Download Macrophages, pathology and parasite persistence in

Review
TRENDS in Parasitology
Vol.20 No.11 November 2004
Immunoparasitology series:
Macrophages, pathology and parasite
persistence in experimental visceral
leishmaniasis
Christian R. Engwerda1, Manabu Ato2 and Paul M. Kaye2
1
Immunology and Infection Laboratory, Queensland Institute of Medical Research and Australian Centre for International and
Tropical Health and Nutrition, 300 Herston Road, Herston, Queensland, 4029, Australia
2
Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street,
London, WC1E 7HT, UK
Many infectious diseases are associated with parasite
persistence, often restricted to certain tissue sites, yet
the determinants of such persistence are poorly understood. Infection with the protozoan parasite Leishmania
donovani has proved a useful experimental tool to
address how immune responses can be differentially
effective in clearing parasites from different tissues and,
conversely, it might also provide a good model for
understanding the basis of parasite persistence. This
article reviews recent studies on the determinants and
consequences of persistent parasite infection in the
spleen and suggest that some of the messages to
emerge could have important implications for the
study of a broad range of infectious diseases.
splenomegaly associated with parasite persistence. This
review discusses recent studies on the immunological and
pathological alterations that occur in the spleen, concurrent with the establishment of this chronic infection. In
addition, future directions for research in this field are
suggested, which might help to increase our understanding of the immunological basis for organ-specific immunity
during infectious disease.
Contrasting outcomes of infection in liver and spleen
The diversity in the quality and quantity of host immunity
against pathogen infection is apparent when distinct
target tissues are examined, and experimental models of
Box 1. Visceral leishmaniasis
Many of the major bacteria, protozoa and helminth
infections of humans are associated with systemic pathogen spread and parasitism of multiple organ systems. This
might occur sequentially (as seen in the migrating
helminths) or many tissues could be simultaneously
involved [as seen in visceral leishmaniasis (VL), tuberculosis and malaria]. Whereas it is apparent from
experiments with simple defined antigens that the
immune response could be compartmentalised with
tissue-specific regulation of the quality and quantity of
response, how such factors impact on the outcome of
complex infections is poorly understood. In 2000, we
reviewed our understanding of the events that mediate
organ-specific immune responses during murine infections with the protozoan parasite Leishmania donovani
[1], a model of human VL (Box 1). At that time, the focus
was largely on understanding the acute but ultimately
resolving infection that is observed in the liver of most
mouse strains (including BALB/c and C57BL/6). This
response is analogous to the protective granulomatous
response seen in the majority of humans infected with
L. donovani and which remain sub-clinically infected [2].
In contrast to the self-limiting infection observed in the
liver, however, these same mouse strains develop life-long
Corresponding author: Paul M. Kaye ([email protected]).
Available online 3 September 2004
Unlike the many Leishmania spp. that can cause cutaneous leishmaniasis, Leishmania donovani and Leishmania infantum are
largely responsible for the systemic disease, visceral leishmaniasis
(VL) or kala azar. Approximately 500 000 new cases of VL are
reported annually, mostly from India, the Sudan and Brazil, although
this is probably a considerable underestimate. In the absence of
chemotherapy, most clinical cases of VL result in death. Many
infections (possibly O95%) do not, however, progress to clinical VL;
parasites are contained, although probably not completely removed,
by a self-limiting granulomatous tissue response. Drug resistance
against conventional antimonial drugs is now common in India and,
despite the availability for a new oral drug, miltefosine, vaccines
appear to be the best hope for control of VL. Unfortunately, most
efforts in vaccine research have focused on cutaneous disease and
those trials that have been performed against VL have been largely
unsuccessful. VL is an emergent disease in Europe in association
with HIV and also poses a major veterinary problem in areas where
domestic dogs serve as reservoir hosts.
As with all Leishmania infections, natural transmission occurs
during the bloodmeal of a phlebotamine sandfly, which introduces
metacyclic infective promastigotes into the dermis. These are
resistant to complement attack and they enter local phagocytes
rapidly. Transformation into aflagellate amastigotes then occurs and
the life history of the infection in humans is perpetuated by this life
cycle stage. At what stage of the infection visceralisation occurs is
not clear. Infected macrophages might migrate from the initial site of
infection to the spleen, liver and bone marrow; alternatively, free
promastigotes could enter the bloodstream directly as a result of
the pool-feeding behaviour of the sandfly. Why L. donovani and
L. infantum are so viscerotropic compared with other species is a key
unanswered question.
www.sciencedirect.com 1471-4922/$ - see front matter Q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.pt.2004.08.009
Review
TRENDS in Parasitology
VL is perhaps the clearest representation of this diversity
(Figure 1). In experimental VL, caused by L. donovani [3]
or Leishmania infantum (Leishmania chagasi) [4–6],
hepatic infection is usually self-limiting, and the hepatic
immune response is a good example of a mononuclear celldominated granulomatous inflammatory response, involving resident Kupffer cells, monocytes and CD4C and
CD8C T cells. Many of the cellular and molecular interactions required for efficient hepatic granuloma formation
which are necessary to kill Leishmania have been recently
reviewed [2]. Of the many cytokines involved [including
interferon (IFN)-g, interleukin (IL)-12, IL-4 and tumour
necrosis factor (TNF)], moderate levels of TNF clearly make
a major contribution to host protection in the liver, with focal
TNF production readily observed in hepatic granulomas [7].
By contrast, simultaneously with the resolution of hepatic
infection, control of amastigote growth in the spleen is lost
and destructive pathology is the norm (Figure 1). Strikingly,
excess TNF in this lymphoid environment mediates much of
the architectural damage and immunological dysfunction
associated with this chronic inflammatory state. While the
current literature provides some clues to the basis of this
dichotomous host response, many of the in vitro systems and
host-cell–parasite combinations that dominate the literature might not be most appropriate for a full appreciation of
its complexity.
Vol.20 No.11 November 2004
525
different tissues. Although this diversity is largely ignored
when studying the pathways for uptake and recognition of
Leishmania, it could play a key role in directing many of
the ensuing organ-specific events. Identifying the receptors involved in L. donovani recognition by mononuclear
phagocytes (including monocytes, tissue macrophages,
dendritic cells and their various precursor populations)
will be of fundamental importance for understanding
several stages in the establishment of infection and
initiation of immune responses (Figure 2). One of the
most fascinating features of L. donovani and L. infantum
infections is their systemic nature; although the underlying biology of visceralisation has yet to be carefully
dissected, recent studies are beginning to shed some light
on this matter. The development of a low-dose skin dermal
model of experimental VL [8] provides a new opportunity
to study the visceralisation process in the context of a
biologically relevant parasite dose. Another recent study
indicated that differential expression of the A2 gene locus
between L. donovani and L. major could have a role in
visceralisation. Leishmania major does not normally
express A2 and transgenic expression of A2 in L. major
inhibited the capacity of this parasite to develop local
cutaneous lesions and prevented survival in skin macrophages [9]. Future proteomic and genomic comparisons
between L. major and L. infantum will soon be possible
with the completion of their respective genome sequencing
Uptake and recognition of L. donovani amastigotes
Mononuclear phagocytes are extremely heterogeneous,
which is often evident when isolating these cells from
Blood
MZ
PALS
(b)
Parasite load (LDU)
(a)
1000
(c)
500
(a)
(e)
(d)
3
14
56
28
128
Days p.i.
Parasite load (LDU)
(b)
400
Key:
200
DC
Macrophage
Leishmania
3
14
28
56
128
Days p.i.
TRENDS in Parasitology
Figure 1. Discrete phases of Leishmania donovani infection in the spleen and liver
of inbred mice. The mice were infected with 1!107 to 2!107 amastigotes of
L. donovani and a characteristically different outcome is seen in (a) the liver and
(b) the spleen. In the liver, amastigote growth is evident from the earliest times
post-infection, but is brought under control around Day 28 p.i., with the maturation
of the granulomatous tissue response. By contrast, after an early acute phase where
amastigote replication is not evident, the spleen shows exaggerated splenomegaly,
loss of microarchitecture and persistent presence of amastigotes. Broken green
lines indicate approximate range of outcomes encountered in studies of common
inbred strains of mice. Broken black lines indicate limit of detection for parasite
quantification. Abbreviation: LDU, Leishman Donovan unit; p.i., post-infection.
www.sciencedirect.com
T cell
IL-12p40
TRENDS in Parasitology
Figure 2. Early interactions between Leishmania donovani and spleen mononuclear
phagocytes. After intravenous injection, amastigotes can be rapidly phagocytosed
by blood-borne DC precursors or by monocytes (a). On entry to the MZ, they are
phagocytosed by MZM (b) and, to a lesser extent, MMM (not shown). DC could bind
amastigotes or antigens released by them (c) and, in rare cases, could themselves
be infected (d). Parasite-derived antigens could be passed between macrophages
and various DC subsets (arrows) either in the form of released free antigen, by
exosomes, or by phagocytosis of apoptotic host cells. Acquired immunity is
initiated following traffic of DC into the T-cell zone (PALS) where they encounter
T cells and are further stimulated to secrete IL-12 (e). Abbreviations: DC, dendritic
cells; IL, interleukin; IL-12p40, interleukin-12/23 p40 subunit; MMM, marginal
metallophilic macrophages; MZ, marginal zone; MZM, marginal zone macrophages; PALS, peri-arteriolar lymphoid sheath.
526
Review
TRENDS in Parasitology
projects (http://www.sanger.ac.uk/Projects/L_infantum/
and http://www.sanger.ac.uk/Projects/L_major/), thus
enabling the identification of gene products potentially
involved in tissue tropism or differential recognition by
mononuclear phagocytes.
Using macrophages from both humans and mice,
in vitro studies have produced a plethora of candidate
receptors involved in parasite uptake (reviewed in
Ref. [10]), with more-recent reports emphasizing the
complex interplay between recognition of parasite surface
structures per se [11] and the role of serum opsonins
(including mannose-binding lectin [12] and C-reactive
protein [13]). Much attention has also focussed on the
C-type lectin DC-specific intracellular adhesion molecule
3-grabbing non-integrin (DC-SIGN; CD209) as a determinant of parasite attachment to dendritic cells (DC)
[14,15]. Both L. donovani amastigotes and promastigotes
bind DC-SIGN on transfected fibroblasts, and binding to
DC can be inhibited by anti-DC-SIGN monoclonal antibodies (mAb). DC-SIGN also binds some (Leishmania
pifanoi), but not all (L. major) parasites associated with
cutaneous leishmaniasis [16]. Unfortunately, as commented
earlier, few of these studies take into account the extreme
diversity of tissue mononuclear phagocytes in both
receptor expression and phagocytic capacity.
Immunohistochemical analysis has been used to localize L. donovani to specific sub-populations of splenic
macrophages [17]. Following intravenous high dose infection, the model currently used for most studies of VL,
amastigotes are rapidly removed from circulation by
marginal zone macrophages (MZM) and marginal metallophilic macrophages (MMM) which are found in the splenic
marginal zone (MZ) (Box 2). The removal of amastigotes
by MMM is surprising, given that MMM are normally
regarded as having limited phagocytic capacity. Red pulp
F4/80C macrophages also avidly phagocytose L. chagasi
amastigotes [18]. Rarely, amastigotes are observed within
Box 2. The marginal zone of the mouse spleen
The spleen is a major secondary lymphoid organ, as well as a site for
the clearance of damaged red blood cells and blood-borne
pathogens. The specialized areas of lymphocyte accumulation are
known as the white pulp, which contains B cells that accumulate in
follicles, and the peri-arteriolar lymphoid sheath (PALS), which
contain T cells that accumulate around the central arteriole. The
white pulp is separated from the erythrocyte-rich red pulp by the
marginal zone (MZ). The MZ of the mouse spleen comprises sinuslining reticular cells, MZ B cells, dendritic cells (DCs), marginal
metallophilic macrophages (MMM) and MZ macrophages (MZM).
The MMM are located on the inner border of the MZ, adjacent to the
white pulp, whereas the MZM are present at the outer boundary of
the MZ, adjacent to the red pulp (reviewed in Ref. [49]). In the MZ,
blood flows from terminal arterioles into open sinuses, where flow is
slowed down and blood-borne particles are removed by MZM and
DCs with high efficiency. In addition to removing antigens from the
blood, the MZ also plays a key role in directing lymphocyte traffic
from the blood into the white pulp. Interest in the function of the MZ
has undergone a recent resurgence following recognition of the key
role this structure must play in generating acquired immune
responses against blood-borne pathogens. Specialized DC subpopulations have been found within this structure, placing them in a
location where antigens can be captured, and where T- and B- cell
responses can be readily modulated.
www.sciencedirect.com
Vol.20 No.11 November 2004
the T-cell zone of the spleen, suggesting that they are
phagocytosed by migratory DCs [17,19], although the
frequency with which this is observed makes further
characterisation of this potential interaction difficult.
Studies using green-fluorescent protein (GFP)-transgenic
parasites have confirmed that much of the DC response
occurs in cells containing no obvious intracellular amastigotes [19]. While most studies on Leishmania uptake in
the liver have focused on phagocytosis by Kupffer cells, the
role of other hepatic mononuclear cells needs to be
investigated.
Modulation of host cell function by L. donovani
Following recognition and uptake of Leishmania, mononuclear phagocytes could elect to produce a functional
response. A new paradigm in immunology is that
conserved microbial structures or pathogen-associated
molecular patterns (PAMPs) can be recognized by a
broad array of host cellular pattern-recognition receptors
(PPRs). Such recognition is believed to serve both for
immediate activation of innate immune defences and as a
bridge for the instruction of acquired immunity (reviewed
in Ref. [20]). Of the many PPRs expressed by mononuclear
phagocytes, those of the Toll-like receptor (TLR) family
have been under the most recent scrutiny. Pathogens as
diverse as mycobacteria, virus, fungi and protozoa are all
variably recognized by one or more of the ten TLRs
identified to date. As a consequence of TLR recognition,
most of these pathogens stimulate the production of
inflammatory (TNF, IL-12p70) and/or anti-inflammatory
(IL-10) cytokines, and trigger a maturation process in DCs
which endows these cells with an enhanced antigenpresenting capacity and the ability to promote functional
T-cell differentiation [21,22]. Leishmania donovani stands
out among the range of pathogens studied, including its
Leishmania relatives, as a consummately quiet invader of
mononuclear phagocytes, initiating limited cytokine
release from macrophages and DCs, and largely failing
to activate DC maturation in vitro [23]. Whether this
equates to an absence of TLR ligands on the surface of
L. donovani promastigotes and/or amastigotes, or the
action of a potent suppressive mechanism is unclear.
Intracellular amastigotes actively inhibit cytokine
responses of macrophages (at least those populations of
macrophages studied in vitro) to third party stimuli,
through pathways including; (i) activation of the suppressor of cytokine signalling (SOCS)-3 [24]; (ii) inhibition
of p38 mitogen-activated protein kinase (MAPK) [25];
(iii) local activation of latent transforming growth factor
(TGF)-b [26]; and/or (iv) through ceramide-induced modulation of extracellular signal-related kinase (ERK), activated protein-1 (AP-1) and NFkB transactivation [27].
Whether such pathways are also activated in the few DCs
that become targets of infection in vivo and whether
different macrophage populations have differing susceptibility to these parasite evasion tactics are important gaps
in our knowledge. Examination of DCs ex vivo at various
times after infection indicates that maturation occurs at
the population level, although most mature DCs are
apparently not infected by parasites [19]. DC maturation
following infection has also been demonstrated to be at
Review
TRENDS in Parasitology
least partly dependent on Myd88 signalling [19] and, at
least for L. major infection, Myd88K/K mice are more
susceptible to infection than that by wild-type mice
[28–30]. However, because Myd88 is involved in signalling
through other key cytokine receptors including IL-1R [31],
and TLRs have endogenous ligands such as heat-shock
proteins [32] and defensins [33], these experiments do not
conclusively demonstrate TLR-mediated DC activation
in vivo. A recent report indicates that lipophosphoglycan
of L. major might however be the first defined TLR ligand
in these parasites [29].
T cell–DC interactions and the spleen immune response
Although a low dose model of infection has recently been
established, most studies on the early immune response to
L. donovani and L. infantum have employed high dose
(107) inoculation of amastigotes. Under these conditions,
there are a few indications that initiation of immune
responses to this parasite are anything other than would
have been expected from studies of simple non-microbial
antigens delivered with weak adjuvant. Following infection, there is a rapid and transient burst of IL-12p70
secretion by splenic DC, which is localized within the
T-cell-rich periarteriolar lymphoid sheath (PALS)
(Figure 2). The appearance of IL-12p70-producing DC in
the PALS parallels an increase in high affinity IL-2
receptors on CD4C T cells, suggesting a classical model
of DC-mediated T-cell activation [17]. Both IL-12p40 and
IL-12p70 production peaks at five hours post-infection
(p.i.) and then diminishes, with IL-12p70 being produced
exclusively by CD8C DC in a T cell-dependent, yet surprisingly CD40-independent, manner. Early DC-derived
IL-12p70 production is important for the activation of
host-protective CD4C and/or CD8C T cells because IL-12
neutralisation during the first few days of infection results
in enhanced parasite growth [34]. The data also suggest
that there is rapid induction of IL-10 in the MZ [18], and
that this might contribute significantly to the establishment of infection [35]. CD8C T-cell responses are also
prominent in the early response to L. donovani, but
pathways for the induction of CD8C T cells specific for this
exogenous parasite have only recently become apparent.
Examination of the cell biology of L. donovani phagocytosis by macrophages has demonstrated that the loading
of parasite-derived antigens onto major histocompatibility
complex (MHC) Class I molecules might be facilitated by
endoplasmic reticulum-mediated phagocytosis [36,37]. In
addition, CD8C DC might acquire Leishmania antigens
for cross-presentation by phagocytosis of infected macrophages or their remnants [38]. The extent to which
responses by non-classical T cells [e.g. gd T cells and
natural killer (NK) T cells] are activated in the spleen
following infection is not yet known. However, a recent
report suggests that, among the many other cell surface
molecules whose expression can be manipulated by
Leishmania, L. donovani infection of DCs inhibits
expression of CD1 [39]. It should be considered that
in vivo, these events occur in a complex microenvironment, increasing the extent to which various cell types can
communicate together (Figure 2). Furthermore, whether
these conclusions concerning the initiation of the immune
www.sciencedirect.com
Vol.20 No.11 November 2004
527
response to L. donovani and L. infantum will be confirmed
following further studies using low-dose needle infections
or natural sandfly infections remains to be seen.
The spleen as a site of chronic inflammation
Following high-dose intravenous infection with L. donovani, the first 14 days progress normally in immunological
terms. There is evidence of extensive T-cell activation as
germinal centres develop rapidly, antibody titres begin to
increase albeit slowly and recall in vitro T-cell responses
can be readily detected. From Day 1–14 p.i., the growth of
amastigotes appears minimal or is matched by death.
Nevertheless, immune responses generated over this time
are clearly ineffective at eliminating amastigotes; within
three weeks p.i., the situation changes dramatically. To
date, three main events have been characterised that
occur between Day 14 and Day 21 p.i.: (i) alterations to MZ
structure; (ii) altered stromal cell function; and (iii) loss of
DC migration (see Figure 3). The functional consequences
of these changes are dramatic. A major advance in
understanding and unifying these events came with the
identification of TNF as a primary mediator of splenic
pathology and immune dysfunction.
Alterations to MZ structure
The splenic MZ is a primary site for parasite clearance and
is also involved in the regulation of lymphocyte homing
(Box 2). At the onset of the chronic phase of infection, the
MZ is subject to extensive, but selective remodelling.
Technically, it is often difficult to show that a cell type is
lost from a tissue using histological or flow cytometric
analysis based on phenotypic markers because it can
always be argued that downregulation of such markers
has occurred. However, in the case of MZM, it is possible to
specifically label these cells and in a stable manner by
virtue of their uptake of injected Indian ink. By this
approach, MZM, but not MMM, were rapidly depleted
from the MZ. This process was mediated by TNF because
TNFK/K mice or mice receiving TNF blockade largely
retained their MZM. As predicted, the loss of MZ
macrophages resulted in severely restricted migration of
lymphocytes into the white pulp during VL [40].
Altered stromal cell function
The PALS comprises a reticular network of collagen fibres
ensheathed by fibroblastic reticular cells, which extend to
connective tissue spaces of the arterioles [41]. This
network of tubular stromal cells is a conduit system that
is accessible for small molecules from the blood. The
stromal cells in the PALS express gp38 [42] and ER-TR7
[43], and are a major source of the constitutively expressed
lymphoid chemokines CCL21 and CCL19, which mediate
recruitment of CCR7-expressing naı̈ve T cells and mature
DCs. These chemokines are present within the tubular
system, as well as on the surface of the stromal cells that
enfold it, providing a framework along which T cells and
DCs can migrate within the PALS. At around the same
time as MZMs are lost from the spleen following
L. donovani infection, gp38C stromal cells in the PALS
also disappear. Again, TNF mediates the loss of gp38C
stromal cells in the PALS, with a resultant significant
Review
528
TRENDS in Parasitology
Vol.20 No.11 November 2004
(a)
(b)
(iv)
(v)
(ii)
(i)
MZ
(iii)
PALS
MZ
PALS
Key:
gp38+ stromal cell
CCL21 and/or CCL19
MMM
TNF-α
T cell
Central arteriole
Reticular fibers
Endothelial cell
MZM
IL-10
DC
Leishmania
TRENDS in Parasitology
Figure 3. Changes to splenic architecture during chronic infection. (a) The structure of the spleen remains normal during acute stages of infection. The MZ comprises two
discrete populations of macrophages: (i) the MZM at the outer boundary; and (ii) the MMM in close proximity to the PALS. Within the PALS, T-zone gp38C stromal cells and
the endothelium of the central arteriole are rich sources of the constitutive chemokines CCL19 and CCL21, which attract CCR7C DCs and T cells from the MZ into the PALS.
(b) The disrupted architecture of the spleen during chronic infection. Excess TNF production, probably from immigrant macrophages (i) leads to destruction of gp38 stromal
cells, loss of the reticular matrix they produce, and a loss of CCL19 and CCL21 expression (ii). The high levels of TNF production also positively regulate IL-10 production. It is
not known whether TNF induces IL-10 production by different subsets of macrophages (e.g. MMM) (iii) or whether the same macrophages make both TNF and IL-10 (iv). High
levels of IL-10 downregulate CCR7 expression on DC, inhibiting their ability to migrate into the PALS (v). Arrows indicate likely cellular sources and targets of TNF and IL-10.
Abbreviations: DC, dendritic cells; IL, interleukin; IL-12p40, interleukin-12/23 p40 subunit; MMM, marginal metallophilic macrophages; MZ, marginal zone; MZM, marginal
zone macrophages; PALS, peri-arteriolar lymphoid sheath; TNF, tumour necrosis factor.
decrease in production of the constitutively expressed
chemokines CCL19 and CCL21 [44].
Loss of DC migration
The increased level of TNF in the spleen also results in
increased production of the anti-inflammatory cytokine
IL-10, and this cytokine induces downregulation of the
crucial chemokine receptor CCR7 on the surface of DC,
preventing their migration into the PALS [44]. When
mature DCs from naı̈ve mice were administered to mice
with VL, significant reductions in splenic parasite burdens were observed [44], indicating that the failure of DC
migration into the PALS during L. donovani infection has
an important impact on disease outcome. These findings
provide a rationale for delivering vaccines by antigenloaded DCs or immunotherapy using DCs.
In addition to these structural changes, there is
other evidence of a changing immune response, as follows:
(i) T helper cell type (Th) 1 and Th2 cytokine profiles could
shift in their balance slightly (although they remain far
from polarised) [45]; (ii) local TGF-b levels are increased
and with apparent differences in kinetics in spleen and
liver [46,47]; (iii) monocyte chemoattractant protein 1
(MCP-1) synthesis is sustained, compatible with increased
macrophage recruitment [5]; and (vi) local haematopoietic
www.sciencedirect.com
activity is dramatically increased [48]. What is less clear
and important to understand is to what extent such events
are the consequence of, or precede the breakdown in,
lymphoid microarchitecture, and to what extent each of
these factors contributes to the relative levels of organspecific immunity versus persistence (alone or in combination with other factors)?
Concluding remarks
Several issues will need to be addressed before the full
basis for Leishmania persistence and the varied immunological responses seen in different tissues is fully understood (Box 3). Among these many outstanding questions,
the issue of mononuclear phagocyte heterogeneity is probably paramount. However, studying these cells in situ in
their unique locations poses great experimental challenges. Such studies will be important to ensure that
future development of vaccines, immunomodulators and
chemotherapy takes into account the various requirements for host protection in different tissue sites. The
development of new infection models and new tools to
study immune responses to Leishmania is progressing
fast, such that investigators will soon have the benefit of
the many tools (e.g. TCR transgenic models) cherished by
those who study viral and bacterial immune responses
Review
TRENDS in Parasitology
Box 3. Outstanding questions
† What receptors govern amastigote internalisation by the heterogeneous subpopulations of mononuclear phagocytes found in the
spleen, liver and other target tissue of Leishmania infection?
† Do all Leishmania parasites express Toll-like receptor ligands?
† What is the relative importance of each of the numerous evasion
strategies that have been identified in vitro for long-term parasite
persistence in vivo?
† Will our view of the early immune response to Leishmania
donovani hold true upon examination of infection by natural
transmission?
† What are the key immunological events that switch the spleen into
a state of chronic inflammation?
† What governs the local regulation of and/or cellular responsiveness to tumour necrosis factor (TNF)? It is host protective in the liver,
but in the spleen it causes pathology?
† Can therapeutic targeting of TNF aimed at limiting pathology be
balanced with the need to retain some TNF function for optimal
immunity against both Leishmania and possible latent infections
(e.g. tuberculosis)?
in vivo. On a more practical note, our hypothesis that
pathology is intimately linked to parasite persistence, and
the observations that such pathology results from the
action of TNF together suggest that intervention to block
TNF, as with other chronic inflammatory diseases [50],
could have some beneficial effects. Indeed, TNF blockade
with pentoxyfylin has some remarkable clinical effects in
drug-resistant cases of chronic cutaneous leishmaniasis
[51]. However, given the requirement for TNF for protection against Leishmania in the liver of mice with
experimental VL, and the potential for reactivation of
tuberculosis in patients with TNF blockade, caution would
be needed in evaluating this approach in countries where
both VL and tuberculosis are common.
Acknowledgements
Work in the authors’ laboratories is funded by grants from the British
Medical Research Council, The Wellcome Trust, the Australian National
Health and Medical Research Council, and the UNDP/World Bank/WHO
Special Programme for Research and Training in Tropical Diseases
(TDR). M.A. was a recipient of a Wellcome Trust International Fellowship; C.E. is a recipient of a NHMRC Career Development Fellowship.
References
1 Engwerda, C.R. and Kaye, P.M. (2000) Organ-specific immune
responses associated with infectious disease. Immunol. Today 21,
73–78
2 Murray, H.W. (2001) Tissue granuloma structure-function in experimental visceral leishmaniasis. Int. J. Exp. Pathol. 82, 249–267
3 Smelt, S.C. et al. (1997) Destruction of follicular dendritic cells during
chronic visceral leishmaniasis. J. Immunol. 158, 3813–3821
4 Leclercq, V. et al. (1996) The outcome of the parasitic process initiated
by Leishmania infantum in laboratory mice: a tissue-dependent
pattern controlled by the Lsh and MHC loci. J. Immunol. 157,
4537–4545
5 Rousseau, D. et al. (2001) Sustained parasite burden in the spleen of
Leishmania infantum-infected BALB/c mice is accompanied by
expression of MCP-1 transcripts and lack of protection against
challenge. Eur. Cytokine Netw. 12, 340–347
6 Wilson, M.E. et al. (1996) Local suppression of IFN-gamma in hepatic
granulomas correlates with tissue-specific replication of Leishmania
chagasi. J. Immunol. 156, 2231–2239
7 Tumang, M.C. et al. (1994) Role and effect of TNF-alpha in
experimental visceral leishmaniasis. J. Immunol. 153, 768–775
www.sciencedirect.com
Vol.20 No.11 November 2004
529
8 Ahmed, S. et al. (2003) Intradermal infection model for pathogenesis
and vaccine studies of murine visceral leishmaniasis. Infect. Immun.
71, 401–410
9 Zhang, W.W. et al. (2003) Comparison of the A2 gene locus in
Leishmania donovani and Leishmania major and its control over
cutaneous infection. J. Biol. Chem. 278, 35508–35515
10 Stafford, J.L. et al. (2002) Macrophage-mediated innate host defense
against protozoan parasites. Crit. Rev. Microbiol. 28, 187–248
11 Chava, A.K. et al. (2004) Identification of sialic acids on Leishmania
donovani amastigotes. Biol. Chem. 385, 59–66
12 Santos, I.K. et al. (2001) Mannan-binding lectin enhances susceptibility to visceral leishmaniasis. Infect. Immun. 69, 5212–5215
13 Bodman-Smith, K.B. et al. (2002) C-reactive protein-mediated phagocytosis of Leishmania donovani promastigotes does not alter parasite
survival or macrophage responses. Parasite Immunol. 24, 447–454
14 Colmenares, M. et al. (2002) Dendritic cell (DC)-specific intercellular
adhesion molecule 3 (ICAM-3)-grabbing nonintegrin (DC-SIGN,
CD209), a C-type surface lectin in human DCs, is a receptor for
Leishmania amastigotes. J. Biol. Chem. 277, 36766–36769
15 Geijtenbeek, T.B. et al. (2002) DC-SIGN, a C-type lectin on dendritic
cells that unveils many aspects of dendritic cell biology. J. Leukoc.
Biol. 71, 921–931
16 Colmenares, M. et al. (2004) The dendritic cell receptor DC-SIGN
discriminates among species and life cycle forms of Leishmania.
J. Immunol. 172, 1186–1190
17 Gorak, P.M. et al. (1998) Dendritic cells, but not macrophages, produce
IL-12 immediately following Leishmania donovani infection. Eur.
J. Immunol. 28, 687–695
18 Melby, P.C. et al. (2001) Leishmania donovani: evolution and
architecture of the splenic cellular immune response related to control
of infection. Exp. Parasitol. 99, 17–25
19 De Trez, C. et al. (2004) Myd88-dependent in vivo maturation of
splenic dendritic cells induced by Leishmania donovani and other
Leishmania species. Infect. Immun. 72, 824–832
20 Janeway, C.A., Jr. and Medzhitov, R. (2002) Innate immune
recognition. Annu. Rev. Immunol. 20, 197–216
21 Reis e Sousa, C. (2004) Toll-like receptors and dendritic cells: for whom
the bug tolls. Semin. Immunol. 16, 27–34
22 Sher, A. et al. (2003) Shaping the immune response to parasites: role of
dendritic cells. Curr. Opin. Immunol. 15, 421–429
23 McDowell, M.A. et al. (2002) Leishmania priming of human dendritic
cells for CD40 ligand-induced interleukin-12p70 secretion is strain
and species dependent. Infect. Immun. 70, 3994–4001
24 Bertholet, S. et al. (2003) Leishmania donovani-induced expression of
suppressor of cytokine signaling 3 in human macrophages: a novel
mechanism for intracellular parasite suppression of activation. Infect.
Immun. 71, 2095–2101
25 Junghae, M. and Raynes, J.G. (2002) Activation of p38 mitogenactivated protein kinase attenuates Leishmania donovani infection in
macrophages. Infect. Immun. 70, 5026–5035
26 Gantt, K.R. et al. (2003) Activation of TGF-beta by Leishmania
chagasi: importance for parasite survival in macrophages.
J. Immunol. 170, 2613–2620
27 Ghosh, S. et al. (2002) Leishmania donovani suppresses activated
protein 1 and NF-kappaB activation in host macrophages via
ceramide generation: involvement of extracellular signal-regulated
kinase. Infect. Immun. 70, 6828–6838
28 Debus, A. et al. (2003) High levels of susceptibility and T helper 2
response in MyD88-deficient mice infected with Leishmania major are
interleukin-4 dependent. Infect. Immun. 71, 7215–7218
29 de Veer, M.J. et al. (2003) MyD88 is essential for clearance of
Leishmania major: possible role for lipophosphoglycan and Toll-like
receptor 2 signaling. Eur. J. Immunol. 33, 2822–2831
30 Muraille, E. et al. (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
31 Hawn, T.R. et al. (2002) Leishmania major activates IL-1 alpha
expression in macrophages through a MyD88-dependent pathway.
Microbes Infect. 4, 763–771
32 Vabulas, R.M. et al. (2002) HSP70 as endogenous stimulus of
the Toll/interleukin-1 receptor signal pathway. J. Biol. Chem. 277,
15107–15112
Review
530
TRENDS in Parasitology
33 Biragyn, A. et al. (2002) Toll-like receptor 4-dependent activation of
dendritic cells by beta-defensin 2. Science 298, 1025–1029
34 Engwerda, C.R. et al. (1998) Neutralization of IL-12 demonstrates the
existence of discrete organ-specific phases in the control of Leishmania donovani. Eur. J. Immunol. 28, 669–680
35 Murphy, M.L. et al. (2001) IL-10 mediates susceptibility to Leishmania donovani infection. Eur. J. Immunol. 31, 2848–2856
36 Houde, M. et al. (2003) Phagosomes are competent organelles for
antigen cross-presentation. Nature 425, 402–406
37 Gagnon, E. et al. (2002) Endoplasmic reticulum-mediated phagocytosis is a mechanism of entry into macrophages. Cell 110, 119–131
38 Jung, S. et al. (2002) In vivo depletion of CD11c(C) dendritic cells
abrogates priming of CD8(C) T cells by exogenous cell-associated
antigens. Immunity 17, 211–220
39 Amprey, J.L. et al. (2004) Inhibition of CD1 expression in human
dendritic cells during intracellular infection with Leishmania donovani. Infect. Immun. 72, 589–592
40 Engwerda, C.R. et al. (2002) A role for tumor necrosis factor-alpha in
remodeling the splenic marginal zone during Leishmania donovani
infection. Am. J. Pathol. 161, 429–437
41 Nolte, M.A. et al. (2003) A conduit system distributes chemokines and
small blood-borne molecules through the splenic white pulp. J. Exp.
Med. 198, 505–512
42 Farr, A.G. et al. (1992) Characterization and cloning of a novel
glycoprotein expressed by stromal cells in T-dependent areas of
peripheral lymphoid tissues. J. Exp. Med. 176, 1477–1482
Vol.20 No.11 November 2004
43 Van Vliet, E. et al. (1986) Reticular fibroblasts in peripheral lymphoid
organs identified by a monoclonal antibody. J. Histochem. Cytochem.
34, 883–890
44 Ato, M. et al. (2002) Defective CCR7 expression on dendritic cells
contributes to the development of visceral leishmaniasis. Nat.
Immunol. 3, 1185–1191
45 Mukherjee, P. et al. (2003) Infection pattern and immune response in
the spleen and liver of BALB/c mice intracardially infected with
Leishmania donovani amastigotes. Immunol. Lett. 86, 131–138
46 Wilson, M.E. et al. (2002) The TGF-beta response to Leishmania
chagasi in the absence of IL-12. Eur. J. Immunol. 32, 3556–3565
47 Gomes, N.A. et al. (2000) TGF-beta mediates CTLA-4 suppression of
cellular immunity in murine kalaazar. J. Immunol. 164, 2001–2008
48 Cotterell, S.E. et al. (2000) Enhanced hematopoietic activity accompanies parasite expansion in the spleen and bone marrow of mice
infected with Leishmania donovani. Infect. Immun. 68, 1840–1848
49 Kraal, G. (1992) Cells in the marginal zone of the spleen. Int. Rev.
Cytol. 132, 31–74
50 Feldmann, M. and Maini, R.N. (2003) Lasker Clinical Medical
Research Award. TNF defined as a therapeutic target for rheumatoid
arthritis and other autoimmune diseases. Nat. Med. 9, 1245–1250
51 Bafica, A. et al. (2003) American cutaneous leishmaniasis unresponsive to antimonial drugs: successful treatment using combination of
N-methilglucamine antimoniate plus pentoxifylline. Int. J. Dermatol.
42, 203–207
World Health Organisation targets leishmaniasis in Afghanistan
The World Health Organisation (http://www.who.int/en/), in conjunction with the Massoud Foundation and HealthNet International
(http://www.heathnetinternational.org/), has stepped in to curb the current epidemic of cutaneous leishmaniasis in Afghanistan before
it escalates beyond control. A donation from the Belgian government made this intervention possible and the initiative aims to reduce
the incidence of the disease within the next two years.
The past two decades of conflict have eroded away any pre-existing leishmaniasis control programmes in the country. Environmental
damage and poor sanitary conditions have resulted in the proliferation of breeding sites for the vector of the Leishmania parasite, the
sand fly, and the influx of large numbers of displaced people, who possess little or no immunity to the disease, threatens to worsen the
situation.
As an immediate measure, WHO and its partners are combining drug treatment with the distribution of 16 000 insecticide-treated
bednets which will help to protect >30 000 people from sandflies. Initially, the program will focus on Kabul, the largest centre of
cutaneous leishmaniasis in the world and accountable for one-third of the 200 000 cases reported in Afghanistan.
For more information, please go to: http://www.who.int/mediacentre/releases/2004/pr55/en/
Written by Anthony Li ([email protected])
www.sciencedirect.com