K. J., Martiney, J., Cerami, A., and Slater, A. F. (1995). Malaria-specific
metabolite hemozoin mediates the release of several potent endogenous pyrogens (TNF, MIP-1 alpha, and MIP1 beta) in vitro, and altered thermoregulation in vivo. J. Inflamm. 45:85-96.
Shibuya, A., Sakamoto, N., Shimizu, Y., Shibuya, K., Osawa, M., Hiroyama, T., Eyre, H. J., Sutherland,
G. R., Endo, Y., Fujita, T., Miyabayashi, T., Sakano, S., Tsuji, T., Nakayama, E., Phillips, J. H., Lanier, L.
L., and Nakauchi, H. (2000). Fc alpha/mu receptor mediates endocytosis of IgM-coated microbes. Nat.
Immunol. 1:441-446.
Shiloh, M., and Nathan, C. F. (2000). Reactive nitrogen intermediates and the pathogenesis of Salmonella and
mycobacteria. Curr. Opin. Microbiol. 3:35-42.
Shim, T. S., Turner, O. C., and Orme, I. M. (2003). Toll-like receptor 4 plays no role in susceptibility of mice
to Mycobacterium tuberculosis infection. Tuberculosis 83:367-371.
Shimada, S., Kawaguchi-Miyashita, M., Kushiro, A., Sato, T., Nanno, M., Sako, T., Matsuoka, Y., Sudo,
K., Tagawa, Y., Iwakura, Y., and Ohwaki, M. (1999). Generation of polymeric immunoglobulin receptordeficient mouse with marked reduction of secretory IgA. J. Immunol. 163:5367-5373.
Silva, M. T., Silva, M. N., and Appelberg, R. (1989). Neutrophil-macrophage cooperation in the host defence
against mycobacterial infections. Microb. Pathog. 6:369-380.
84
Skorohod, O. A., Alessio, M., Mordmuller, B., Arese, P., and Schwarzer, E. (2004). Hemozoin (malarial
pigment) inhibits differentiation and maturation of human monocyte-derived dendritic cells: a peroxisome
proliferator-activated receptor-gamma-mediated effect. J. Immunol. 173:4066-4074.
Slater, A. F., Swiggard, W. J., Orton, B. R., Flitter, W. D., Goldberg, D. G., Cerami, A., and Henderson,
G.B. (1991). An iron-carboxylate bond links the heme units of malaria pigment. Proc. Natl. Acad. Sci. USA.
88:325-329.
Smiley, S. T., King, J. A., and Hancock, W. W. (2001). Fibrinogen stimulates macrophage chemokine
secretion through toll-like receptor 4. J. Immunol. 167:2887-2894.
Smith, S. M., and Dockrell, H. M. (2000). Role of CD8 T cells in mycobacterial infections. Immunol. Cell Biol.
78:325-333.
Snow, R. W., Guerra, C. A., Noor, A. M., Myint, H. Y., and Hay, S. I. (2005). The global distribution of
clinical episodes of Plasmodium falciparum malaria. Nature 434:214-217.
Sollid, L. M., Kvale, D., Brandtzaeg, P., Markussen, G., and Torsby, E. (1987). Interferon-γ enhances
expression of secretory component, the epithelial receptor for polymeric immunoglobulins. J. Immunol.
138:4303-4306.
Sousa, A. O., Mazzaccaro, R., Russell, D. G., Lee, F. K., Turner, O. C., Hong, S., Van Kaer, L., and
Bloom, B. R. (1999). Relative contributions of distinct MHC class I-dependent cell populations in protection to
tuberculosis infection in mice. Proc. Natl. Acad. Sci. USA 97:4204-4208.
Steinman, R. M. (1991). The dendritic cell system and its role in immunogenicity. Ann. Rev. Immunol. 9:271296.
Stenger, S., Hanson, D. A., Teitelbaum, R., Dewan, P., Niazi, K. R., Froelich, C. J., Ganz, T., ThomaUszynski, S., Melian, A., Bogdan, C., Porcelli, S. A., Bloom, B. R., Krensky, A. M., and Modlin, R. L.
(1998). An antimicrobial activity of cytotoxic T cells mediated by granulysin. Science 282:121-125.
Stenger, S., Mazzaccaro, R., Uyemura, K., Cho, S., Barnes, P., Rosat, J., Sette, A., Brenner, M., Porcelli,
S., Bloom, B, and Modlin, R. (1997). Differential effects of cytolytic T cell subsets on intracellular infection.
Science 276:1684-1687.
Sterne, J. A., Rodrigues, L. C., and Guedes, I. N. (1998). Does the efficacy of BCG decline with time since
vaccination? Int. J. Tuber. Lung Dis. 2:200-207.
Stevenson, M., Lemieux, S., and Skamene, E. (1984). Genetic control of resistance to murine malaria. J. Cell.
Biochem. 24:91-102.
Stevenson, M. M., and Riley, E. M. (2004). Innate immunity to malaria. Nat. Rev. Immunol. 4:169-180.
Stevenson, M., and Urban, B. C. (2006). Antigen presentation and dendritic cell biology in malaria. Parasite
Immunol. 28:5-14.
Sturgill-Koszycki, S., Schaible, U. E., and Russell, D. G. (1996). Mycobacterium-containing phagosomes are
accessible to early endosomes and reflect a transitional state in normal phagosome biogenesis. EMBO J.
15:6960-6968.
Sturgill-Koszycki, S., Schlesinger, P. H., Chakraborty, P., Haddix, P. L., Collins, H. L., Fok, A. K., Allen,
R. D., Gluck, S. L., Heuser, J., and Russell, D. G. (1994). Lack of acidification in Mycobacterium phagosomes
produced by exclusion of the vesicular proton-ATPase. Science 263:678-681.
Sugawara, I., Yamada, H., Li, C., Mizuno, S., Takeuchi, O., and Akira, S. (2003). Mycobacterial infection in
TLR2 and TLR6 knockout mice. Microbiol. Immunol. 47:327-336.
85
Sullivan Jr., D. J., Gluzman, I. Y., and Goldberg, D. G. (1996a). Plasmodium hemozoin formation mediated
by histidine-rich proteins. Science 271:219-222.
Sullivan Jr., D. J., Gluzman, I. Y., Russell, D. G., and Goldberg, D. G. (1996a). On the molecular mechanism
of chloroquine's antimalarial action. Proc. Natl. Acad. Sci. USA. 93:11865-11870.
Suss, G., Eichmann, K., Kury, E., Linke, A., and Langhorne, J. (1988). Roles of CD4- and CD8-bearing T
lymphocytes in the immune response to the erythrocytic stages of Plasmodium chabaudi. Infect. Immun.
56:3081-3088.
Tachado, S. D., Gerold, P., Schwaz, R., Novakovic, S., McConville, M., and Schofied, L. (1997). Signal
transduction in macrophages by glycosylphosphatidylinositols of Plasmodium, Trypanosoma, and Leishmania:
activation of protein tyrosine kinases and protein kinase C by inositolglycan and diacylglycerol moieties. Proc.
Natl. Acad Sci. USA. 94:4022-4027.
Tascon, R. E., Stavropoulos, E., Lukacs, K. V., and Colston, M. J. (1998). Protection against Mycobacterium
tuberculosis infection by CD8 T cells requires production of gamma interferon. Infect. Immun. 66:830-834.
Tailleux, L., Schwartz, O., Herrman, J. L., Pivert, E., Jackson, M., Amara, A., Legres, L., Dreher, D.,
Nicod, L. P., Gluckman, J. C., Lagrange, P. H., Gicquel, B., and Neyrolles, O. (2003). DC-SIGN is the major
Mycobacterium tuberculosis receptor on human dendritic cells. J. Exp. Med. 197:121-127.
Takeuchi, O., Hoshino, K., Kawai, T., Sanjo, H., Takada, H., Ogawa, T., Takeda, K., and Akira, S. (1999).
Differential roles for TLR2 and TLR4 in recognition of gram-negative and gram-positive bacterial cell wall
components. Immunity 11:443-451.
Tan, B. H., Meinken, C., Bastian, M., Bruns, H., Legaspi, A., Ochoa, M. T., Krutzik, S. R., Bloom, B. R.,
Ganz, T., Modlin, R. L., and Stenger, S. (2006). Macrophages acquire neutrophil granules for antimicrobial
activity against intracellular pathogens. J. Immunol. 177:1864-1871.
Taub, D. D., Turcovski-Corrales, S. M., Key, M. L., Longo, D. L., and Murphy, W. J. (1996). Chemokines
and T lymphocyte activation: I. Beta chemokines costimulate human T lymphocyte activation in vitro. J.
Immunol. 156:2095-2103.
Taylor-Robinson, A. W., and Phillips, R. S. (1993). Protective CD4+ T-cell lines raised against Plasmodium
chabaudi show characteristics of either Th1 or Th2 cells. Parasite Immunol. 15:301-310.
Taylor-Robinson, A. W., Phillips, R. S., Severn, A., Moncada, S., and Liew, F Y. (1993). The role of TH1
and TH2 cells in a rodent malaria infection. Science 260:1931-1934.
Taylor-Robinson, A. W., and Smith, E. C. (1991). A role for cytokines in potentiation of malaria vaccines
through immunological modulation of blood stage infection. Immunol. Rev. 171:105-123.
Teitelbaum, R., Cammer, M., Maitland, M. L., Freitag, N. E., Condeelis, J., and Bloom, B. R. (1999).
Mycobacterial infection of macrophages results in membrane-permeable phagosomes. Proc. Natl. Acad. Sci.
USA 96:15190-15195.
Teitelbaum, R., Glatman-Freedman, A., Chen, B., Robbins, J. B., Unanue, E., Casadevall, A., and Bloom,
B. R. (1998). A mAb recognizing a surface antigen of Mycobacterium tuberculosis enhances host survival. Proc.
Natl. Acad. Sci. USA. 95:15688-15693.
Teitelbaum, R., Schubert, W., Gunther, L., Kress, Y., Macaluso, F., Pollard, J. W., McMurray, F., and
Bloom, B. R. (1999). The M cell as a portal of entry to the lung for the bacterial pathogen Mycobacterium
tuberculosis. Immunity 10:641-650.
Tenner-Racz, K., Racz, P., Myrvik, Q. N., Ockers, J. R., and Geister, R. (1979). Uptake and transport of
horseradish peroxidase by lymphoepithelium of the bronchus-associated lymphoid tissue in normal and bacillus
Calmette-Guerin-immunized and challenged rabbits. Lab. Invest. 41:106-115.
86
Termeer, C., Benedix, F., Sleeman, J., Fieber, C., Voith, U., Ahrens, T., Miyake, K., Freudenberg, M.,
Galanos, C., and Simon, J. C. (2002). Oligosaccharides of Hyaluronan activate dendritic cells via toll-like
receptor 4. J. Exp. Med. 195:99-111.
Theander, T. G., Pedersen, B. K., Bygbjerg, I. C., Jepsen, S., and Larsen, P. B. (1987). Enhancement of
human natural cytotoxicity by Plasmodium falciparum antigen activated lymphocytes. Acta. Trop. 44:415-422.
Theuer, C. P., Hopewell, P. C., Elias, D., Schected, G. F., Rutherford, G. W., and Chassion, R. E. (1990).
Human immunodefiency virus infection in tuberculosis patients. J. Infect. Dis. 162:8-12.
Thorel, M. F. (1980). Isolation of Mycobacterium africanum from monkeys. Tubercle 61:101-104.
Thurnher, M., Ramoner, R., Gastl, G., Radmayr, C., Bock, G., Herold, M., Klocker, H., and Bartsch, G.
(1997). Bacillus Calmette-Guerin mycobacteria stimulate human blood dendritic cells. Int. J. Cancer 70:128134.
Thoma-Uszynski, S., Kiertscher, S. M., Ochoa, M. T., Bouis, D. A., Norgard, M. V., Miyake, K.,
Godowski, P. J., Roth, M. D., and Modlin, R. L. (2000). Activation of Toll-like receptor 2 on human dendritic
cells triggers induction of IL-12, but not IL-10. J. Immunol. 165:3804-3810.
Toossi, Z. (2003). Virological and immunological impact of tuberculosis on human immunodeficiency virus
type 1 disease. J. Infect. Dis. 188:1146-1155.
Toossi, Z., Gogate, P., Shiratsuchi, H., Young, T., and Ellner, J. J. (1995). Enhanced production of TGF-β by
blood monocytes from patients with active tuberculosis and presence of TGF-β in tuberculous granulomatous
lung lesions. J. Immunol. 154:465-473.
Toossi, Z., Kleinhenz, M. E., and Ellner, J. J. (1986). Defective interleukin 2 production and responsiveness in
human pulmonary tuberculosis. J. Exp. Med. 163:1162-1172.
Torres, M., Mendez-Sampeiro, P., Jimenez-Zamdio, L., Teran, L., Camarena, A., Quezada, R., Ramos, E.,
and Sada, E. (1994). Comparison of the immune response against Mycobacterium tuberculosis antigens
between a group of patients with active pulmonary tuberculosis and healthy household contacts. Clin. Exp.
Immunol. 96:75-78.
Trager, W., and Jensen, J. B. (1976). Human malaria parasites in continuous culture. Science 193:673-675.
Troye-Blomberg, M., Berzins, K., and Perlmann, P. (1994). T-cell control of immunity to the asexual blood
stages of the malaria parasite. Crit. Rev. Immunol. 14:131-155.
Tsenova, L., Bergtold, A., Freedman, V. H., Young, R. A., and Kaplan, G. (1999). Tumor necrosis factor
alpha is a determinant of pathogenesis and disease progression in mycobacterial infection in the central nervous
system. Proc. Natl. Acad. Sci. USA 96:5657-5662.
Tsuji, S., Matsumoto, M., Takeuchi, O., Akira, S., Azuma, I., Hayashi, A., Toyoshima, K., and Seya, T.
(2000). Maturation of human dendritic cells by cell wall skeleton of Mycobacterium bovis Calmette-Guerin:
involvement of Toll-like receptors. Infect. Immun. 68:6883-6890.
Turner, J., Frank, A. A., Brooks, J. V., Gonzalez-Juarrero, M., and Orme, I. M. (2001). The progression of
chronic tuberculosis in the mouse does not require the participation of B lymphocytes of interleukin-4. Exp.
Gerontol. 36:537-545.
Ueki, T., Mizuno, M., Uesu, T., Kiso, T., and Tsuji, T. (1995). Expression of ICAM-I on M cells covering
isolated lymphoid follicles of the human colon. Acta. Med. Okayama. 49:145-151.
Ulevitch, R. J. (2004). Therapeutics targeting the innate immune system. Nat. Rev. Immunol. 4:512-520.
Ulrichs, T., and Kaufmann, S. H. (2002). Mycobacterial persistence and immunity. Front. Biosci. 7:d458d469.
87
Urban, B. C., Ferguson, D. J., Pain, A., Willcox, N., Plebanski, M., Austyn, J. M., and Roberts, D. J.
(1999). Plasmodium falciparum-infected erythrocytes modulate the maturation of dendritic cells. Nature 400:7377.
Urban, B. C., Willcox, N., and Roberts, D. J. (2001). A role for CD36 in the regulation of dendritic cell
function. Proc. Natl. Acad. Sci. USA. 98:8750-8755.
Vabulas, R. M., Ahmad-Nejad, P., Ghose, S., Kirschning, C. J., Issels, R. D., and Wagner, H. (2002).
HSP70 as endogenous stimulus of the Toll/interleukin-1 receptor signal pathway. J. Biol. Chem. 277:1510715112.
Vabulas, R. M., Braedel, S., Hilf, N., Singh-Jasuja, H., Herter, S., Ahmad-Nejad, P., Kirschning, C. J., Da
Costa, C., Rammensee, H. G., Wagner, H., and Schild, H. (2002). The endoplasmic reticulum-resident heat
shock protein Gp96 activates dendritic cells via the Toll-like receptor 2/4 pathway. J. Biol. Chem. 277:2084720853.
van Egmond, M., van Garderen, E., van Spriel, A. B., Damen, C. A., van Emersfoort, E. S., van
Zandbergen, G., van Hattum, J., Kuiper, J., and van de Winkel, J. G. (2000). FcalphaRI-positive liver
Kupffer cells: reappraisal of the function of immunoglobulin A in immunity. Nat. Med. 6:680-685.
van Gisbergen, K. P., Sanchez-Hernandez, M., Geijtenbeek, T. B., and van Kooyk, Y. (2005). Neutrophils
mediate immune modulation of dendritic cells through glycosylation-dependent interactions between Mac-1 and
DC-SIGN. J. Exp. Med. 201:1281-1292.
Velema, J. P., Alihonou, E. M., Gandaho, T., and Hounye, F. H. (1991). Childhood mortality among users
and non-users of primary health care in a rural west African community. Int. J. Epidemiol. 20:474-479.
Vordermeier, H. M., Venkataprasad, N., Harris, D. P., and Ivanyi, J. (1996). Increase of tuberculosis
infection in the organs of B cell-deficient mice. Clin. Exp. Immunol. 106:312-316.
Walker, D. (2001). Economic analysis of tuberculosis diagnostic tests in disease control: how can it be modelled
and what additional information is needed? Int. J. Tuberc. Lung Dis. 5:1099-1108.
Wang, C-H., Liu, C-Y., Lin, H-C., Yu, C-T., Chung, K. F., and Kuo, H. P. (1998). Increased exhaled nitric
oxide in active pulmonary tuberculosis due to inducible NO synthase upregulation in alveolar macrophages. Eur.
Respir. J. 11:809-815.
Weinbaum, F. I., Weintraub, J., Nkrumah, F. K., Evans, C. B., Tigelaar, R. E., and Rosenberg, Y. J.
(1978). Immunity to Plasmodium berghei yoelii in mice. II. Specific and nonspecific cellular and humoral
responses during the course of infection. J. Immunol. 121:629-636.
Weir, M. R., and Thornton, G. F. (1985). Extrapulmonary tuberculosis. Experience from a community hospital
and review of the literature. Am. J. Med. 79:467-478.
WHO. (2007). Global Tuberculosis Control, Geneva, Switzerland, Report no. WHO/HTM/TB/2007.376.
Wijburg, O. L., Uren, T. K., Simpfendorfer, K., Johansen, F. E., Brandtzaeg, P., Strugnell, R. A. (2006).
Innate secretory antibodies protect against natural Salmonella typhimurium infection. J. Exp. Med. 203:21-26.
Wilkinson, R. J., Haslov, K., Rappuoli, R., Giovannoni, F., Narayanan, P. R., Desai, C. R., Vordermeier,
H. M., Paulsen, J., Pasvol, G., Ivanyi, J., and Singh, M. (1997). Evaluation of the recombinant 38-kilodalton
antigen of Mycobacterium tuberculosis as a potential immunodiagnostic reagent. J. Clin. Microbiol. 35:553-537.
Williams, A., Reljic, R., Naylor, I., Clark, S. O., Falero-Diaz, G., Singh, M., Challacombe, S., Marsh, P. D.,
and Ivanyi, I. (2004). Passive protection with immunoglobulin A antibodies against tuberculous early infection
of the lungs. Immunology 111:328-333.
Winslow, G. M., Yager, E., Shilo, K., Volk, E., Reilly, A., and Chu, F. K. (2000). Antibody-mediated
elimination of the obligate intracellular bacterial pathogen Ehrlichia chaffeensis during active infection. Infect.
Immun. 68:2187-2195.
88
Wright, S. D., and Siverstein, S. C. (1983). Recptors for C3b and C3bi promote phagocytosis but not the
release of toxic oxygen from human phagocytes. J. Exp. Med. 158:2016-2023.
Yan, H., Lamm, M. E., Bjorling, E., and Huang, Y. T. (2002). Multiple functions of immunoglobulin A in
mucosal defense against viruses: an in vitro measles virus model. J. Virol. 76:10972-10979.
Yang, D., Chen, Q., Chertov, O., and Oppenheim, J. J. (2000). Human neutrophil defensins selectively
chemoattract naive T and immature dendritic cells. J. Leukoc. Biol. 68:9-14.
Youngman, K. R., Fiocchi, C., and Kaetzel, C. S. (1994). Inhibition of IFN-γ activity in supernatants from
stimulated human intestinal mononuclear cells prevents upregulation of the polymeric Ig receptor in an intestinal
epithelial cell line. J. Immunol. 153:675-681.
Zhang, M., Gong, J., Iyer, D. V., Jones, B. E., Modlin, R. L., and Barnes, P. F. (1994). T cell cytokine
responses in persons with tuberculosis and human immunodeficiency virus infection. J. Clin. Invest. 94:24352442.
Zhang, Y., Pacheco, S., Acuna, C. L., Switzer, K. C., Wang, Y., Gilmore, X., Harriman, G. R., and
Mbawuike, I. N. (2002). Immunoglobulin A-deficient mice exhibit altered T helper 1-type immune responses
but retain mucosal immunity to influenza virus. Immunology 105:286-294.
Zimmerli, S., Edwards, S., and Ernst, J. D. (1996). Selective receptor blockade during phagocytosis does not
alter the survival and growth of Mycobacterium tuberculosis in human macrophages. Am. J. Respir. Cell. Mol.
Biol. 15:760-770.
89