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Retrospective Theses and Dissertations
1991
Pathogenesis of respiratory infections caused by
Mycoplasma dispar
Raul Antonio Almeida
Iowa State University
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Almeida, Raul Antonio, "Pathogenesis of respiratory infections caused by Mycoplasma dispar " (1991). Retrospective Theses and
Dissertations. Paper 9626.
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Pathogenesis of respiratory infections caused by Mycoplasma
dispar
Almeida, Raul Antonio, Ph.D.
Iowa State University, 1991
UMI
300N.ZeebRd.
Ann Arbor, MI 48106
Pathogenesis of respiratory infections caused by Mycoplasma dispar
by
Raul Antonio Almeida
A Dissertation Submitted to the
Graduate Faculty in Partial Fulfillment of the
Requirements for the Degree
of
DOCTOR OF PHILOSOPHY
Department: Microbiology, Immunology, and
Preventive Medicine
Major: Veterinary Microbiology
Approved:
Signature was redacted for privacy.
In Charge of Major Work
Signature was redacted for privacy.
FdHhe Major Program
Signature was redacted for privacy.
Signature was redacted for privacy.
^feâu^Coîié^
Iowa State University
Ames, Iowa
1991
L
ii
TABLE OF CONTENTS
Page
INTRODUCTION
1
EXPLANATION OF THE DISSERTATION FORMAT
3
LITERATURE REVIEW
4
Defense Mechanisms of the Respiratory Tract
Extracellular Mechanisms
The Mucociliary Apparatus
Response of the mucociliary apparatus to irritants
4
5
6
10
Interaction of pathogenic microorganism with the
respiratory mucociliary apparatus
12
Bacteria
12
Viruses
16
Mycoplasmas
19
Cellular Factors
28
Interaction of pathogenic microorganisms with alveolar
macrophages
36
Bacteria
36
Viruses
39
Mycoplasmas
42
PART I. "IMPAIRED FUNCTION OF THE MUCOCILIAriY
APPARATUS OF CALVES INFECTED WITH
Mycoplasma dispar"
48
SUMMARY
50
INTRODUCTION
51
METHODS
53
RESULTS
57
DISCUSSION
68
REFERENCES
73
PART II.- "INTERACTION OF Myœplasma dispar\N\TH BOVINE
ALVEOLAR MACROPHAGES"
78
ABSTRACT
80
INTRODUCTION
81
MATERIALS AND METHODS
83
RESULTS
89
DISCUSSION
101
REFERENCES
105
GENERAL CONCLUSIONS
110
REFERENCES
115
ACKNOWLEDGEMENTS
140
1
INTRODUCTION
The development of Infectious disease can be understood as the failure
of the host's defense mechanism to control proliferation, colonization, and
invasion of healthy tissue by pathogenic organisms. Establishment of the
infectious process can be the result of the damaging effect of pathogenic
microorganisms on cellular elements of the host defense or be enhanced by the
host's constitutive defects. Aside from these major determinants, other factors
that cause progressive detenoration of the host's defense mechanisms may
contribute to the development of disease (Mims, 1982; Roitt, 1988). Stressful
situations, deficient nutrition, poor management, and adverse environmental
conditions are included among these factors that have been proven to reduce
the efficiency of the respiratory defensive mechanisms (Abraham et al., 1986).
Microorganisms differ in their capacity to cause disease. Some non­
pathogenic microorganisms infect the host soon after birth and cannot
overcome natural host defenses and they do not produce disease even when
the host defense mechanisms are impaired by predisposing factors. Other
microorganisms may coexist with the host and they will produce disease only in
the event of profound deterioration of the host's defenses. In contrast, highly
virulent microorganisms usually produce disease when they infect. Pathogenic
microorganisms differ in their persistence in the infected host. Some
microorganisms are usually eradicated after the acute disease but other
microorganisms may also persist after recovery in healthy carriers; others can
lead to dormant infections that may relapse. Still others are present only during
active disease. In contrast, infectious microorganisms with moderate virulence.
2
such as some pathogenic mycoplasmas, cause disease characterized by
chronicity and long term colonization. The progressively deteriorating condition
that results from such infections determines a rupture of the balance between
host defenses and microorganism, allowing opportunistic proliferation of
microorganisms generally regarded as nonpathogenic.
Mycoplasma dispar is a pathogenic mycoplasma species commonly
associated with enzootic pneumonia of calves . The infection with this
mycoplasma species is characterized by colonization on the surface of
respiratory mucous membranes (Gourlay et al., 1970; Thomas et al., 1987). In
vitro experiments have indicated that M. d/spar restricts its colonization to the
apical surface of respiratory tract ciliated epithelial cells, causing profound
alteration of ciliary activity (Thomas et al., 1987). In addition, M. d/spar induced
a poor immune response which could help it in evading respiratory defense
mechanisms, resulting in long term colonization of these membranes (Howard,
1980; Tanskanen, 1984).
The aim of the present research was to characterize the strategy used by
M. dispar \o evade respiratory defense mechanisms. The approach used was
to determine the effect that M. d/spar infection has on the activity of two major
elements of the respiratory defense mechanism of the lungs, the mucociliary
apparatus and the alveolar macrophage.
Explanation of the Dissertation Format
This dissertation was written using the alternate format and consists of an
introduction, a literature review, two manuscripts, a general conclusion and a
list of references cited in the literature review. The manuscripts were written
following the format required by the journal to which these articles will be
submitted (American Review of Respiratory Diseases and Infection and
Immunity). The remaining sections of this dissertation were written following the
format established by the Iowa State University thesis office.
The doctoral candidate, Raul Antonio Almeida, is the senior author and
principal investigator for each of the manuscripts.
LITERATURE REVIEW
Defense Mechanisms of the Respiratory Tract
The capacity of the respiratory system to eliminate airborne agents is
determined by a complex and Interdependent set of extracellular and cellular
factors (Phipps, 1981; Dungworth, 1985; Roitt, 1988; Sleigh et al., 1988).
Extracellular factors involved in the defense of the respiratory membranes are
non-specific and effective as a first defense barrier. These include the
mucociliary apparatus, cough, bronchoconstriction reflex, and antibacterial
substances present in the secretions that line the airways and alveolar walls
(Sleigh et al., 1988). These secretions contain components such as surfactant,
bacteriostatic iron-containing compounds, complement fractions, and gamma
globulin (Roitt, 1988).
Cellular factors involved in the defense of the respiratory tract, include
several types of lymphoid cells and their secretory products (German et al.,
1990). It was described that the activity of cellular factors was initially
nonspecific, but a high degree of specificity was reached soon after the
beginning of infectious processes. The achievement of this specific response
results from complex and intricate interactions among these cellular elements,
which require the synergistic effect of extracellular non-specific factors (Roitt,
1988; Phipps, 1981). The mucociliary apparatus through Its constant
neutralization and removal of inhaled infectious and irritant particles constitutes
the first and the main defense mechanism of the airways mucosa. In the alveoli,
this role is performed by the alveolar macrophage. These cells engulf and
inactivate the particulate matter that escapes the mucociliary apparatus, present
processed antigens to accesory cells (e.g., B and T lymphocytes), and through
the secretion of cytokines, direct the immune response against infectious agents
(Sleigh et al., 1988; Khandom et al.,1985; Green et al.,1977)
Extracellular Defense Mechanisms
Effective removal of harmful agents that are deposited in the respiratory
membranes occurs mainly in the upper respiratory tract above the
bronchoalveolar junction. Deposition of inhaled particles occurred at different
anatomic locations according to their aerodynamic diameter. Those larger than
10 |im are cleared above the larynx and contact with the mucous layer occurred
mainly by inertial Impaction (Dungworth, 1985; Sleigh et al., 1988). These
large particles impacted against the mucous surfaces of airway passages
where air turbulence occurs (i.e., turbinates, airway branching), and the
efficiency of such trapping was determined by the size of the particle, by the
anatomic complexity of the nasal passages, and by the pattern of respiration
(Dungworth, 1985).
Particles with aerodynamic diameter between 1 to 3 |im were subject to
gravitational settlement. This form of deposition was favored by the relatively
still air of deeper parts of the respiratory system (e.g., bronchoalveolar junctions,
alveoli). The critical feature of this mechanism, from the point of view of
pulmonary homeostasis, was that infectious droplet nuclei and other irritant
particles of 1-2 ^m of diameter were more affected than particles of 3 p.m of
diameter, and this occurred where the linear velocity of the air stream fell near
zero (e.g., the bronchoalveolar junction). In that way, these anatomical regions
were exposed to the damaging activity of inhaled infectious agents and irritants
(Dungworth, 1985). Deposition of particles with aerodynamic diameters of 0.1
to 0.2 nm occurred by molecular collision which resulted in the settling of these
small particles in the alveoli (Dungworth, 1985).
Removal of particles from the upper respiratory tract was achieved by the
activity of the mucociliary apparatus (McDermott et al., 1982). Small particles
which contact the respiratory mucosa at deeper anatomical regions (i.e.,
bronchoalveolar junction and alveoli) were removed by the combined activity of
alveolar macrophages and the mucociliary apparatus (McDermott et al., 1982;
Dungworth, 1985). Microorganisms that reach these regions were readily
ingested by alveolar macrophages. These cells then leave the alveolar lumen
via the tissue and migrate to distant sites through lymphoid follicles, bronchus
associated lymphoid tissue (BALT), lymphatics and the blood circulation
(Bienenstock etal., 1983; McDermott et al., 1982; Roitt ,1988; Berman et al.,
1990) Additionally, it has been described that alveolar macrophages leave the
respiratory system via the mucociliary escalator and were ultimately swallowed
and destroyed in the gastrointestinal tract (McDermott et al., 1982).
The mucociliary apparatus
The mucociliary apparatus constitutes one of the most important
defenses of the airways against inhaled particles (dust, irritants, bacteria,
viruses and allergens) and against changes in the humidity and temperature of
the inspired air (Veit and Farrell, 1978; Sleigh et al., 1988). The mucus fraction
of this apparatus is secreted onto the mucosal surfaces of the respiratory tract,
where it can trap inhaled particles and neutralize irritant gases. This fraction
and trapped particles on it are then removed from the airways by coordinated
beating of the cilia present on the surface of the respiratory epithelium (Veit and
Farrell, 1978; Dungworth, 1985; Sleigh et al., 1988).
The mucus fraction of this apparatus was described to be composed of a
outer gel and a inner sol fraction (Veit and Farrell, 1978; Sleigh et al., 1988).
The trapping of inhaled particles occurred in the outer gel layer and was
favored by the stickiness of this fraction. The propelling of this outer fraction and
the trapped particles was mediated by the ciliary beating which, in turn, was
facilitated by the low viscosity of the inner sol fraction. The viscoelastic and gellike-propieties of the mucus were essential for the transport of trapped particles
(Veit and Farrell, 1978; Sleigh et al., 1988). The airway mucus was described
as a complex mixture of water, inorganic salts, sulphomucins, sialomucins,
neutral mucosubstances, proteins and lipids which were produced and
secreted by goblet cells and mucous glands. The inter-and intramolecular
cross-linking between these components, mainly through disulphide bonds,
largely determined the properties of the mucus (Dungworth, 1985; Phipps,
1981; Sleigh et al., 1988).
The mucous surface of the tracheobronchial tree of most mammals was
described to be lined by a pseudostratified, ciliated, columnar epithelium which
rests on a basal membrane and elastic lamina (Dungworth, 1985; Sleigh et al.,
1988). Mucosubstances were locally synthesized and secreted by goblet cells
and by serous acini in glands present in the submucosa of cartilage-containing
airways (Phipps, 1981 ; Sleigh et al.,1988). The amount of glandular tissue in
the submucosa varies among species, ranging from complete absence in birds
to one gland per square millimeter in humans (Walsh and McLelland, 1974;
Phipps, 1981). When present, these glands accounted for the majority of
secretions. Goblet cells have been shown to be irregularly distributed along the
respiratory tree. Most of them were in cartilage-containing airways, but some
were also found in bronchioles (Thurlbeck et al., 1975; Sleigh et al., 1988).
These cells contained large electron-transparent secretory granules composed
mainly of acidic glycoproteins (i.e., sulphomucins), although neutral
glycoproteins and sialomucins may be also present (Thurlbeck et al., 1975;
Sleigh et al., 1988).
Epithelial serous cells were found to have a similar pattern of distribution
to that described for goblet cells. These cells contained a variable number of
electron-dense secretory granules and synthesized neutral glycoproteins,
lysozyme and secretory component (Brandtzaeg, 1974; Jeffrey and Reid, 1977;
Spiceret al., 1977).
Clara cells are another common cell type in the respiratory mucosa
(Weibel et al., 1976; Kuhn, 1976; Sleigh et al., 1988). These cells were
described as non-ciliated cells presenting a uniform distribution along the
respiratory tract. It was also described that the secretory activity of these lipids
and glycoprotein-secreting cells responded to environmental conditions (Kuhn,
1976; Phipps, 1981). Lipid secretion might act as a type of bronchiolar
surfactant, while glycoprotein secretion could be part of the bronchiolar mucus
layer which was described as the beginning of the ainvay mucociliary escalator
(Phipps, 1981). In addition, Clara cells were reported to have the central role of
stem cells for renewal of other cell types after irritation and damage of the
respiratory epithelium (Evans et al., 1978; Kuhn, 1976; Sleigh et al., 1988).
The mucosa of the airways was reported to contain various cells types.
Among these, ciliated epithelial cells were the most common type. These cells
were found down to the terminal bronchioles, increasing their relative number
from peripheral to central airways (Breeze and Wheeldon, 1977; Sleigh et al.,
1988). On the apical surface of each cell there were up to 250 cilia of 6 |im of
length and 0.2-0.3 ^m in diameter, which were anchored to basal bodies
located in the cytoplasm of the epithelial cells (Phipps,1981 ; Sleigh et
al.,1988). The free portion of each cilia was covered with cell membrane, and
inside of the cilia a complex of filamentous contractile elements, composed by
nine peripheral double and two central adjacent fibrils was described{Phipps,
1981: Sleigh et al., 1988). This constitutes a highly conserved structure since it
was found in all other cilia throughout the animal kingdom (Sleigh, 1977;
Sleigh et al., 1988). Claw-like structures have been detected on the tip of the
cilia and it was suggested that these structures may play a role in propelling the
mucus toward the larynx (Kuhn, 1976; Sleigh et al., 1988). On the apical cell
surface of the ciliated cells, a carpet of short non-motile microvilli was described
to occur in the spaces between cilia.
Mucociliary clearance is performed by the additive effect of an external
gel layer of highly viscoelastic mucus that traps inhaled particles and ride on the
tip of the cilia, and a internal sol layer of low viscoelastic fluid in which the cilia
beat. The propelling effect caused by the cilia beating determines the
movement of trapped particles and external mucus toward the larynx. (Veit and
Farrell, 1976; Dungworth, 1985; Sleigh et al., 1988). It is not known if these
layers represent two separate secretions with independent secretion control
mechanisms or if they represent a spontaneous two-phase separation of a
polymer mixture (Phipps, 1981 ). As support of the latter concept, it was
postulated that the movement of the cilia breaks down the structure of the
10
mucus gel creating a less viscous perclllar fluid (Edwards, 1978). The
consistency and depth of this Internal layer determines the efficiency of the
propelling activity of the cilia. It was proposed that microvilli of both ciliated and
brush cells (an accessory cell type found in low numbers In bronchial and
alveolar epithelium) had an absorptive function that helps to control these two
features of the perclllar fluid (Jeffrey, 1978; Phlpps, 1981; Sleigh et al., 1988).
In addition to the propelling activity, it was described that ciliated cells may also
synthesize and secrete sulfated glycoproteins (Sleigh et al., 1988), particularly
In response to airway Irritation (Coles, 1987).
Response of the mucociliary apparatus to irritants
Acute Irritation of the airways with ammonia vapor (100-1000 ppm) or
sulfur dioxide (10 ppm) were characterized by a rapid Increase in the secretion
of mucosubstances mainly from submucosal glands and from epithelial
secretory cells (Richardson et al., 1976; Coles, 1987). It was proposed that this
effect was mediated through two pathways which were: A) a reflex neural
pathway Involving secretory responses and the stimulation of the cough reflex;
and B) a direct action on the mucus secreting cells which Included the release
of metabolites of the arachldonic acid pathway (Phlpps, 1981 ; Coles, 1987).
The Irritant substances mentioned above also reduced the frequency of the
ciliary movement. Thus, the reduced ciliary activity In addition to the decreased
viscoelastic properties caused by altered and increased secretions, determined
a reduction of the mucus transport (Veit and Farrell, 1978; Sleigh et al., 1988).
This effect caused inefficient neutralization and removal of the irritant which
resulted in a nflammatory response. Serum also can act as an irritant since it
11
caused stimulation of the secretion of mucin glycoproteins (Phipps, 1981). It
was proposed that this effect was mediated through the direct release of luminal
surface glycoproteins and increased secretion of the submucosal glands
(Phipps,1981).
Responses to chronic insult were characterized by increased mucus
production and decreased mucociliary function as a consequence of the
repeated exposure to irritants or long term infections. These changes were
brought about in part, by the loss of cilia and by the hyperplastic reaction of
epithelial mucous cells and submucosal glands (Jeffrey and Reid, 1977; Cole,
1987). Changes in the secretory function of the respiratory mucosa were
elicited in response to direct and indirect stimulation of the secretory cells. In
one study on the effect of cigarette smoke in animals, it was shown that the anti­
inflammatory agent phenylmethyloxadiazole prevented smoke-induced
hypertrophic changes in the epithelium but not in submucosal glands (Jones
and Reid, 1978). This was taken as evidence of the direct effect of the irritant on
the respiratory epithelium. However, there was also evidence that the
metabolically active respiratory mucosa may modulate the function of the
underlying smooth muscle by metabolites and the production of relaxant,
constrictor, or chemotactic factors (Cuss and Barnes, 1987). The involvement of
indirect stimulation in the smoke-induced hypertrophic changes was highlighted
in an experiment in which these hypertrophic changes were reproduced by
repeated injections of pilocarpine and isoprenaline (Phipps, 1981). In addition,
chronic inflammatory processes elicited an increase in the relative number of
secretory cells and caused a shift in the composition of secretions of
submucosal glands and goblet cells. This resulted in a net increase of the
12
population of cells containing sialomucins (Reid, 1965; Wheeldon et al., 1976).
The increased activity of goblet cells and submucosal glands, and the
production of secretions of lowered viscosity was coupled with an inability to
remove such excess of mucus production by ciliary epithelium that was
impaired by the direct damaging effect of the irritant (Jeffrey and Reid, 1977). In
addition, this scenario was worsened by the effects of these changes on small
airways, which become blocked with mucus, increasing the exposure of the
respiratory mucosa to the irritant (Woods et al., 1976; Sleigh et al., 1988).
Under these predisposing conditions, bacterial pathogens proliferated and
caused disease, such as has been described in human cystic fibrosis. In cystic
fibrosis, the basic defect was described as a general dysfunction of exocrine
glands which caused changes in the chemical composition of the mucus with
alteration of its physical properties (Woods et al., 1976). The mucus of patients
with cystic fibrous was reported to contain more mucus glycoproteins, plasma
transudate, and DNA than mucus from healthy controls (Woods et al., 1976). It
was proposed that physicochemical changes in the mucus may alter the
interaction between mucus and cilia, or may alter the bonding between
glycoproteins present in the mucus, responsible for their consistency. These
changes, in addition to the loss of cilia, lead to impairment of the mucociliary
function and to the proliferation of secondary pathogens (Woods et al., 1976).
Interaction of pathogenic microorganism with the respiratory mucociliary
apparatus
Bacteria
Pseudomona aeruginosa is the most common cause of
morbidity and mortality in cystic fibrosis patients. The bacteria presented tropism
13
for the respiratory tract and adhered to tracheal ciliated cells and
tracheobronchial mucins by means of pili or, in mucoid strains, through the
bacterial exopolysaccharide (Vishwanath, 1988). The marked tropism and
adherence capabilities of P. aeruginosa, in addition to the failure of the host to
physically or immunologically remove the bacteria from colonized cells,
determined the persistence of the infection and the non-favorable outcome of
the disease (Rampai, 1987; Vishwanath, 1988). Pseudomona aeruginosa was
also reported to induce ciliary and epithelial necrosis, which worsen even more
the prognosis of cystic fibrosis. Similarly, Haemophilus influenzae caused
dyskinesia and destruction of the ciliary epithelium. Encapsulated H. influenzae
strains were shown to reduce the ciliary activity more than the unencapsulated
variant of the bacteria (Cole, 1987; Reynolds, 1987).
Several members of the Bordetella genus also exhibited a marked
affinity for the ciliated epithelium. Of the various toxins and virulence-related
factors produced by Bordetella pertussis, only one has been demonstrated to
reproduce the specific respiratory epithelial cytopathologic changes
characteristic of the pertussis syndrome (Goldman and Herwaldt, 1985). This
factor, known as tracheal cytotoxin (TCT), was released by B. pertussis during
log phase growth. Intratracheal exposure to purified TCT specifically damages
ciliated epithelial cells, causing ciliostasis and extrusion of these cells
(Goldman and Henwaldt, 1985). The particular disaccharide-tetrapeptide
structure of TCT reveals that it was apparently formed by cleavage of
peptidoglycan (Goldman and Cookson, 1988). Unlike other gram-negative
bacteria, B. pertussis appears to be very selective in the release of cell wall
fragments, since more than 95% of soluble peptidoglycan in culture
14
supernatants was TCT. The structure of TCT places it in the "muramyl peptide"
family, a group of structurally related molecules that were responsible for a
diverse array of biological activities (Goldman and Cookson, 1988). The
selective biological activity of B. pertussis TCT was studied in tracheal organ
cultures by light and electron microscopy. From this study it was reported that
TCT only affected ciliated cells, and these underwent a series of pathological
changes that preceded their eventual extrusion (Goldman and Herwaldt, 1985).
In addition to TCT, two other proteins secreted by B. pertussis mediated
the adherence of this or other bacterial pathogens to mammalian respiratory
cilia (Tuomanen, 1986). When either ciliated cells or other pathogenic bacteria,
such as Streptococcus pneumoniae, H. influenzae, and Staphylococcus aureus
were treated with these adhesins, they acquired the ability to adhere to cilia in
vitro or in vivo. Such piracy of adhesins may contribute to superinfection of
mucosal surfaces by secondary pathogens as is commonly seen in respiratory
diseases such as whooping cough (Tuomanen, 1986). Muramyl-peptide
molecules (one of which is identical to TCT) were described as being released
by Neisseria gonorrhoeae. This compund can cause ciliated cell-specific
damage like that described during gonococcal infection of fallopian tube
mucosa (Goldman and Cookson, 1988).
Inoculation of lambs with Bordetella parapertussis also caused alteration
in the mucociliary apparatus (Chen et al., 1988). In the first 24 hours after
infection, tracheal and bronchial epithelium was covered by a large amount of
inflammatory exudate. Later, the tracheobronchial epithelium showed focal
extrusions of ciliated cells, which were occasionally associated with B.
parapertussis organisms (Chen et al., 1988). Cytopathological changes
F
16
associated with B. parapertussis infection were mild focal degeneration of
airway epithelium with slight loss of cilia, and focal inflammation of the lungs
with moderate to severe degeneration of type I and type II pneumocytes. In
addition to these cells, alveolar macrophages presented pathological changes
which included moderate to severe cytoplasmic vacuolization (Chen et al.,
1988).
Bordetella bronchiseptica, has been described as one of the etiological
agents of atrophic rhinitis in pigs and "kennel pneumonia" in dogs (Dungworth,
1985). This bacterium showed tropism for the ciliated epithelium of the
respiratory tract and possesed readily detectable somatic pili. This was the only
consistent phenotypic characteristic associated with the ability to induce
ciliostasis (Bemis and Wilson, 1985). Filiated strains of B. bronchiseptica
treated with proteases lost their capacity to attach to cilia and the capacity to
induce ciliostasis (Bemis and Wilson, 1985). In another work, ciliated epithelial
cell outgrowths from canine tracheal expiants were used to study the interaction
between B. bronchiseptica and respiratory cilia (Bemis and Kennedy, 1981).
Phase I and intermediate-phase B. bronchiseptica isolates caused reductions
(more than or equal to 50%) in ciliary beat frequencies within 5 minutes of
infection, and nearly complete ciliostasis within 3 hours after infection. Rough
B. bronchiseptica and heat and formalin-killed preparations of the phase I
isolate had no ciliostatic effect. Phase I and intermediate-phase isolates
attached to cilia, whereas the rough variant did not. The ciliostatic effects of the
phase I isolate could not be reproduced with endotoxin or culture supernatants
from the organism. From this experiment it was concluded that attachment
alone does not produce ciliostasis but mediation of ciliostatic effects of
16
Bordetella may require the close association between metabolically active
organisms and cilia (Bemis and Kennedy, 1981).
The avian pathogen Bordetella avium also exhibited affinity for the
ciliated epithelium. In one study, one-day-old turkeys were infected intranasally
with B. avium and tracheas were examined by scanning and transmission
electron microscopy 1 to 5 weeks post-inoculation. From these it was described
that infection with this pathogen caused progressive loss of ciliated epithelium
with replacement by non-ciliated cells. In addition, bacterial colonization of
ciliated cells was associated with membrane-bound crystalline inclusions in the
cytoplasm of epithelial cells, depletion of secretory granules, and distortion of
tracheal rings and of the mucosal surface (Fagerland and Arp, 1987).
Viruses
Bovine herpes virus-1 (BHV-1) caused profound alterations of
the respiratory epithelium of the upper regions of the respiratory tract upon
infection. In experimental cases, rhinitis, laryngitis and tracheitis developed
with pinpoint haemorrhages on the mucosal surface 4, 5 or 7 days after
infection (Allan and Msolla, 1980). There was neutrophilic infiltration of the
hypertrophied epithelium with extensive loss of cilia, leaving areas of epithelium
covered by microvilli (Allan and Msolla, 1980). The submucosa of these areas
was infiltrated with lymphocytes and the tracheal submucosal glands were
dilated and slightly hypertrophied. Lesions were of similar severity on day 5
and 7 after infection and virus was re-isolated from nasal passages from days 412 after infection (Allan and Msolla, 1980). The importance of the infection by
this viral agent in the development of more severe respiratory processes was
stressed by Jericho (1983). This author described that the main histopathologic
17
changes observed in a mixed infection with BHV-1 and P. hemolytica were
hyperemia of pharyngeal tonsils and the lamina propria of air passages and
alveolitis associated with edema and fibrin deposition in lumen and septa.
Initially, eosinophilic granulocytes were numerous in turbinates, bronchi and
lung, but 3 days after exposure, polymorphonuclear leukocytes predominated.
Bacterial colonies were seen in nasal turbinates, tonsils, trachea and lung.
Necrosis was seen in the nasal, tracheal, and pulmonary epithelium.
Necropurulent changes were particularly evident in tonsils and alveoli. Blood
vessels, walls of bronchioles, and major air passages were generally free from
inflammation (Jericho, 1983).
Parainfluenza-3 virus (PI-3) was also described to induce damage to the
respiratory epithelium. In the early acute stage of the pneumonia, PI-3
replication was observed within epithelial cells of the respiratory tract and within
alveolar macrophages (Bryson et al., 1983). Morphological changes included
loss of cilia and of ciliated cells in small bronchi and bronchioles. Necrosis was
noted in both bronchiolar and alveolar epithelium. Proliferation of non-ciliated
bronchiolar cells (Clara cells) produced zones of hyperplastic bronchiolar
epithelium. Within the alveoli, widespread proliferation of type 2
pneumonocytes led to alveolar epithelization. Organization of bronchiolar
exudate involving fibroblasts and macrophages was widespread in calves killed
between 7 and 12 days after Infection and in calves killed at 12 days and
subsequently, bronchiolitis obliterans was widespread ( Bryson et al., 1983).
Moreover, in another study, electron microscopic demonstration of aggregates
of viral nucleocapsids in the cytoplasm, alterations of cilia and basal bodies,
dissolution of cytoplasmic membranes, and shedding of virus into luminal
18
spaces confirmed that epithelial cells of the respiratory tract were the primary
target cells for PI-3 replication (Tsai and Thompson, 1975). These observations
revealed that productive infection was more efficient in the bronchioalveolar
junctions and destructive changes were more pronounced in the tracheal
epithelium. The finding that PI-3 virus replicates in alveolar type II cells, which
are considered to be the producers of pulmonary surfactant, suggests that
changes in surfactant production may occur during the peak of infection of these
cells (Tsai and Thompson, 1975). The demonstration of virus penetration
through the basolateral aspect of epithelial cells in small bronchioles and the
presence of virus particles in the interstitial regions imply that host defense
barriers may be impaired by virus invasion.
Bovine respiratory syncytial virus (BRSV) also had a marked tropism for
ciliated respiratory epithelium (Castleman et al., 1985). In experimental
infections of calves with BRSV, there was hyperplasia of basal epithelial cells in
association with other epithelial morphological changes . These lesions
resulted in the loss of normal ciliated epithelium in these airways 5 to 7 days
after infection (Castleman et al., 1985). Regeneration of airway epithelium was
largely completed by 10 days after inoculation, except in 1 of 4 calves that
developed secondary bacterial pneumonia. The pulmonary ultrastructure of
BRSV inoculated calves 30 days after inoculation was indistinguishable from
that of controls. These results demonstrated that BRSV can induce reversible
alterations in airway epithelium, which may cause depression of mucociliary
clearance and thereby enhance susceptibility to bacterial infection (Castleman
et al., 1985). In another work, 33 cattle with fatal respiratory tract diseases were
examined for gross and histopathologic lesions and presence of viral and
19
bacterial pathogens in the lungs. Fifteen animals had lesions that were
classified as atypical interstitial pneumonia ( AlP), and the histologic features of
these included interstitial edema, thickening of alveolar walls, hyaline
membrane formation and hyperplasia of type -II pneumonocytes (Collins et al.,
1988). Bovine respiratory syncytial virus was detected in 11 of these 15 cattle
with AlP. The significant association found between these lesions and the
presence of BRSV led these researchers to postulate BRSV as an etiologic
factor of AlP (Collins et al., 1988).
Mvcoplasmas
Several species of Mycoplasmas can produce alteration
of the ciliated respiratory epithelium of humans or domestic animals. These
alterations were observed under in vitro conditions, using tracheal expiants, or
under in vivo conditions. For instance, Jones et al. (1985) using tracheal
expiants from fetal lambs or newborn kids showed that Mycoplasma
ovipneumoniae and Mycoplasma arginini were capable of stopping ciliary
beating and causing the loss of these appendages. Direct observation using
scanning electron microscopy showed clusters of microorganisms attached to
epithelial surfaces and in association with cilia (Jones et al., 1985).
In calves naturally infected with M. dispar and Ureaplasma diversum, the
respiratory tract presented alterations of the mucociliary escalator (Allan et al.,
1977). Among these, morphological changes of the bronchial epithelial cells
included hyperplasia, loss of cilia, cytoplasmic protrusion into the airway lumen,
and distended and dismpted mitochondria (Allan et al., 1977). In addition,
changes in the composition of the mucus were reported, including a net
increase of sialomucin production. In later stages of the infection, some of the
20
cells lost their characteristic histochemical staining due to exhaustion or
replacement by a different or immature cell type (Allan et al., 1977). Using
tracheal organ cultures, it was shown that M. dispar was capable of stopping
ciliary motility and destroying the ciliated epithelium (Thomas and Howard,
1974; Howard and Thomas, 1974; Thomas et al., 1987). In these studies it
was observed that the inhibitory activity on ciliary motility caused by M. dispar
was unaffected by catalase, ruling out the production of H2O2 as mediator of
this effect. It was shown, however, that the inhibitory effect on the cilia was
dependent on the concentration of fetal bovine serum (FBS) in the culture
medium (Howard and Thomas, 1974), In a comparative study of the growth rate
of M. dispar m serum-free media or with the addition of 5 % of FBS, these
authors reported that in spite of the similar final mycoplasma titer observed
under both conditions, faster growth of M. dispar was observed when serum
was added, compared with that observed in the serum-free condition. Based on
this observation, it was postulated that a toxic substance could be synthesized
and released into the medium, and this putative molecule could be derived
either from FBS or produced at high concentration in the presence of FBS.
However, no changes were observed when filtered supernatants from M. dispar
infected cultures were used. (Howard and Thomas, 1974).
The effect of porcine mycoplasmas on the ciliated respiratory epithelium
was studied using tracheal organ cultures (Pijoan et al., 1972). In this work, it
was shown that Mycoplasma granularum, Mycoplasma hyorhirjis and
Mycoplasma hyosynoviae were able to cause more than 50 % cilia-stopping
effect (CSE) after 7 days of inoculation. In contrast, in the same system
Mycoplasma hyopneumoniae caused CSE that was below or slightly above the
21
effect observed in the non-inoculated controls. The titer of these porcine
mycoplasmas in the pig tracheal organ cultures used in this study remained
constant at levels of IQS-IO? color changing units/ml (ccu/ml) in these
mycoplasmas with the exception of M. hyopneumoniae. This indicated that the
CSE exerted by the latter mycoplasma species was dependent on the titer of
the microorganism. This observation was confirmed in the work of Williams and
Gallagher (1977), in which these researchers, using porcine tracheal rings
supplemented with monolayer cultures of fetal lung fibroblasts, were able to
demonstrate severe cellular damage and CSE in tracheal rings infected with M.
hyopneumoniae. Using this organ culture system, the M. hyopneumoniae titer
increased from 101 to 10® ccu/ml after 3 days of inoculation (Williams and
Gallagher, 1977).
Using chicken tracheal cultures. Cherry and Taylor-Robinson (1970)
showed that Mycoplasma galiisepticum was capable of stopping ciliary activity
and that the effect was not related with the infecting dose. It was also shown
that the addition of neuraminidase or catalase to the cultures did not inhibit the
CSE. These observations were taken as evidence to postulate that neither
cytoadsorption nor peroxide production were important factors in the virulence
of this mycoplasma. However, these authors reported the presence of
molecule(s) toxic for the ciliated epithelium (Cherry and Taylor-Robinson,
1970b). In contrast, the same authors using a similar model, reported that the
CSE exerted by Mycoplasma mycoides Msx.capri was directly associated with
the number of organism inoculated. The observed CSE was dependent upon a
close association of multiplying mycoplasma, and the addition of catalase or
glucose protected the cilia from such effect. No demonstrable toxic factors were
22
related with these changes in the ciliary epithelium (Cherry and TaylorRobinson, 1970a).
Mycoplasma pneumoniae is a pathogen of the human respiratory tract.
This mycoplasma species was described as the principal cause of atypical
pneumonia in children and young adults (Denny et al., 1971; Foy et al., 1979).
Clinical manifestations of the disease range from asymptomatic conditions to
bronchitis and pneumonia. The damage originated by M. pneumoniae infection
has been studied using tracheal organ cultures and the changes detected
included gradual loss of the ciliary activity, nuclear swelling, cytoplasmic
vacuolization and exfoliation of ciliated cells (Carson et al., 1980). These
changes were accompanied by metabolic abnormalities, which included
decreases in nucleotide content, intracellular levels of ATP, carbohydrate
utilization, amino acid transport, macromolecular synthesis, and oxidative
metabolism (Gabridge and Polisky, 1977; Upchurch and Gabridge,1982; Hu et
al., 1976; Gabridge, 1975). In another study, chicken-embryo tracheal organ
cultures were inoculated with M. arginini, M. equigenitalium, 2 strains of M.
subdolum, Acholeplasma laidlawii and 3 strains of A. oculi (Moorthy and
Spradbrow, 1985). All these strains established and multiplied in the expiant
cultures, but only M. subdolum and A. oculi produced a cytopathic effect on
ciliated epithelial cells, that ended with the sloughing of these cells after 6 days.
There was a positive correlation between ciliostasis and increased in the titer of
both M. subdolum and A. oculi, but such a relationship was not detected with M.
equigenitalium and A. laidlawii (Moorthy and Spradbrow, 1985). Cells infected
with M. subdolum showed sloughing of cilia, vacuolization, and increase in size
of mitochondria, followed by disorganization of epithelium and marked
destruction of cellular organelles (Moorthy and Spradbrow, 1985).
Mycoplasmas were attached to the epithelial surface of the tracheal expiant 8
days after infection (Moorthy and Spradbrow, 1985). Even when these results
indicated that these equine mycoplasma species can induce cytopathic effects
on chick embryo tracheal expiants, the same can not be taken as valid for
equine ciliated respiratory epithelium. Using a similar in vitro approach but
complemented with in vivo observations, Dykstra et al. (1985) studied the
pathological effect induced by M. gallisepticum on ciliated respiratory
epithelium of chickens. These workers described that the changes in the
tracheal epithelial surfaces induced by the mycoplasma infection under in vivo
and in vitro conditions, included release of granules containing mucus, followed
by exfoliation of ciliated and non-ciliated epithelial cells. Loss of cilia from
individual cells was infrequent, but in those cells in which this change was
detected, this was followed by loss of the epithelial cell's intercellular
connections. These events were then followed by rounding up and exfoliation
of cells with degradation of their membranes (Dystkra et al., 1985). Thus, this
process gave rise to a population of released cellular organelles, such as
mitochondria, which along with the sloughed cilia and mucus formed the
exudate present within the tracheal lumen (Dystkra et al., 1985). Repair of the
epithelial surface was effected by basal epithelial cells that began to
differentiate and fill the spaces left by exfoliated cells. Fourteen days after
infection, these cells were hypertrophic and non-ciliated but covered with
microvilli (Dystkra et al., 1985). Histologically, there was increased epithelial
thickness due to cellular infiltration and edema. Results from experiments with
tracheal rings showed similar changes, with the exception that exfoliation of the
24
epithelial cells occurred sooner after infection and was characterized by more
severe ultrastuctural changes than that observed in vivo (Dystkra et al., 1985).
Finally, using a similar in vitro model, it was shown that two strains of M.
synoviae and one strain of M. gallisepticum exerted pathological effects on the
ciliary respiratory epithelium of chickens (Hirano et.al., 1976). Mycoplasma
gallisepticum was more effective in stopping ciliary activity than the two M.
synoviae strains, and electron microscopic examination showed the
mycoplasma attached to cilia and epithelial cells (Hirano et.al., 1976).
Mycoplasma hyorhinis was shown to produce alterations on the ciliated
epithelium under in vivo conditions (Baskerville and Wright, 1973). Twenty-four
4-week-old pigs were infected intranasally with M. hyorhinis isolated from lungs
of animals with enzootic pneumonia. All the animals that were slaughtered
between 15 days to 8 weeks after infection showed widespread pulmonary
consolidation (Baskerville and Wright, 1973). Electron microscopic
examinations revealed mycoplasmas in the lumen of airways. Ultrastructural
changes included large masses of cytoplasm projected from the apex of many
ciliated bronchiolar cells, and in these, cilia lost their upright position, became
matted, and appeared intertwined (Baskerville and Wright, 1973).
The affinity of mycoplasmas for ciliated stmctures is well documented.
Mycoplasmas not only show affinity to respiratory ciliated cells, but also to
similar stmctures present in other anatomical locations. Scanning electronmicroscopic observation of the mucous membrane of an infraorbital turkey sinus
infected with M. gallisepticum revealed a swollen corrugated membrane surface
and swollen dome-shaped epithelial cells. All cilia were lost, although microvilli
remained. No necrosis of mucosal surfaces was detected (Sanger, 1973). In
another study, gnotobiotic mice were inoculated intranasally with M. pulmonis
(Haliiwell et al., 1974). Lesions were detected in the middle ear, and among
these, proliferation of mucus-secreting cells in the medial wall of the timpanic
cavity and areas of ciliary fusion and ciliary loss were described. It was noted
that increased numbers of mononuclear cells were accompanied by edema and
proliferation of submucosal connective tissue (Haliiwell et al., 1974).
Ureaplasma species isolated from the human genital tract and from the
genital and respiratory tract of cattle stopped the ciliary activity of the ciliated
genital epithelium when cultured in organ cultures of bovine oviduct (Stalheim
et al., 1976). This effect was noticed between 24 and 144 hours after
inoculation and cellular changes detected by SEM were the collapse and
sloughing of cila. In addition, vacuolization, disorganization, necrosis and
sloughing of the ciliated epithelium was detected. The mycoplasmal tropism for
ciliated cells was well documented by Kohn et al. (1977). In this report, these
authors communicated the induction of hydrocephalus as consequence of
intracerebral inoculation of M. pulmonis. This congenital teratogenic defect was
induced in 116 of 120 rats (96%) and in 23 of 28 hamsters (82%). Electron
microscopic examinations revealed that an amorphous material covered
portions of the ependymal surface and fusion of the cilia on ciliated cells was
also seen. It was suggested that the hydrocephalus was due to ciliary
dysfunction or to an imbalance of cerebrospinal fluid secretion and absorption
(Kohn et al., 1977). In another work porcine lateral ventricular ependymal cells
were used to assert the effect of M. hyopneumoniae and M. hyorhinis on the
central nervous system of pigs. It was reported that these mycoplasma species
caused degenerative processes which included loss of microvilli and cilia.
26
membrane disruption, and colonization of these ciliated cells by mycoplasmas
(Williams and Gallagher, 1981).
It was previously shown that ciliary structure was highly conserved
throughout the animal kingdom (Sleigh, 1977). It Is possible that the tropism
shown by several species of mycoplasma to ciliated cells, reflects the affinity of
these microorganisms to a common ciliary surface structure. The attachment of
mycoplasmas to host cells was described as a prerequisite for colonization and
induction of pathogenic changes in these cells (Hansen et al., 1979; Razin,
1978; Lipman et al., 1969; Sobeslavsky et al., 1968). Flask- shape
mycoplasmas (e.g., M. pneumoniae and M. gallisepticum) have been described
to possess protein receptors localized in the tip of these microorganisms which
specifically interact with neuraminidase-sensitive receptors in host cell
membranes (Bredt et al., 1981). It was shown that M. pneumoniae cells interact
through their receptor with sialo-oligosaccharide branched structures (sialosyl-l
and sialosyl-i) as well as with other related oligosaccharide structures of poly-A/acetylglucosamine type. In the respiratory mucosa, these receptors were only
expressed in ciliated cells (Loveless and Feizi, 1989). Long-chain
oligosaccharides were found highly polarized at the luminal aspects of the
ciliated cells, where the branched structures (sialosyl-l and sialosyl-i) were
detected both at the epithelial apical border and on the cilia (Loveless and
Feizi, 1989). The distribution and the density of these receptors on the luminal
surface of the ciliated epithelium may explain the close association of this
mycoplasma species with the cilia and the apical surface of the ciliated cells
(Hu et al., 1977). In addition, it was shown that M. pneumoniae could rearrange
its shape and increase the amount of binding sites on the specialized tip upon
proximity to target cells (Kahane, 1984; Kahane et al., 1984). These changes
provide the mycoplasma with an advantageous shape and surface receptor
distribution to pass between cilia and microvilli, reaching the membrane surface
of ciliated respiratory cells where this mycoplasma attaches (Hu et al., 1977). In
contrast, other non-flask shaped mycoplasma which affect the respiratory tract
(e.g., M. pulmonis, M. dispar, M. hyopneumoniae), did not show these binding
sites on their membrane surfaces, nor specialized tips or ability to rearrange
surface receptor density and shape, which can explain the fact that they were
only seen in close association with the microvilli present on the apical surface of
the ciliated respiratory cells (Baskerville and Wright, 1973; Allan, 1978; Razin,
1978; Kirchhoff, et al., 1984; Kahane, 1984).
There appear to be several possible mechanisms that contribute to host
cell dysfunction during mycoplasma infection. One of these was the production
of hydrogen peroxide and superoxide with oxidative damage and alteration of
the redox potential, which was supported by the data reported by Almagor et al.
(1983; 1986; 1984), and by the work of Cherry and Taylor Robinson (1970a).
Host cell membrane alteration induced by mycoplasma membrane components
was reported and proposed as a pathogenic mechanism and production of toxic
compounds with CSE was described in M. gallisepticum infection of tracheal
expiants (Cherry and Taylor-Robison,1970a;1970b; Gabridge et al., 1974).
Finally, the drainage of nutrients from the cell caused by the mycoplasma
parasitism was proposed and supported by the protective effect of adenine
sulfate in the prevention of cytopathic effects and CSE caused by M.
pneumoniae (Gabridge and Garden Stahl, 1978). It is possible that the
combination of all these and not the isolated effect of an individual factor are
28
likely to contribute to the induction of pathogenic effects detected on the ciliated
epithelium infected with mycoplasmas.
Cellular Factors
The respiratory tract, as well as skin and the gastrointestinal tract, can be
considered as an interface between the sterile internal sanctuary of the body
and the environment. It would be advantageous to have, in addition to a non­
specific clearing mechanism, an antigen-responsive cellular component in the
lung that serves as a first-line defense as well as for local antigen sampling.
Such cellular factors could allow the adjustment and fine tuning of the specificity
of the immune response to antigens present in the environment. This function is
fulfilled by the lung lymphoid tissue and the alveolar macrophage.
Normally, the lung parenchima contains lymphocytes at defined sites in
the lung. Among these sites are the bronchus-associated lymphoid tissue
(BALT), lymphoreticular aggregates, and the hilar and paratracheal lymph
nodes. There were also a small number of lymphocytes residing within the lung
interstitium (German et al., 1990).
The relative number of lymphocytes increases upon the exposure of the
lung to chemoattractants released by the presence of antigens, and the
entrance of these cells to the infected parenchima is not a random process but
rather involves a series of defined steps. These included adherence of blood
lymphocytes to the vascular endothelium and subsequent directed
extravasation and ameboid motion through the tissue in response to locally
produced chemotactic factors (Freitas and Bognacki, 1979; German et al.,
1990). The exit of lymphocytes from blood to organized lymphoid tissue occurs
F
29
at the post-capillary venules, known as high endothelial venules (HEV)
(Stamper and Woodmff, 1976). The venular endothelial cells were described
as having a plump shape protruding into the vessel lumen, in contrast to normal
flattened endothelium (Stamper and Woodruff, 1976). Under non-inflammatory
conditions, high endothelial venules were usually observed in lymphoid tissue,
but these structures were widely found in tissue undergoing inflammatory
processes with lymphocytic infiltration (Freemont, 1983). Gamma interferon
(ylFN) appears to possess a dual inducer role of HEV and surface adhesive
molecules on endothelial cells (Diuvestijn et al., 1986; Diuvestijn and Hamann,
1989). Adherence of lymphocytes to HEV was described to be mediated by the
interaction of specialized vascular addressins on HEV which included
intercellular adhesion molecule-1 (ICAM-1) and class II major histocompatibilty
complex (MHC) antigens such as HLA-DR (Diuvestijn and Hamann, 1989;
Hamann et al., 1988; Haskard et al., 1986). Neither ICAM-1 nor class II MHC
antigens were present in normal vascular endothelium, but appeared to be
induced by treatment with inflammatory cytokines, chiefly ylFN (Haskard et al.,
1986; Masuyama et al., 1986; Pober, 1986). Endothelial cells stimulated with
ylFN have been described as "activated", and under this state they were
capable of expressing adherence molecules and synthetizing cytokines
(German et al., 1990). Thus, local secretion of cytokines induced during
inflammation can alter the traffic through the endothelium by regulating the
presence of lymphocyte adherence molecules (Curtis et al., 1988; German et
al., 1990). After adherence, translocation of lymphocytes occurred through
ameboid movements. This involved changes in the cytoskeleton, particularly in
actin filaments and microtubules which, in turn, were originated from the
interaction of a chemoattractant with its receptor in the membrane surface of the
target cell (German et al., 1990). As a result of this interaction, biochemical
signals were generated that resulted in not only changes in the cytoskeleton but
also induced lymphocytes to enter into an "activated" state that included the
progression of the cell from Go to Gi, increased synthesis and secretion of
chemoattractants, and increased expression of surface receptors, including
lymphocyte function-associated antigen-1 (LFA-1) and CD-4 (Allen et al., 1988;
German et al., 1990).
Organized mucosal lymphoid follicles in close apposition to the bronchial
epithelium have been noted for over a hundred years. These lymphoid
aggregates were named BALT by Bienenstock et al. (1973), due to the
similitude with the gut-associated lymphoid tissue (GALT). Studies on the
distribution of BALT in different species, indicated that rats, rabbits, and guinea
pigs showed the most extensive distribution of these accumulations, whereas its
presence has been disputed in humans (German et al., 1990). The BALT was
described to be absent at birth, developing later at sites of particle deposition in
response to postnatal antigen exposure (Plesch, 1983). The main characteristic
of BALT was the presence of overlying lymphoepithelium in which epithelial
cells were attenuated and flattened and ciliated cells were absent. This
lymphoepithelium allowed the passage of soluble and particulate material from
the bronchial lumen to the underlying lymphoid tissue (Berman et al., 1990).
Under this modified epithelium, a meshwork of lymphocytes and cells that
express class II MHC antigens were present. The class II antigen-expressing
cells were macrophage-like antigen-processing and-presenting cells needed to
activated T cells (Simecka et al., 1986). This lymphoepithelial network had
been shown to contain specialized capillary endothelial cells that facilitate the
transit of lymphocytes from the blood to BALT (Bermann et al., 1990). Both B
and T lymphocytes were found In BALT. In contrast with lymphonodules, no T
cell-dependent areas were found in BALT. However, there were areas that
predominantly contained B or T cells. (Breel et al., 1988). Studies performed in
young rats and in rabbits, indicated the propensity of B cells to express IgA, but
as the animal aged, the numbers of these cells decreased In comparison with
IgM- and IgG-bearing cells. Nonetheless, BALT together with GALT was though
to be a major source of IgA producing B cells (Rudzik et al., 1975). Most of the
cells types that were found in BALT, including lymphoepithelial cells, expressed
class II MHC antigens (Simecka, 1986). The presence of this structure was in
concordance with the proposed function of BALT as a local response site for
antigen introduced with the inhaled air (Berman et al., 1990). The proposed
theory was that inhaled antigens can percolate through the lympoephitelium
and be taken up and be processed by class II MHC-expressing antigen
processing cells. These cells then would be capable of presenting processed
antigens to helper T-cells which could provide the necessary stimuli for humoral
and cytotoxic responses to such foreign molecules (Bermann et al., 1990).
There was evidence of traffic of antigen-activated lymphocytes through the
lymph to blood and hence, to other organized lymphoid tissue. Thus, cell
clones expanded through contact with inhaled antigen might recirculate
preferentially to mucosal sites, including GALT, BALT, salivary and cervical
mucosa (Bienenstock et al., 1983).
Eighty percent of the cells present in bronchoalveolar lavage (BAL) were
macrophages and 5 to 10 % were lymphocytes. From the latter, approximately
60% were T cells and 10% B-cells. This leaves as much as 40% of the
lymphocytes in BAL unaccounted for as "null cells" (Hunninghake et al., 1979;
Young et al., 1984). Lymphocytes showing the phenotype of natural killer cells
(NK) were found in normal lungs and this organ has been shown to be a major
source of NK activity. In this regard, it was shown that NK-cells derived from
bone marrow precursors and they matured in major NK reservoirs such as
spleen and lungs. This suggested that precursors were recruited to and
matured in the lung much like tissue macrophages (Bender et al., 1987).
Lymphocytes obtained from BAL were shown to possess functional
activity of every type, including immunoglobulin production, secretion of
lymphokines, cytotoxicity, expression and activation of adhesion molecules, and
NK function (Berman et al., 1990). However, studies of lymphocytes from
normaJ lungs provided the view that these cells were hyporeactive in NK activity
and proliferative and antibody production responses to mitogens when
compared with blood-derived lymphocytes (Robinson et al., 1984; Kaltreider
and Salmon, 1973). In this regard, it was found that surfactant lipids have a
profound suppressive effect on lymphocyte proliferation and effector cell
function (Shimizu et al., 1988; Wilsher et al., 1988). In regards to T cells, it was
found that 8 to 20% of the CD3+ cells in the lung, expressed yS T cell receptor.
This type of T cell receptor was also described to be present in high numbers in
intraepithelial lymphocytes of the intestine and dendritic cells of the skin, and
cells showing this surface antigen were also highly represented in responses
following the administration of protein purified derivative (Augustin et al., 1989).
These results suggested the presence of a lung-specific population of T cells
(Berman, 1990).
It was established that alveolar macrophages were unique among other
macrophages. They differ in their restricted residence in the lung and by their
metabolism particularly adapted to the aerobic conditions of the lung alveoli
(Khandom et al., 1985; Henson et al., 1984). Alveolar macrophages were
described as the principal free resident cells of the distal airspaces and
responsible for maintaining a microorganism-free environment in the lower
respiratory tract (Glasgow et al., 1989). In addition, this cell was thought to play
a role in the mediation of acute lung injury (Brieland et al., 1987), as well as in
resolving pulmonary inflammation (Dal Nogare and Towens, 1990).
The initial inflammatory events originated as a result of the proliferation of
pathogenic microorganisms in the lower regions of the respiratory tract were
characterized by increased vascular permeability and accumulation of
inflammatory cells, which at an early step (6 hours) were leukocyte
polymorphonuclear cells (PMN) (Henson, 1984; German et al., 1990). By 48
hours and beginning as early as 6 hours after the initiation of the inflammatory
changes, the cellular infiltrate has shifted to lymphocytes and mononuclear
monocytes (phagocytes). By day 7 to 10, in uncomplicated cases, the lesion
was resolved and a solid immune response was established (Henson, 1984).
This final state was achieved by the interplay of macrophages, lymphocytes and
cytokines produced and secreted by these cells types (Berman et al., 1990).
After the initial increase of vascular permeability and migration of PMNs,
probably elicited by the complement fractions Cab and Cga, the activity of
macrophages was characterized by engulfment and processing of antigens.
Concomitantly, macrophages secreted interleukin 1 (IL-1) which activated T
cells that migrated to the foci of inflammation (Roitt, 1988). Activated T cells
produced interlukin 2 (IL-2) which activated additional T-cells, amplifying the T
cell response. Activated T- cells also secreted interleukin 4 (B-cell stimulatory
factor) interleukin 6 (8 cell growth factor), and B cell differentiation factors which,
synergistically, caused the activation and maturation of B cells responsible for
the humoral phase of the immune reaction (Roitt, 1988). In addition, activated T
helper cells secreted macrophage chemotactic factors, macrophage migration
inhibition factors, and macrophage activating factors which mediated the
activation and recruitment of monocytes from the blood circulation to the foci of
inflammation (Roitt, 1988; Fuchs et al., 1989). Even when most of the cellular
amplification and recruitment was mediated via T helper cells and their
interleukins, the triggering factor (IL-1) is launched by alveolar macrophages. It
was described that monocyte cells recruited from the blood undergo an
activation process during which these cells developed increased functional
capabilities compared to those of the alveolar macrophages. These activated
monocytes, through an increased capacity of synthesis and secretion of IL-1,
originated a positive feed-back mechanism, which amplified the inflammatory
response even more (Fuchs et al., 1989). These activated macrophages were
"armed" with receptors that mediated the removal of aged PMNs and cellular
debris from the area of inflammation (Garret et al., 1984, Henson et al., 1984,
Dal Nogare and Towens, 1990). These activated cells presented increased
capabilities for the production and secretions of factors needed for the healing
and the repair of the lung lesions. In this sense, recruited monocytes differ from
resident macrophages (Dal Nogare and Towens, 1990). Recruited
macrophages (monocytes) were capable to take up aged PMNs or their
enzymes after a maturation step. These recruited macrophages also showed
35
increased fibrinolytic activity and less procoagulant activity than resident
macrophages. These changes would maximize fibrin removal (Dal Nogare and
Towens, 1990). The fact that fibrin could be easily removed had direct
implications in resolving pulmonary inflammation since it permitted more
efficient macrophage motility, thus enhancing their cleansing function. Also
decreased formation of fibrin deposits, which may act as foci for collagen and
fibroblast attachment, reduces the risk of generating lung fibrosis (Dal Nogare
and Towens, 1990).
Macrophage activation is not always a beneficial response. It was clear
that activation may also result in injury to the lung parenchima and induce
interstitial fibrosis (Brieland et al., 1987). Secretory products of macrophages,
including degradative enzymes such as acid hydrolases and reactive oxygen
metabolites were reported to both directly and indirectly initiate acute lung injury
(Brieland et al., 1987). In addition, other potential mechanisms of macrophagemediated lung injury include: production of chemotactic and vasoactive
complement fractions (Cab), generation of arachidonic acid metabolites, release
of plasminogen activator, secretion of cytokines, and release of lysosomal
degradative enzymes (i.e., elastase, mucopolysaccharidases, collagenase)
(Fantone and Ward, 1984). It was proven that most of the damage detected in
lungs during the initial phase of the inflammatory process was due to increased
production of Oz by these cells, and this was caused by a direct increase in the
membrane-bound oxidase activity in these cells (Brieland et al., 1987). In
addition, an increase in the secretion of acid hydrolases (A/-acetyl -p-D glucosaminidase) was observed (Brieland et al., 1987). It was also proven that
mononuclear macrophage cells that arrived from the blood exhibited increased
generation of oxygen free radicals as well as acid hydrolases, when compared
with the activated resident pulmonary macrophages (Brieland et al., 1987).
Abnormal macrophage activation was described as the central alteration
in the pathogenesis of several diseases, such as sarcoidosis and
granulomatous inflammation of the lungs (Pinkston et al., 1983; Robinson and
McLemore,1985; Bitterman et al., 1986). Sarcoidosis was described as a
disorder characterized by excessive macrophage-mediated helper T cell
activation, excessive ylFN synthesis, and secretion of profibrotic and destructive
substances by activated macrophages (Pinkston et al., 1983; Robinson and
McLemore, 1985; Bitterman et al., 1986). In granulomatous inflammation of the
lungs, the presence of highly irritant, indigestible or at least poorly degradable,
persistent unanimated or living substances, elicited the accumulation and
activation of macrophages (Adams, 1983). Activation of these cells , as it was
mentioned above, determined the generation of substances destructive to the
lung parenchima, secretion of cytokines, accumulation of T cells, and synthesis
of substances (e.g., fibronectin and alveolar macrophage-derived growth factor)
that promoted the migration and proliferation of fibroblasts (Adams and
Hamilton, 1988).
Interaction of pathogenic microorganism with alveolar macrophages
Bacteria
The effect of bacterial species on the alveolar macrophages
are mediated, preferentially through the secretion of toxic products. There are
reports that link bacterial endotoxin and dysfunction of alveolar macrophages.
For instance, in one experiment, Sprague-Dawley rats were injected
intravenously with either placebo or 5 mg/kg of E. coli lipopolysaccharide B.
37
Two hours after treatment, the rats were challenged with S. aureus and
bacteriologic examination was performed immediately and at 4 hours after
bacterial challenge (Harris et al., 1988). Rats pre-treated with endotoxin
showed a significant decrease in pulmonary bactericidal activity when
compared to aerosol-challenged control rats, and this was interpreted as a
defect in alveolar macrophage function mediated by the application of
endotoxin. Increased production of free radical oxygen species In endotoxintreated alveolar macrophages was also shown, when compared with control
untreated cells. (Harris et al., 1988). In another experiment, dogs were given 2
or 20 mg/kg of Escherichia coii endotoxin and several cellular functions of
alveolar macrophages were compared with those from control dogs (Jacobs et.
al.,1986). Adherence of alveolar macrophages from the endotoxin-treated dogs
was significantly reduced. In addition, alveolar macrophages from endotoxintreated dogs showed significantly greater production of hydrogen peroxide,
greater peaks in chemiluminescence, reduced phagocytosis and a diminished
ability to kill cell-associated S. aureus and E. coii overtime (Jacobs et al.,
1986). In another experiment it was proven that generation of oxygen radicals,
especially hydrogen peroxide (H2O2), inhibited phagocytosis and superoxide
anion production by alveolar macrophages in a irreversible and concentrationand exposure time- dependent fashion. These authors suggested that ATP
depletion may play an important role in the described H2O2 toxicity for alveolar
macrophages (Jacobs et al., 1986). This suggestion was proven later (Oosting
et al.,1990). These authors showed that comparable concentrations of H2O2
caused an irreversible depletion of cellular ATP, reduction of phagocytosis, and
production of superoxide by alveolar macrophages . In addition, the time
38
course of ATP depletion and induction of impaired alveolar macrophage
function were similar to that described by Jacobs et al. (1986). These authors
also indicated that in view of the fact that H2O2 may react with a large variety of
biological substances, other possible toxic lesions may not be excluded as
underlying mechanism for HaOa-induced inhibition of the phagocytic and killing
functions of these cells (Oosting et al.,1990).
Several respiratory bacterial pathogens caused impairment of alveolar
macrophages by resisting killing. These microorganism are able to multiply
inside of the macrophages causing the death of the cell. Legionella
pneumophila, the causative agent of Legionnaires' disease, is a Gram-negative
bacterium and a facultative intracellular parasite that multiplies in human
monocytes and alveolar macrophages. It was shown that under in vitro
conditrans, this bacterium was capable of multiplying to 2.5-4.5 logs/ml, and
result in the destruction of the monocyte monolayers (Horwitz, 1987). It was
reported that L pneumophila inhibited the phagosome-lysosome fusion, thus
evading the killing mechanisms of macrophages (Horwitz, 1987). Respiratory
bacterial pathogens of cattle such as Pasteurella hemoiytica and Haemophilus
somnus exerted their pathogenic effect on alveolar macrophages through the
production of species specific cytotoxins (Corbeil and Gogolewski, 1985). The
cytotoxin of P. hemoiytica impaired phagocytosis, killed macrophages, was
specific for ruminant leukocytes and was more cytotoxic if opsonization took
place. In addition,P. hemoiytica also avoided phagocytosis by means of their
capsule. In effect, a high molecular weight capsular fraction of this bacterial
species has been shown to adversely affect bovine neutrophil phagocytic
function (Corbeil and Gogolewski, 1985).
39
Viruses
The effect that virus infection has on alveolar macrophage
activity was well documented by Bryson et al. (1983). These authors described
parainfluenza-3 (PI-3) virus replication in bovine alveolar macrophages under
in vivo conditions and together with the information reported by Breeze and
Liggitt (1989), a detailed information on the cellular events induced by the viral
replication in BAM is now available . For instance, it was shown that upon
infection with PI-3, changes occurred in the arachidonate acid (AA) metabolism
(Breeze and Liggitt, 1989). These changes included the elevated production of
some of the AA derivatives, most notably PGE2 . It was postulated that these
changes were due either to increased phospholypase A activity or to interaction
of the virus with the membrane of the cell (Breeze and Liggitt, 1989). It was also
shown^ that some of these metabolites, especially PGE2, were inhibitory to
phagocyte functions (Laegreid et al., 1989). By using primary cultures of BAM
infected in vitro with PI-3 virus, these authors showed that PI-3 virus-infected
BAM killed significantly fewer Staphylococus epidermidis than uninfected
controls (Laegreid et al., 1989). This inhibitory effect was reverted by the
addition of cycloxygenase pathway inhibitors (i. e., indomethacine, pyroxicam )
1 hour prior to the assay. In addition, they showed that phagosome-lysosome
fusion was severely impaired in virus-infected BAM and, pre-treatment of virusinfected BAM with indomethacine significantly enhanced the percentage of cells
expressing phagosome-lysosome fusion activity (Laegreid et al., 1989).
Other researchers have shown that the blockage of b-1 surface receptors
caused the inhibition of reactive oxygen species-dependent chemiluminescent
reactions in BAM. Infection of BAM by PI3 virus or BHV-1 was shown to cause
impairment of b1-receptor function (Ogunbiyi et ai., 1988). Based on these
findings, it was postulated that infection by these virus species render the BAM
unable to generate oxygen reactive products, inhibiting the oxygen-dependent
killing mechanism of these macrophages (Ogunbiyi et al., 1988). Treatment of
BAM with levamisole, reverted the blockage of the b-1 receptors, rendering the
macrophages as functionally active as the non-infected control cells (Ogunbiyi
et al., 1988). Similarly, it was shown that infection of porcine alveolar
macrophages with pseudorabies vims (PVR) caused depression in some of the
macrophage functions (Iglesias et al., 1989). It was shown that phagocytosis of
yeast was significantly reduced in the alveolar macrophage cultures. It was
also shown that phagosome-lysosome fusion and O2 release after stimulation
with opsonized zymosan was significantly reduced in all the PRV-infected
cultures (Iglesias et al., 1989). These authors concluded that the inhibitory
effect of PRV infection on alveolar macrophage bactericidal functions may
explain the increased susceptibility of PRV-infected pigs to bacterial
pneumonias (Iglesias et al., 1989).
In another work it was shown that infection of BAM with PI-3 or BHV1
caused a significant reduction in the percentage of Fc and Cab receptors . The
alveolar macrophages obtained after virus inoculation were significantly
impaired in their ability to phagocytize opsonized S.epidermidis, but were able
to kill ingested bacteria (Brown and Ananaba, 1988). In other work, the same
group of researchers obtained similar results under in vitro and they reported a
significant reduction in the Csb mediated phagocytic index in alveolar
macrophages inoculated with BHV-1 or PI-3 (Brown and Shin, 1990).
41
The overall effect of BHV-1 infection on BAM and their function on the
immune response in calves was documented by Tsevtkov (1990). Results from
his research indicated that the number, viability and differential distribution of
the BAM and their phagocytic and microbicidal ability showed a tendency to
decrease during the acute phase of infection (2nd-5th days after infection). This
was followed by a slow restoration during the period of convalescence (by the
15th day after infection). Some parameters of systemic immunity.(e.g., number
of plaque-forming cells, rosette-forming cells, differential and absolute number
of leucocytes in the peripheral blood) also showed similar dynamics
(Tsevtkov,1990). It was stated that changes in the local and systemic, and in
the cell and humoral immunity were connected to the immune suppressive
properties of BHV-1 (Tsevtkov, 1990).
Perhaps the best example to understand the central role of the alveolar
macrophage in lung defense and the effect of some virus infections is given by
a disease where the alveolar macrophage's function is missing. Good
examples are the respiratory complications in patients with acquired
immunodeficiency syndrome (AIDS). In this disease, the frequency of
pulmonary infections suggested that pulmonary host defenses were
compromised (Beck and Shellito, 1989). The defense capabilities of the upper
and lower airways, which prevent the majority of infectious organisms from
reaching the lungs in healthy individuals, were diminished in AIDS patients. In
the lower respiratory tract, direct infection of alveolar macrophages with HIV
altered clearance of microorganisms (Beck and Shellito, 1989). In addition to a
direct effect on the ingestion and killing events, it was shown that soluble
signals secreted by activated alveolar macrophages required to activate other
macrophages and accessory cells were deficient (Cox et al.,1990). Without an
adequate amount of these signals, lung defenses cannot be mounted properly.
In addition, peripheral T and B lymphocytes from AIDS patients do not respond
to antigen normally, and the resulting defects in cellular and humoral immunity
may impair defenses against a variety of pulmonary pathogens (Beck and
Shellito, 1989). In addition, the defective microbicidal function of PMN's from
AIDS patients, and the reduced migration of blood leukocytes into the lungs
result in an insufficient cellular response to an infectious challenge (Beck and
Shellito,1989).
Mycoplasmas
One of the most intriguing aspect of the interaction
between mycoplasmas and macrophages is their ability to colonize the surface
membrane of the macrophages and actively proliferate in this location without
being ingested. This phenomenon was initially described by Jones and Hirsch
(1971) and Jones et al. (1972) with M. pulmonis, and it was noticed that the
mycoplasmas bound to the phagocyte surface proliferated and were not
phagocytized unless anti-mycoplasma opsonizing antibodies were added.
These authors proposed the presence of an antiphagocytic mycoplasmal
surface protein (Jones et al.,1972). Similar observations were reported with M.
arthhtidis, but with this mycoplasma species, only partial opsonization was
achieved upon the addition of anti-mycoplasma antibodies (Cole and
Ward,1973; Davis et al.,1980). It also was reported that incubation of
macrophages with M. hominis and M. arthritidis resulted in diminished
opsonization of Escherichia coli, a microorganism that was readily ingested by
these cells. This was taken as indication of direct impairment of the phagocytic
functions of these cells by the mycoplasma species used in this experiment
(Cole et al., 1985).
Suppression of lymphoid cell function has been reported in several
mycoplasma species. The first report on this effect was described with M.
pulmonis. It was shown that the addition of this mycoplasma to human
peripheral blood treated with phytohemagglutinin (PHA) resulted in inhibition of
the mitogenic effect detected otherwise (Copperman and Morton, 1966). This
effect was reversed upon removal of the mycoplasma and supplementation with
fresh medium and PHA. It was demonstrated that this effect was due to
depletion of arginine by mycoplasmas from the growth medium, rather than a
direct effect of the microorganism of lymphocyte functions (Barile and Levinthal,
1968). The same authors documented the inhibition of PHA stimulation on
lymphocytes by non-fermentative mycoplasma species (e.g., M orale and M.
arthritidis), and these results were confirmed by Morton et al. (1968) and
Simberkoff et al. (1969), who reported that the inhibitory effect was promoted by
extracts obtained from non-fermentative species of mycoplasma. It was also
shown that the inhibitory moiety was heat-labile and papain sensitive,
suggesting a proteinaceous composition (Simberkoff et al., 1969). Mycoplasma
species involved in enzootic pneumonia in calves, (i.e., M. dispar and M. bovis)
have been reported to be immunosuppressive. Such property has been
supported by several experimental and in vivo observations (Bennet and
Jasper, 1977; Howard et al., 1987). Among these, it was reported that
subcutaneous inoculation of killed M. dispar Induced a poor immune response
in calves compared to that obtained with M. bovis (Howard and Gourlay, 1983).
In addition, the Inoculation of killed preparations of M. bovis and M. dispar
resulted in a reduced antibody response to M. bovis, lower than that observed
when M. bovis was inoculated alone (Howard et al., 1987). In another study,
mycoplasma species pathogenic for the respiratory tract of cattle {M. dispar, M.
bovis ), and a non-pathogenic respiratory mycoplasma species {M. bovirhinis ),
were studied for their effect on the response of lymphocytes to mitogens (Finch
and Howard, 1990). It was found that live M. dispar and M. bovis had inhibitory
effects on the stimulation of these cells by PHA. When killed M. dispar was
used, little effect was noticed, which was in contrast with the elevated inhibitory
effect shown with killed M. bovis. Neither live nor killed M. bovirhinis had a
demonstrable effects on lymphocyte responses to PHA. In experiments with live
M. dispar and M. bovis it was noticed that there was no alteration of the
lymphocyte viability, and there was no evidence of mycoplasma replication.
Live M. dispar also inhibited the mixed leucocyte response (MLR) (Finch and
Howard, 1990). Similarly, live M. bovis also showed the same effect on MLR,
but in contrast with the previous experiment, heat- killed and formalin-killed M.
dispar and formalin-killed M. bovis were also inhibitory. As before, there were
no alterations in leucocyte viability and no indication of mycoplasma replication
in these experiments containing viable mycoplasma (Finch and Howard,1990).
In another set of experiments, the effect of filtered supernatants from bovine
peripheral mononuclear cells inoculated with M. dispar and M. bovis was
tested. It was observed that supernatants from cultures containing viable
mycoplasma inhibited the MLR. The same effect was observed with formalin
killed but, in contrast, no effect on MLR was observed whit supernatant from
mononuclear cells with heat killed M. bovis. Supernatants from mononuclear
cells treated with heat or formalin killed M. dispar d\d not show any suppressive
effect on MLR (Finch and Howard, 1990). The results obtained from these
experiments indicated that viable M. dispar or M. bows was required for the
inhibition of MLR and the molecule(s) responsible for such effect were heat
sensitive in both mycoplasma species (Finch and Howard, 1990). The
correlation between mitogenicity, induction of peribronchial cuffing, and
lymphocyte accumulation was previously shown with M. pulmonis (Cole et al.,
1985). The results reported by Finch and Howard (1990) did not agree with this
observation, since peribronchial cuffing and lymphocyte accumulations were
described in M. bovis pneumonias, yet this mycoplasma had inhibitory effects
on lymphocyte proliferation. These authors proposed that these pathologic
changes could by induced by induction of TNF and interleukins, which in turn,
caused necrosis and accumulation of leucocytes around the airways (Finch and
Howard,1990). While the induction of TNF by macrophages and reproduction
of the pathological changes seen in the natural disease was proven with TNF or
a carbohydrate moiety produced by Mycoplasma mycoides subsp. mycoides
(LC) (Rosendal, 1990), it is not known if macrophages infected with M. bovis
can be induced to produceTNF. The induction of a inhibitory state by the
supernatants of cultures with viable mycoplasma (M dispar or M. bovis) without
changes in the viability of leucocytes was taken as evidence of the presence of
a heat-labile factor(s) produced by the mycoplasma that modified the cellular
function of the cells (Finch and Howard,1990). Similar observations were made
with M hyopneumonia, where azide-treated mycoplasma were found to
suppress the response of porcine lymphocytes to mitogens, an effect that was
also heat-labile (Kishima and Ross, 1985).
46
Various mycoplasma species have been shown to possess T cell and B
cell mitogenic activity (Cole et al., 1981; Cole et al., 1985; Naot et al., 1987). It
also was reported that mycoplasma induced the cytotoxic activity of NK cells,
cytotoxic T-cells and macrophages (Wayner et al., 1984; Lowenstein and
Gallily, 1984). Recent reports indicated that several mycoplasmas may induce
production of tumor necrosis factor by macrophages. Such stimulation was
mediated in a Ca2(+)-dependent but not protein kinase C-dependent pathway
and the induction of this cytokine occurs at the transcription level (Gallily et al.,
1989; Sugama et al., 1990). It was also reported that some mycoplasma
species can induce expression of MHC class I and II antigens, alter the
production of IIL-1, induce production of IL-2, and induce the production of
granulocyte-macrophage colony-stimulating factor from human lymphocytes
(GM-CSF). It was shown that purified membranes of M. arginini and M.
arthritidis but not M. pulmonis induced the proliferation of macrophages
throughout the production of GM-CSF by lymphocytes (Cole et al., 1990). In this
study, it was shown that the factor that mediates this effect was a mycoplasmal
membrane protein of approximately 60 kDa in size. The significance of these
host responses to mycoplasma products was documented by the involvement of
mycoplasmas in several animal diseases, including atypical pneumonia and
arthritis. It was postulated that the mechanisms for the pathogenic effects of
mycoplasmas included the activation of host immune cells. The fact that M.
arthritidis produces a superantigen that induces autoimmune reactions through
the activation of T cell clones, suggests the direct participation of mycoplasma in
the pathogenesis of these autoimmune conditions (Cole et al., 1990).
47
It is not clear at this point whether the activation of the host immune cells
by mycoplasma products leads to development of autoimmunity or if they simply
represent an exacerbated inflammatory response.
I
48
PART i. Impaired Function of the Mucociliary Apparatus of Calves Infected with
Mycoplasma dispar
ï
49
Impaired Function of the Mucociliary Apparatus of Calves Infected with
Mycoplasma dispar
Raul A. Almeida, D.V. M., M. S.
Ricardo F. Rosenbusch, D. V. M. , Ph. D.
From the Veterinary Medical Research Institute, and the
Department of Microbiology, Immunology and Preventive
Medicine, Iowa State University, Ames, Iowa, 50011.
Supported in part by Iowa Livestock Health Advisory
Council, grant No.109-05-17
50
SUMMARY
Infection of tracheal organ cultures with Mycoplasma dispar results in
degeneration of respiratory epithelial cells with loss of ciliary activity. To assess
the effect that these changes have on the clearance of bacteria from the
respiratory tract, the tracheobronchial clearance of a suspension of Serratia
marcescens was determined in calves before and after infection with M. dispar.
Tracheobronchial swab samples were obtained at various times after
deposition of the marker bacteria. The clearance was significant by 3 hours
after inoculation and only few colonies were detected 4 hours after challenge
with the marker bacteria. In contrast, in the same animals, 5 days after
intratracheal exposure with M. dispar, clearance of the marker was impaired
beyond 4 hours after inoculation. Histopathological and scanning electron
microscopy (SEM) studies of infected lungs revealed that as a result of infection
with M. dispar, areas of degeneration and destruction of the respiratory ciliated
epithelium occur in intermediate and small airways. These lesions may be
responsible for the altered clearance observed in these mycoplasma-exposed
calves.
51
INTRODUCTION
The sterility of the internal portions of the respiratory tract is achieved and
maintained by the synergistic interaction of numerous defense mechanisms.
The mucociliary apparatus is one among these, and in this regard, its function is
to prevent the contact of harmful substances and infectious agents with the
epithelial surfaces of the airways by removing these harmful agents from the
respiratory tract. These effects are achieved by the interaction of the trapping
capacity of the mucous layer and the propelling effect of the ciliary blanket.
The initial steps in the pathogenesis of pneumonic processes are the
multiplication of pathogenic microorganisms in the upper regions of the
respiratory system, which is followed by the overcoming of the mucociliary
apparatus and dissemination into deeper regions of the lungs. Pathogenic
bacteria have developed means to circumvent this first defense barrier by
reducing the viscoelastic properties of the mucous layer and by impairing ciliary
activity (6, 7). Some bacterial species such Pasteurella multocida and
Actinomyces pyogenes break down the mucus layer through the production of
neuraminidase or extracellular proteases, thus rendering the mucus less
viscous and ineffective for the trapping and removal these or other infectious
agents (4, 5). Other respiratory pathogens, such as mycoplasmas, may cause
ciliostatic and ciliotoxic alterations which impair the mucociliary clearance (6, 7).
Using a bovine tracheal organ culture system it was shown that Mycoplasma
dispar, a bovine respiratory pathogen, caused alterations of the ciliary
apparatus which include degeneration of the respiratory epithelial cells and
loss of the ciliary activity (9, 10). Since this ciliostatic action of M. of/spar may be
52
an important predisposing factor in the developing of lung infection in cattle, in
the present report we extend these observations to in vivo conditions by
investigating the tracheobronchial clearance of a marker bacteria in calves
experimentally inoculated with M. dispar.
i'
53
METHODS
Animals
Ten calves of 2 to 2.5 months of age were procured from a herd negative to
mycoplasmas by serology and by culture of nasal swabs. Calves were housed
in individual isolation rooms and daily inspected during 7 days. Inspection
consisted in verification of the general condition of the calves, presence of signs
of respiratory infections (increased respiratory rates, nasal discharge, cough),
presence of gastrointestinal alterations (feces consistency), and apetite.
Microorganisms.
Mycoplasma dispar SD-0 (11) at passage level 7 was used in this experiment.
Mycoplasmas were grown in modified Friis broth medium (12), washed three
times with PBS, resuspended at 100 X concentration in 1 ml aliquots of Friis
broth medium, frozen and kept at -70 C until use. To challenge calves, aliquots
of the mycoplasma suspension were resuspended in sterile PBS to a titer of
10^° color changing units/ml (ccu/ml). Serratia marcescens strain NIMA used in
this experiment was grown as was described by Veit et al., 1978 (13). Briefly,
sterile tryptose broth was inoculated withS. marcescens and incubated at room
temperature for 96 h. Aliquots of the broth were used to inoculate tryptose agar
plates which were incubated for 36 h at room temperature. One single red
colony was picked from a selected agar plate and used to inoculate 100 ml of
tryptose broth. After 24 h of incubation at 37 C, cultures were centrifuged at
5000 X g for 20 min, resuspended in 20 ml of lactose-glutamate diluent (14),
and 1 ml aliquots of the broth were quick-frozen and kept at •70°C until use.
54 a
To challenge calves, one aliquot of the suspension was thawed at 37°C and
resuspended in PBS (pH 7.2) to an OD of 1.045 at 600 nm (Spectronic, Milton
Roy, Rochester, NY).
Experimental Design.
Calves were assigned at random to each of 3 groups. Group A consisted of
calves that were inoculated transtracheally (between the upper and mid-third of
the tracheal length) with 10 ml of a suspension of S. marcescens. These
calves were put under sedation with 0.5 mg/lb Xylazine given I.M. (Mobay Corp.
Shawnee, KA). Mucosal swab and tracheal samples were obtained with 27
inch-long occluded swabs (Accu-Med Corporation, Pleasantville, NY) with the
aid of a laryngoscope. Briefly, while holding the head and the neck of the calf in
a horizontal position, the swab was carefully introduced through the larynx into
the trachea. Once the occluded end of the swab contacted the tracheal
bifurcation, it was retracted 2-3 cm, the tip exposed and kept in that position for
15 to 20 seconds. Rotational movement of the swab was avoided to reduce
chances of producing physical trauma to the tracheal mucosa. Four samples
were obtained at one hour intervals, and after the last sampling time, calves
were inoculated transtracheally with 10 ml of a M d/spar suspension in PBS
(lO'io ccu/ml). After 5 days, calves were challenged again with S. marcescens
following the routine described previously. Tracheal swab samples were
placed in dry tubes and kept on ice until processing, which occurred 5 to 10 min
after obtain the sample. Calves were euthanized 24 hours after the last
sampling. Group B consisted of 2 calves that were subjected to a routine similar
to that described above, but in this case no experimental infection with M dispar
54 b
was performed. Calves were euthanized 24 hours after the last sampling.
Group C consisted of 2 calves that were inoculated transtracheally with 10 ml of
a M. d/spar suspension in PBS (1010 ccu/ml). These calves were euthanized 5
days after infection.
Determination of the tracheobronchial clearance of S. marcescens.
To enumerate the number of S. marcescens, swabs taken from calves of group
A and B were placed in microtubes with 1 ml of diluent solution (0.5 M sucrose,
50 mM phosphate buffer pH 7.0, and 50 mM MgS04 ), briefly centrifuged (1 min
at 14,000 X g) and 10 -, 100 -, 1000 - fold dilutions were performed in the same
diluent. An additional dilution ( 10000-fold) was added to samples obtained
from M. d/spar infected calves. One hundred microliters of undiluted samples
and of .each dilution were plated by triplicate on tryptose agar plates using
standard spread plate methodology and incubated at room temperature for 48
hours. Pigmented colonies were counted and the logarithm of the number of
colonies/swab was calculated. Comparisons were made between the data
obtained before and after infection with M. dispar.
Necropsy Procedures.
All the calves were euthanized by IV administration of an overdose of sodium
pentobarbital (Fort Dodge Laboratories Inc., Fort Dodge, la). Lungs were
aseptically removed from the thoracic cavity and after recording the percentage
of lung consolidation, samples from several predetermined areas were
processed for histopathology, SEM, and mycoplasma and bacterial isolation.
Samples were placed in 10% buffered-formalin or vigorously washed with
55
0.075 M Sorensen's phosphate buffer (pH 7.2) to remove mucus and placed in
ice-cold and freshly prepared 3%glutaraldehyde solution for histopathology and
SEM, respectively. Samples for mycoplasma and bacterial isolation were
placed in sterile containers and kept on ice until processing.
Histopathological Procedures.
Formalin fixed lung tissue specimens were dehydrated, embedded in paraffin,
sectioned at 5 ^m, mounted on glass slides and stained with hematoxilyn-eosin.
SEM procedures.
Tissues fixed overnight in 3% glutaraidheyde were sequentially treated with two
changes of osmium tetroxide and 5% tannic acid solution, using the method
described by Sweney and Shapiro (8). Tissues were dehydrated in ethanol,
critical point dried from absolute alcohol, and sputter-coated with goldpalladium.
Microbiology Procedures.
Lung tissues were suspended in PBS (pH 7.2) and triturated in a tissue grinder.
The homogenates were centrifuged at 100 x g for 10 min and ten fold dilutions
of the supernatants were inoculated in Hayflick broth (pH 6.8) and Modified Friis
broth tubes (12). In addition, 0.2 ml of undiluted lung homogenate was placed
in Friis and Hayflick agar plates and spread with the aid of a Drigalsky spatula.
Tubes and agar plates were placed at 37' C in a CO2 incubator and checked for
mycoplasma growth during 10 days. Suspect broth cultures were transferred to
the corresponding agar plates and colonies of mycoplasmas were identified by
56
the IFA technique. For bacterial isolation, 7% bovine blood agar plates and
tryptose agar plates were inoculated with 0.2 ml of lung homogenates and
incubated at 37 C for 48 hours. Bacterial isolates were identified using a
Minitek identification system (BBL, Cockeysville, IVID).
Statistical Analysis
Data are reported as mean ± standard deviation. Data were analyzed by paired
f tests. Significance was accepted for a comparison when the p value was less
than 0.05.
57
RESULTS
Clearance of Serratia marcescens
Recoveries of S. marcescens at different times before and after the infection
with M disparare shown in Figure 1A and 1 B. The tracheobronchial
elimination of S. marcescens was significantly slower after infection with M.
d/spar compared with that observed in animals not infected with mycoplasma.
Before infection with M. dispar, rapid elimination of the bacterial marker was
detected 3 hours after inoculation and at 4 hours, only few colony forming units
were detected. In contrast, in M. dispar infected calves, recovery of the marker
was high at 3 hours and unchanged 4 h after inoculation with S. marcescens.
The pattern of elimination observed after M of/spar infection, suggested that
clearance of the marker bacteria would extend well beyond 4 hours after
inoculation. Differences in recoveries of S. marcescens before and after
infection with M. of/spar were significant (p< 0.05). In addition to these changes,
increased bacterial counts were observed overall on M. dispar infected calves
when compared to those obtained in the non-mycoplasma infected status. The
pattern of tracheobronchial elimination of the bacterial marker in calves given
saline instead of mycoplasma (group B) was similar after the first and second
inoculation (Fig. 1 B), and was similar to that detected in principal calves before
infection with M. dispar.
Clinical Signs
On day 2 after inoculation with M. d/sparcalves were dull and depressed.
These signs remained for 24 hours affecting 6 calves (5 calves of group A and
Figure 1. Means of the number of CPU of S, marcescens recovered from the
principal and control calves at each sampling time.
A) Lower line (open squares) corresponds to determinations made before
infection with Mycoplasma dispar and upper line (close squares) correspond to
those made after infection with Mycoplasma dispar.
B) Colony forming units of S. marcescens detected on the control calves at each
sampling time. Open circles correspond to determinations made after the first
challenge and closed circles line correspond to those made after the second
challenge with the bacterial marker.
59
B) Recoveries in caives from group B
81
A)Recoveries in caives from group A
7-
6-
I
« 4
3-
T—'—I
—r
2
3
Time (hours)
4
-I
r—'
1
1
2
1
3
'
Time (hours)
1
4
'
1
5
60
one of group C) of the 8 calves inoculated with M. dispar. No other clinical
signs of disease were observed.
Gross Post-Mortem Findings
At necropsy, macroscopic lesions were confined to the lungs. The lungs of the calves
infected with M. d/spar contained consolidated areas which occupied up to 1-2% of
the lung parenchyma (Table 1). These areas of consolidation were located mainly in
the cranial lobes and were dark red. No exudate was expressed when these areas
were pressed.
Table 1- Histopathological and Microbiological Findings in Experimental Calves.
Calf
Qoup
Pathology
Consolidation
Microbiology
BronchiolUs
Emphysara
M
dispar Smanxsoem
f/o)
1
a
1
+
+
+
+
2
a
2
+
+
+
+
3
a
0.5
+
-
+
+
4
a
1
+
-
+
+
5
a
1
+
+
+
+
6
a
1
+
-
+
-
7
b
0
-
-
-
-
8
b
0
-
-
-
-
9
c
1
+
+
+
-
10
c
2
+
+
+
-
61
Histopathological and SEM findings
Histopathological studies performed in calves infected with M. of/spar showed
that early changes were detected mainly in the respiratory epithelium of
medium and small bronchioles. These alterations included degeneration of
ciliated cells with loss of cilia, pyknosis and cytoplasmic shrinkage. The lumen
of bronchioles and smaller airways from M. cA'spar infected calves contained a
considerable amount of cellular debris and polymorphonuclear cells (Fig. 2).
Also, a moderate infiltrate of inflammatory cells, mainly PMN's and
macrophages, was noticed in the submucosa of these airways. In contrast,
ciliated epithelium from large airways was not altered. No lesions were
detected in calves of group B (Fig. 3). Scanning electron microscopy of the
airways of calves infected with M. dispar showed ciliary clumping with presence
of inflammatory cells (Fig. 4). Also patchy areas with loss of the ciliated
epithelium and presence of inflamatory cells were seen in these airways (Fig.
5). Extruded ciliated cells were observed in the lumen of the airways. No
alterations were detected in the epithelium of large airways (Fig. 6). No lesions
were observed in animals challenged with S. marcescens alone (data not
shown).
Microbiology Findings.
Mycoplasma dispar was isolated from the lungs of all the calves of group A and C. In
addition, S. marcescens was isolated from the lungs of 5 calves belonging to group A.
No other bacteria was isolated from any of the lungs of the calves in these
experiments. No mycoplasmas were isolated from the respiratory tract of calves
belonging to group B (Table 1 ).
Figure 2. Small bronchiole from a M. d/spar infected calf showing alteration of
the ciliated eipthelium and presence of polymorphonuclear cells in the lumen.
(H&E, 450 X).
Figure 3. Small bronchiole from a calf of group B without degenerative
changes. (H&E, 450X).
63
Fîg. 4. Scanning electron micrograph of small bronchiole of calves
infected with M. d/spar showing clumping of the ciliary blanket with
inflammatory cells on surface (SEM, 940 X).
Figure 5. Scanning electron micrograph of a small bronchus of a calf
infected with M cf/spar showing areas with loss of the ciliated epithelium
and presence of inflamatory cells. (SEM, 600 X).
Figure 6. Scanning electron micrograph of a large bronchus of a calf
Infected with M. d/spar showing no apparent damage of the ciliated
epithelium (SEM, 3600 X),
67
I
68
DISCUSSION
The study of lung clearance has been used to delineate factors and conditions
that determine the retention of organic or inorganic particulate markers in the
respiratory tract. In some of these studies, clearance of the particulate marker
has been studied using the technique of sequential slaughter (13,15,16,
17,18,19,20). This method has a major disadvantage, which Is the slaughter of
a large number of animals and the obtaining of multiple samples from each
individual, adding more cost and source of variation to the system. Using a
different approach, mechanical clearance has been assessed by external
monitoring of inhaled, inert radioactive aerosols (7,21,22,23). A variation of this
technique was described by Viso et al. (24), In which the clearance of a
opportunistic bacterial species of low pathogenic potential was sequentially
determined in the same animal during a period of time. In the present
investigation, a modification of this method was used to determine the effect of
M d/spar infection on clearance of S. marcescens and it could be shown that
animals infected with this mycoplasma species presented reduced clearance of
the bacterial marker. Four hours after Inoculation of M. dispar-Uee calves, only
a few colonies of S. marcescens were recovered. In contrast, in the same
calves and after infection with M. dispar, the tracheobronchial elimination of S.
marcescens was prolonged beyond 4 hours after inoculation of the bacterial
marker. In addition, the isolation of S. marcescens was possible from the lungs
of calves from group A but not from group B, which was indicative of the
impaired tracheobronchial clearance and increased retention of the bacterial
marker in calves infected with M. dispar.
L
69
It was previously shown that exposure to virus (25) or to mycoplasmal agents
(20) will retard the clearance of inhaled bacteria. In these studies the
impairment of the clearance was presumed to be due to lesions of the
mucociliary apparatus caused by these pathogens. The results presented here
are comparable with these previous reports, in that the infection with M. dispar
alters the normal tracheobronchial clearance in calves. Similarly, it is possible
that the decreased clearance could be the result of the lesions that M. dispar
produced to ciliated epithelium of the respiratory tract.
In previous studies, conventionally raised and gnotobiotic calves were
experimentally infected with M dispar and killed between 3 to 5 weeks later.
Histophatological results from these studies included the presence of exudative
bronchitis and interstitial alveolitis centered around the bronchioles with
moderate to negligible production of peribronchial cuffs (3, 2 ). The
histopathological descriptions in calves naturally infected with M. dispar
included bronchitis and bronchiolitis obliterans with large cuffs of mononuclear
cells around these ainways. Mycoplasma of/spar colonies were detected in the
surface of the affected airways (11). Changes in the ultrastructure of the
bronchial epithelial cells included loss of the cilia and cytoplasmic protrusion
into the airway lumen (1 ). Lesions described from in vitro studies, in which
expiants of bovine tracheas were inoculated with M. dispar, included profound
damage to the ciliary epithelium with pyknosis, sloughing and flattening of the
cells with consequent loss of the ciliary activity (9). Based on these results, it
was proposed that M. d/spar colonizes the bronchioles in close relationship with
the cilia and microvilli of the epithelial cells, altering their metabolism and
stopping the ciliary movements with necrosis and desquamation of the cells (9).
Results from histopathological and SEM studies of the lungs of calves Infected
with M. dispar alone or in combination with S. marcescens as presented here,
indicated destruction of respiratory ciliated surfaces and degeneration of the
respiratory ciliated epithelial cells mainly in small and medium size airways.
Minimal alveolar reaction surrounded these damaged ainways and no cuffing
reaction was seen. The slow development of the pulmonary lesion which
correlated with the slow growth of M. dispar was previously described (3), and
these can be taken here to explain the mild character of the alveolar lesions
described. Previous descriptions of lung damage caused by M. dispar have
focused on late stages of lesion development, in contrast these experiments
provide a description of the lesions that develop in the early stages of the
infection. Based in the results of the present communication, it is likely that
these circumscript lesions affecting the ciliary blanket are related to the strong
affinity shown by this mycoplasma species with the surface structures of the
respiratory mucosa (9). These lesions were localized mainly in medium and
small airways and not in bronchii or the trachea, contrasting with the lesions
associated with M. hyopneumoniae infections which were detected in epithelial
ciliated cells of trachea and in main bronchii with minimal damage to the
bronchioles or medium size airways epithelium (27).
The decreased clearance of S. marcescens can also be partially explained by
changes in the composition of the mucous layer. It was reported that the
increased numbers of goblet cells detected in the respiratory tract of pneumonic
calves resulted in decreased viscosity of the mucous layer and reduced the
efficinecy of trapping and removing inhaled microorganism or particles out of
the respiratory tree (1). This effect which was considered non-specific for
mycoplasmal pneumonias, may act in conjunction with the destruction of ciliary
cells.
An interesting result from the present study was the finding that damage of the
ciliated epithelium was more evident in small bronchioles than in bronchi or
main ainways. This feature can be explained by the slower mucus transport and
the paucity of ciliated cells present in small and medium airways, which may
allow more close contact between the mycoplasma and ciliated structures.
The retarded mucus flow caused by damage of the ciliary blanket not only will
interfere with the removal of inhaled particles but also will not provide the
adequate mucus flow to assure the clearance and preservation of the
underlaying respiratory mucosa above the damaged segment (21). The final
outcome of this process will be the entire reduction of the mucociliary transport
in the distal segments of the airways allowing the colonization of M. disparas
well as the local proliferation of secondary bacterial invaders.
The importance of this latter observation needs to be considered in reference
with fast growing, highly pathogenic bacterial species for cattle, such as
Pasteurella hemolytica.
i
72
The results presented here and the effect that M. of/spar has on the alveolar
macrophage phagocytic functions (28), are grounds to reconsider the
importance of this agent as a causal and predisposing agent of pneumonic
infection in cattle.
73
REFERENCES
1. Allan EM. Some features of pulmonary mycoplasmosis in groups of naturally
infected calves. In pages 305-316. Respiratory disease of cattle. A seminar of
the EEC programme of coordination of research on beef production held at the
Netherlands. Martinus Nijoff, The Netherlands 1978.
2. Friis RN. Mycoplasma disparas a causative agent in pneumonia of calves.
Acta Vet Scan 1980; 21:34-42.
3. Gourlay RN, Howard CJ, Thomas LH, and Wild SG. Pathogenicity of some
Mycoplasmas and Acholepasma species in the lungs of gnotobiotic calves.
Res Vet Sci 1979; 27:233-237.
4. Mueller HE, Krasemann C. Virulence of Pasteurella multocida strains and
their neuraminidase production. Zentralb Bakteriol 1974; 299A:391-400
5. Lipsky BA, Goderbreg AC, Tompkins LS. Infections causes by nondiphteric
Corynebacteria. Rev Infec Dis 1982; 4:1220-1235.
6. Konietzko N. Mucociliary transport and bronchial inflammation. Z Gesamt
Med 1988; 43:323-327.
7. Jarstrand C, Camner P, Philipson K. Mycoplasma pneumoniae and
tracheobronchial clearance. Am Rev Resp Dis 1974; 110:415-419.
8. Sweney LR, Shapiro BL. Rapid preparation of uncoated biological
specinnens for scanning electron microscopy. Stain Tech 1977; 52:221-227
9. Thomas LH, Howard CJ, Parsons KR, Anger HS. Growth of Mycoplasma
bovis in organ cultures of bovine foetal trachea and comparison with
Mycoplasma dispar. Vet Microbiol 1987; 13:189-200.
10. Howard CJ, Thomas LH. Inhibition by Mycoplasma dispar oi ciliary activity
in tracheal organ cultures. Infect Immun 1974; 10:405-408.
11. Tinant MK, Bergeland ME, Knudtson, WU. Calf pneumonia associated with
Mycoplasma d/spar infection. J Amer Vet Med Assoc 1979; 175:812-814
12. Knudtson WU, Reed DE, Daniels G. Identification of mycoplasmatales in
pneumonic calf lungs. Vet Microbiol 1986; 11:79-91
13. Veit HP, Farrell RL, Troutt HF. Pulmonary clearance of Serrafta
marcescens in calves. Am J Vet Res 1978; 10:1646-1650
14. Rosenbusch RF. Influence of mycoplasma infection on the expression of
Moraxella bovis pathogenicity. Am J Vet Res 1983: 9:1621-1624.
15. Lillie LE, Thompson RG. The pulmonary clearance of bacteria by calves
and mice. Can J Comp Med 1972; 36:129-137.
75
16. Sebunya TNK, Saunders JR. Pulmonary clearance of Haemophilus
pleuropneumoniae and Serratia marcescens in mice. Am J Vet Res 1982;
43:1799-1801.
17. Saunders JR, Sebunya TNK, Osborn AD. Pulmonary clearance of Bacillus
subtilis spores in pigs. Can J Comp Med 1983; 47:43-47.
18. Veit HP, Farrell R. Increased pulmonary clearance of Serratia marcescens
In calves given intravenous Freund's complete adjuvant. Cornell Vet 1984;
74:269-281
19. Rodriguez LM, Lopez A, Merino-Moncada M, Martinez-Burnes J,
Mondragon I. Tracheal versus pulmonary deposition and clearance of inhaled
Pasteurella hemolytica or Staphylococcus aureus in mice. Can J Comp Med
1985; 49:323-326.
20. Slauson DO, Lay JC, Castleman WL, Neilsen NR. Acute inflammatory lung
injury retards pulmonary particle clearance. Inflammation 1989; 13:185-199.
21. Foster WM, Langenback E, Bergofsky, EH. Measurement of tracheal and
bronchial mucus velocities in man: relation to lung clearance. J Appl Physiol
1980; 48:965-971.
76
22. Jones CDR, Bull JR. Deposition and clearance of radiolabelled
monodisperse polystyrene spheres in the calf lung. Res Vet Sci 1986; .42:8291.
23. Davies CP, Webster AJF. Deposition and clearance of monodisperse
aerosols in the calf lung: Effect of particle size and mucolytic agent
(Bromhexine). Can J Vet Res 1987; 51:306-311.
24. Viso M., Espinasse J, Lambert P. Measure de la clairance
tracheonbronchiale des bovins a l'aide de 1' aspiration transtracheale.
Proceedings of the Fourteenth World Congress on Diseases of Cattle. Dublin,
Ireland. World Association for Buiatrics. 1986; 1:509-511.
25. Sellers TF, Schuman J, Boubier C, McCune R, Kilbourne ED. The
influence of influenza virus infection on exogenous staphylococcal infection and
endogenous murine bacterial infection of the bronchopulmonary tissues. J Exp
Med 1961; 114:237-256.
26. Stalheim OHV. Mycoplasmal respiratory disease in ruminants: A review
and update. J Amer Vet Med Assoc 1983; 182: 403-406.
27. Mebus CA, Underdahl NR. Scanning electron microscopy of trachea and
bronchi from gnotobiotic pigs inoculated with Mycoplasma hyopneumoniae. Am
J Vet Res 1977; 38:1249-1254
77
28. Almeida RA, Rosenbusch RF. Interaction between capsulated Mycoplasma
dispar and bovine alveolar macrophages. Proceedings of the 91st General
Meeting of the American Society for Microbiology. 1991 ; Abst.G-40 pp 140.
Dallas, Texas.
78
PART II. Interaction of Mycoplasma disparwWh bovine alveolar macrophages
79
Interaction of Mycoplasma disparwWh bovine alveolar macrophages
Raul. A. Almeida, D. V. M., M. S.
Ricardo. F. Rosenbusch, D. V. M., Ph. D.
Michael. J. Wannemuehler, Ph. D.
From the Veterinary Medical Research Institute and the Department of
Microbiology, Immunology, and Preventive Medicine, Iowa State
University, Ames, Iowa, 50011.
Supported in part by Iowa Livestock Health Advisory Council, grant
No.109-05-17
r
80
ABSTRACT
The capacity to avoid phagocytosis, the impairment of the bactericidal activity
of bovine alveolar macrophages (BAM) and the activation of BAM by
encapsulated Mycoplasma dispar or purified M. d/spar capsule was
investigated. Encapsulated and unencapsulated M. dispar were cocultured
with BAM in the presence or absence of antisera prepared against
unencapsulated M. dispar or purified capsule antiserum. Unopsonized
mycoplasma resisted phagocytosis while only anticapsular antibodies
enhanced the phagocytosis of encapsulated mycoplasmas. Bovine alveolar
macrophages, cultured in presence of viable unencapsulated or encapsulated
M. dispar or purified capsule, were tested for their killing activity on Serratia
marcescens and Staphylococcus aureus. Killing of both bacterial species was
reduced when BAM were treated with purified capsule or encapsulated M.
dispar. In contrast, no reduction in the bactericidal activity was observed when
unencapsulated mycoplasma were used. Bovine alveolar macrophages were
cultured in presence of purified M. dispar capsule or either live or heat killed
encapsulated or unencapsulated M. dispar. The supernatant of these cultures
were assayed for tumor necrosis factor (TNF), interleukin 1 (iL-1), and glucose
consumption, as indicators of macrophage activation. Tumor necrosis factor
and IL-1 were produced by BAM when stimulated with unencapsulated M.
dispar but not when encapsulated M dispar or its purified capsule was used.
Similarly, glucose consumption was increased in presence of unencapsulated
M dispar, but not when BAM were cocultured with encapsulated M. dispar or
purified capsule. These results indicate that encapsulated M. dispar or purified
capsule exert an inhibitory effect on the activity of BAM.
81
INTRODUCTION
Alveolar macrophages are central in the protection of the lower portions of the
lungs, operating in many nonspecific modes of defense and augmented by
specific immunologic mechanisms. Previous studies have shown that alveolar
macrophages ingest, inactivate, and degrade inhaled microorganisms within
eight hours of their entrance into alveolar regions (12 ). Such antimicrobial
ability of macrophages is centered on their phagocytic capabilities and in the
production of potent microbicidal and degradative substances within the
macrophage.
It has been shown that macrophages have innate functional capacities that
extend far beyond phagocytosis. They can act as secretory and regulatory
cells, can initiate and prolong inflammatory processes, and can stimulate
synthesis of extracellular matrix proteins (4, 14). Thus, they can adjust their
response to their microenvironment and exert regulatory activities on other cells
such as neutrophils and lymphocytes mounting the defense system that protects
the respiratory parenchyma from microbial invasion (4). However, if
macrophage function is impaired due to pollutant or microbial exposure, the
host-parasite balance is upset and disease ensues.
In the absence of specific antibodies, several mycoplasma species can
colonize the surface of macrophages without stimulating phagocytosis (2, 5, 8,
18). In contrast, other mycoplasma species were capable of stimulating
macrophage activity, induce secretion of cytokines, expression of MHC
molecules and release of hydrolytic enzymes from these cells (7, 27).
Experiments with M. d/spar have shown that this mycoplasma species can
induce immunosuppression when injected in combination with other antigens,
and it was postulated that the production of soluble inhibitory factor(s) were
responsible for the observed immunosuppression (11). The present studies
examined the interaction of M. d/spar with bovine alveolar macrophages and
found that M. d/spar capsule exerts a suppressive effect. The bactericidal
activity of macrophages as well as the secretory functions of these phagocytic
cells were reduced by the presence of encapsulated M. dispar or
polysaccharide capsule.
MATERIALS AND METHODS
Macrophages. Bovine alveolar macrophages were obtained by
bronchoalveolar lavage of whole lungs. Briefly, 75-90 day-old calves from a
mycoplasma-free herd were sacrificed by intravenous injection of a overdose of
sodium pentobarbital (Fort Dodge Inc., Fort Dodge, la.), and trachea and lungs
were aseptically recovered for lavage. Two consecutive bronchoalveolar
lavages were performed with 400 ml of ice-cold modified Hank's solution (EHBSS: 2.5 mM EDTA, 0.4 gm/l KCI, 0.06 gm/l KH2PO4. 8.0 gm/l NaCI, 0.35
gm/l NaHCOs, 0.09 gm/l Na2HP04.7H20, penicillin G 62 p.g/m, kanamycin 100
|ig/ml,streptomycin 100 p.g/ml, and amphotericin B 50 p.g/ml). The first 10 ml of
bronchoalveolar lavage fluid were discarded and the rest of the fluid was gently
transferred to a sterile container and kept at 4°C. The suspension was then
centrifuged at 200 X p for 10 min at 4°C and the pellet was gently resuspended
in Eagles's minimal essential medium (MEM) supplemented with 5% FBS
(GIBCO Laboratories, Grand Island, N.Y.), and centrifuged at 200 X gf for 10 min
at 4°C. The cell population was depleted of erythrocytes by isotonic lysis with a
0.83% Tris ammonium chloride solution for 5 min at 37°C. Cells were
centrifuged as before and the pellet was resuspended in E-HBSS without
amphothericin B. The proportion of macrophages was estimated by
phagocytosis of latex beads (Poly Sciences, Inc., Warrington, PA), and viability
of the cells was checked by Trypan-Blue exclusion.
The proportion of macrophages was enriched using the selective adherence
of macrophages to plastic surfaces, following the protocol described by
Pennline (21). Briefly, cells were spun down (200 X g for 10 min at 4°C) and
resuspended in MEM containing 15 % FBS with 62 |ig/ml of penicillin G,
['
84a
100 ng/ml of kanamycin, and 100 ^ig/ml streptomycin, and placed in Petri dishes
at a concentration of 3 X 10^ cells/cmZ. The cells were incubated overnight at
a
37°C in a 4 % CO2 atmosphere. Adherent cells were then harvested as follows:
after washing the monolayer to remove non adherent cells, ice cold E-HBSS
was added and allowed to stand at 4°C for 30 min. Cells were then washed
twice with warm E-HBSS, spun down as before and resuspended in MEM 10%
FBS. Viability and percentage of macrophages was measured as described
above. Macrophages were then placed in 96 or 12-well cluster culture plates at
a concentration of 3 X 10^ cells/cm^ and incubated at 37°C in a 5% CO2
atmosphere. Absence of mycoplasma contamination was ascertained by
culturing the BAM without antibiotics, followed by microscopic examination after
staining with the fluorescent dye 4',6'-diamidlno-2-phenylindole dihydrochloride
(DAPI,. Calbiochem, San Diego,Calif.).
Microorganisms. The SD-0 strain of M. dispar was isolated from a
pneumonic calf (29), was cloned twice, and was used at passage level 7.
Mycoplasma dispar was grown in Friis modified broth medium as previously
described (19). Logarithmic phase cultures were centrifuged for 10 min at 500
X flf to remove debris and the resulting supernatant centrifuged at 34,000 X g for
30 min. The pellet was washed three times with 0.01 M phosphate buffered
saline solution (PBS), pH 7.2. Protein concentrations were determined by the
method of Lowry et al. (20 ) with alkali treatment prior to the assay to solubilize
membranes (1 ). Mycoplasma suspensions were stored after quick freezing
L
84b
at -70°C in 1 ml aliquots (30 mg protein/ml). For induction of M. cf/spar capsule,
we followed the methodology described by Almeida and Rosenbusch (3).
Briefly, M. disparwere cocultured with bovine lung fibroblast monolayers for
23 h. Capsulated mycoplasmas were then harvested from the cell culture
medium by differential centrifugation at 200 X g for 10 min at 4°C and washed 3
times by high speed centrifugation at 23,000 X g with cold PBS supplemented
with 1 mM Tris and 1 mM EDTA (E-PBS). Aliquots of unencapsulated and
encapsulated mycoplasmas were heat -killed by placing them in boiling water
for 3 min. For use as a marker Serratia marcescens strain NIMA used in this
experiment was grown on tryptose agar plates (Difco, Detroit, Mich.) and
incubated at room temperature for 36 hours while Staphylococcus aureus
(ATCC 10390) was grown in 7% bovine blood agar plates at 37°C overnight.
The bacterial lawns were harvested and resuspended in lactose glutamate
diluent (22) at a concentration of 6.0 X 10^ and 6.5 X 10^ colony forming units
(CPU) per ml respectively, fractionated in 1 ml aliquots and kept at -70 °C until
use.
Capsule Purification. Capsule was extracted from the mycoplasmas by
placing them in E-PBS (pH 8.0) at 37°C for 1 h. After centrifugation at 34,000 X
g for 30 min, the supernatant was dialyzed for 72 h against the same buffer.
Capsule material was then captured on immobilized Ricinus communis II lectin
(Vector, Burlingame, Calif.), washed in decreasing salt concentrations (2 M, 1.1
M, and 0.2 M NaCI in 1 mM Tris ph 8.0 and 1 mM EDTA), eluted with 0.2 M
lactose, and purified by size exclusion chromatography (Biogel P4, Bio-Pad,
Richmond, Calif.) using E-PBS (pH 8.) as running buffer. Collected fractions
were analyzed by thin layer chromatography (TLC) using TLC silica gel plates
(E. Merck, Darmstadt, Germany) and a mixture of pyridine, ethanol, water, and
n-butanol at a ratio of 4:1:1:1 as solvent system. Presence of capsule was
detected by staining the plates with a saturated solution of silver nitrate diluted
1:10 in a mixture of H2O and acetone (1:1 ratio), followed by washing steps with
0.5 M NaOH in ethanol and then 6 M ammonium hydroxide solution. Fractions
showing presence of capsule material were then analyzed following a
radioimmunoprecipitation protocol previously described (3). Positive fractions
were then pooled, lyophilized, resuspended at 15 ^ig/ml in 10 mM HEPES, 0.85
M NaCI and 0.1 mM CaClg buffer (pH 8.0), and kept in 500 p.1 aliquots at -70°C.
Phagocytosis assay. The phagocytosis protocol described by Davis et al.
(9) was followed. Briefly, 12-well cluster plates containing 3X10® /ml BAM were
treated with 3X10® ccu/ml of encapsulated or unencapsulated M. dispaireaclBd
previously for 10 min at 37°C with or without rabbit anti-capsule or anti-M.
dispar sera at a 1:100 final dilution. Plates were then centrifuged at 800 X gr at
4°Cfor 20 min, and then incubated for 15 min at 37°C to promote mycoplasma
attachment. Cultures were washed 5 times with warm MEM 5 % FBS to remove
non-adherent mycoplasmas and incubated at 37°C in MEM with 10% FBS. At
4 h intervals after the addition of mycoplasmas macrophages of 3 wells per
treatment were scraped from the plastic surface and centrifuged at 14,000 X g
and 4°C for 10 min. The pellets were resuspended in MEM 10% FBS,
sonicated on ice for 60 s (8 mm probe, 120 Watt; Braunsonic 1510, South San
Francisco, Calif.) and the lysate was serially diluted 10-fold and plated in
duplicate modified Friis plates (19) or in modified Friis broth tubes; broth tubes
86
and agar plates were incubated at 37°C in a 5% CO2 atmosphere for 5-7 days
before titer determination or colony counting. Concomitantly with the last
sampling, macrophage viability was checked in non-treated control wells by
Trypan-Blue exclusion.
Bactericidal Assay. Bovine alveolar macrophages in 12-well plates were
exposed for 12 h to unencapsulated or encapsulated M. dispar, or to 7.5 |il/ml
(final concentration) of purified capsule, or left untreated. Macrophages were
then incubated for 4 h in MEM 10% FBS with 60 p.g/ml of tylosin (final
concentration), washed 5 times with MEM 5% FBS and incubated with
preopsonized Serratia marcescens or Staphylococcus aureus at 10:1
bacteria/BAM ratio. Plates were centrifuged at 800 X p for 5 min to assure
contact between bacteria and BAM, incubated at 37°C for 2 h (S. marcescens)
or 15 min (S. aureus) in a 5% CO2 atmosphere, and washed 5 times with warm
MEM 5% PCS. The BAM cultures were then treated with warm MEM containing
lysostaphin (1U/ ml) for 15 min (S. aureus), or 200 |ig/m of kanamycin and 100
|ig/ml of gentamycin for 2 h at 37°C (S. marcescens), followed by 5 washes with
warm MEM 5%PBS to remove extracellular bacteria. Macrophages were then
lysed with H2O and the number of viable intracellular bacteria were calculated
by triplicate using end dilution titration in tryptose agar plates (S. marcescens)
or in brain heart infusion agar plates (Difco, Detroit, Mi.) (S. aureus). Viability of
BAM was checked in non-treated control wells by Trypan-Blue exclusion.
TNF Assay. Bovine alveolar macrophages were treated with live or heat
inactivated encapsulated or unencapsulated M. d/spar or treated with 7.5 ^g/ml
87a
of purified capsule, 2 mg/ml Escherichia coli K235 endotoxin (Sigma, St. Louis,
Mo), or culture medium, and incubated at 37°C in a 5% COg atmosphere. Each
treatment was conducted in triplicate wells and ^0 p.1 samples of the
supernatants were taken at 4, 8, 12, and 24 h after treatment. Samples were
then centrifuged at 14,000 Xg for 10 min at 4°C, and kept at -70°C until use.
Concomitantly with the last sampling, BAM viability was checked in non-treated
control wells by Trypan-Blue exclusion.
For the TNF assay, supernatants from the macrophages were assayed using
the TNF sensitive L929 cell line. Briefly, L929 cells were grown in MEM with 10
% horse serum (HyClone, Logan Ut.), resuspended in the same media and
placed in 96-well culture plates at a concentration of 2.4 XI0^ cells/well, and
incubated at 37°C in a 5% CO2 atmosphere. The medium was then changed to
MEM with 10% horse serum and 3 ng/ml of actinomycin D (Sigma, St. Louis,
Mo.), and after this, wells were inoculated with 100 p,l of 1:10, 1:50 and 1:250
dilutions of macrophage supernatants, and incubated overnight at 37°C in a 5%
CO2 atmosphere. After fixation with 10% formaldehyde, cells were washed 3
times with saline and stained with a 0.2% solution of crystal violet for 20 min.
Plates were then washed to remove crystal violet, and dried for 24 h at 37°C.
One hundred microliters of a 1:1 mixture of PBS (pH 7.2) and 100% ethanol
was added to each well and then the plate was read at 595 nm in an automated
ELISA reader (Bio-Tek Instruments Inc., Winooski, Vt).
IL-1 Assay. Bovine alveolar macrophages were treated with live or heat
killed encapsulated or unencapsulated M. d/spar or with 7.5 ng/ml of purified
capsule, Escherichia coli K235 endotoxin (2 |ig/ml), or culture medium. Each
87b
capsule, Escherichia coli K235 endotoxin (2 |xg/ml), or culture medium. Each
treatment was conducted in triplicate wells and 300 |il samples of the
supernatants were taken at 4, 8,12, and 24 h after treatment. With the last
sampling, macrophage viability was checked in non-treated control wells by
Trypan-Blue exclusion. Samples were then centrifuged at 14,000 X g for 10
min at 4°C, and kept at -70°C until use. For IL-1 activity, supernatants were
assayed using the IL-1 dependent D10G4.1 cell line. Briefly, triplicate wells
were inoculated with 1:5, 1:25 and 1:125 dilutions of macrophage supernatants
in RPIy/ll 1640 suplemented with 5% PCS and incubated at 37°C in a 5% CO2
atmosphere for 48 h and then pulsed for 24 h with 50 |iCi/ml of
- thymidine.
Cells were then harvested on glass microfibre filters (Whatman, Maidstone,
England) which were allowed to dry. Scintillation vials containing individual
glass microfibre filters were filled with 2,5 ml of scintillation cocktail (Ready-Solv
Hp/b, Beckman Instruments, Fullerton, Calif.) and counts per minute were
determined in a scintillation counter (Packard Instruments, Downers Grove, II ).
Glucose consumption. Ten microliter aliquots of supernatant from BAM
treated with live or heat killed encapsulated or unencapsulated M. dispar, 7.5
|ig/ml purified capsule, or incubated with Escherichia coli K235 endotoxin
(2^g/ml), or culture medium, were assayed for glucose consumption as
indication of BAM activation, following the protocol described by Ryan et al.
(24). Briefly, 10 ^il of each supernatant were mixed with 100 |il of a 5 mM NADP
solution, 100 p.1 of a 20 mM ATP solution, and 5 ml of a hexokinase (250 units of
hexokinase/ml) and G6PD mixture (Sigma, St. Louis, Mo), in 0.1 M Tris buffer
ph 7.5. This mixture was added to a cuvette containing 700 p.1 of 0.1 M Tris-HCL
[•
88
(pH 7.5), 64 mM NaCI, 3.5 mM MgCIa, and 0.15 mM CaClg. Two min after the
solutions were mixed, the OD340 was read to determine NADPH production.
Statistical Analysis. The data obtained from each experiment were
analyzed by analysis of variance (ANOVA). When significant differences were
found among groups, data were analyzed by paired f tests. Significance was
accepted for comparison when the p value was less than 0.05.
89
RESULTS
Phagocytosis. The results from the phagocytosis assay are shown In Fig 1A
and B. When unencapsulated or encapsulated M.disparwere cultured with
BAM in absence of antibodies specific for M. d/spar surface antigens, no
reduction of the titer of the microorganisms was observed. The need for specific
antibodies to promote phagocytosis was evidenced by a decrease of 2-3 logio
in the titer of the mycoplasma.
Bactericidal Activity. Bactericidal activity against opsonized S. aureus and
S. marcescens was seen with BAM, as evidenced by reductions of 5 logs in the
titer of these bacteria (Table 1). No effect on this bactericidal activity was
observed when BAM were pre-treated with unencapsulated M. dispar. In
contrast, inhibitory effect was detected when BAM were pre-treated with
encapsulated mycoplasma since the titer of S. aureus or S. marcescens was
over 2 logio higher in BAM treated with encapsulated M. disparthan that
observed with the untreated macrophages. Similar results were observed
when purified M. d/spar capsule was used to treat BAM prior to the assay.
TNF Production. Live and heat killed unencapsulated M. dispar Induced
marked TNF production that presents similar patterns of induction over time as
that observed in endotoxin stimulated BAM (Fig 2A and 2B). Slight differences
between live and heat-killed unencapsulated M. dispar were noticeable since
levels of TNF production continued to increase up to 80 % of cytotoxicity at 20 h
following the addition of heat-killed preparation while they reached 65 % of
cytotoxicity with the live preparation in the same period of time.
I:
90
When BAM were treated with live or heat killed encapsulated M. dispar, TNF
production was significantly less (p< 0.05) than that of BAM stimulated with
endotoxin or unencapsulated M. dispar. Likewise, purified capsule also had an
inhibitory effect on TNF production by BAM.
Fig. 1. Phagocytosis assay conducted with bovine alveolar macrophages:
(A) unencapsulated M. dispar or (B) encapsulated M. dispar 'm presence and
absence of specific antisemm against M.dispar or purified capsule. Symbols:
unencapsulated M. d/spar without antibodies (•); unencapsulated M. dispar
with anti-M d/sparantibodies (A); unencapsulated M. d/sparwith anti-capsule
antibodies(#); encapsulated M. d/spar without antibodies (-•-);
encapsulated M. d/sparwith anti-M d/spar antibodies {-A-y, encapsulated M.
dispar with anti-capsule antibodies (-•-); Colony forming units (cfu). Mean of
three samples are recorded with standard deviation of the mean.
8-
Log cfu/ml
O
O
' '19' '
o-
s-
93
Table 1. Killing of Serratia marcescens or Staphylococcus aureus by bovine
alveolar macrophages pre-treated with unencapsulated M. dispar, encapsulated
M. dispar or purified capsule.
Pre-treatment
None
Microorganisms
Unencapsulated
Encapsulated
Purified
M. dispar
M. dispar
capsule
S. marcescens
1.30®{1.02)b
1.48 (0.48)
3.70 (1.90)
3.02 (2.00)
S. aureus
1.40(0.78)
1.54(0.90)
3.59(3.00)
3.30(2.95)
^Values are the mean of three counts expressed as log colony forming units/well.
A total of 3 X 106 colony forming units of either S. marcescens or
S. aureus was added to each well
^Values in parenthesis represent the standard deviation of the mean of three
observations.
IL-1 Production. Bovine alveolar macrophages produced lL-1 upon
stimulation with unencapsulated M dispar, but the level reached was
significantly lower than that observed with endotoxin stimulated BAM (Fig 3A).
In contrast, the IL-1 production induced by encapsulated M. dispar or purified
capsule was negligible and was no different from that detected with untreated
macrophages. When heat killed encapsulated or unencapsulated M. dispar
was used, it was observed that IL-1 was produced and at similar levels. Still,
these were significantly lower than endotoxin stimulated macrophages but
significant higher than untreated macrophages (Fig SB).
r
94
Glucose Consumption. Glucose consumption was measured as an
indicator of macrophage activation. In presence of unencapsulated M. disparthe
rate of glucose consumption detected was similar to that observed in BAM
stimulated with endotoxin (Fig. 4). In presence of encapsulated M. dispar,
however, BAM showed only minimal consumption of glucose, significantly lower
than that observed in endotoxin treated macrophages. Even more, when purified
capsule was used, glucose consumption was less than the observed with
encapsulated M. dispar and significantly different from that of BAM stimulated with
endotoxin or treated with unencapsulated M dispar.
1
Fig 2. Production of tumor necrosis factor activity by bovine alveolar
macrophages treated with live (A), or heat killed (B) encapsulated and
unèncapsulated M. cf/spar Symbols: Media (A); untreated BAM (O); endotoxin
(•); purified capsule (•); encapsulated M. dispar{xy, heat killed capsulated M.
dispar, (-x-); live unencapsulated M.dispar {%)•, heat killed unencapsulated M.
dispar
96
(A)
(B)
100
100
80
>
60
u 60
M
e
>>
u
40
20
"O
0
10
TIm# (h)
20
30
20
10
Tlm*(h)
30
Fig. 3. Production of interleukin-1 {IL-1) by bovine alveolar macrophages
(BAM) treated with live (A), or heat killed (B) encapsulated and unencapsulated
M. dispar. Symbols as in Fig.2
98
(B)
40000 1
40000 1
30000 -
30000
&
&
o
S
•
Z
1
• 20000 -
m"
20000
10000
10000 -
—I
T
10
20
TIm# In hour*
10
1—
20
TIm# In hours
—I
30
Fig 4. Glucose consumption of bovine alveolar macrophages treated with
unencapsulated M. dispar, encapsulated M. dispar or purified capsule.
Symbols: endotoxin (•); unencapsulated M. dispar (9), encapsulated
M. dispar {xy, purified capsule (•), untreated BAM
100
100 1
80 -
at
c
E
2
I
t»
o
20
40
60
80
Time (h)
100
120
140
101
DISCUSSION
Pathogenic microorganisms have evolved several strategies to avoid
phagocytosis by interfering at distinct steps of the attachment, ingestion, and
killing process. Among these, microbial capsules were considered the most
important and ubiquitous anti-phagocytic substance (15). Some
microorganisms posses surface structures that enable them to attach to
phagocytes without being ingested. Neisseria gonorrheae and several
mycoplasmas have been reported among these microorganisms and in N.
gonorrheae such property was linked to their capsule (10). While it is not
known if the capsule of M. dispar p\ays a role in attachment, it has been shown
that capsule production occurs in vivo or in coculture with bovine cells (3).
The results presented in this report indicated that the capsule of M dispar
exerts antiphagocytic activity. When encapsulated or unencapsulated M. dispar
were exposed to alveolar macrophages in absence of specific antibodies, no
loss of viability of the mycoplasma was detected. In contrast, in presence of
antibodies specific to capsule or surface proteins, significant loss of viability to
encapsulated or unencapsulated M. dispar, respectively, was detected. The
protective role of the capsule can be deduced by the fact that complete loss of
viability of the mycoplasma was detected only when encapsulated
mycoplasmas were exposed to BAM in presence of specific anti-capsule
antibodies. The failure to obtain the same reduction in mycoplasma viability
observed in the system which included unencapsulated mycoplasmas BAM and
M. d/spar antiserum, can be explained by the persistence of a low percentage of
encapsulated M dispar \n unencapsulated mycoplasma grown in broth media
(3).
102
This fraction of the mycoplasma population will resist phagocytosis in absence
of specific anti-capsule antibodies.
It was previously reported that mycoplasmas can inhibit the phagocytosis and
killing of other microorganisms (17, 26, 28). It was postulated that this effect was
independent of the attachment of the mycoplasmas to the phagocyte membrane
and this was supported by the finding that Mycoplasma arthritidis did not attached
to cells but still inhibited phagocytosis (17). Also, It was reported that the capsular
polysaccharide of Pseudomona aeruginosa Inhibited phagocytosis by blocking
mannose receptors on the surface of the macrophages (6). The results presented
here are in agreement with these previous reports. Capsulated M. dispar or
purified capsule inhibited the killing of S.maKescens or S.aureus by 2 logs
compared to the killing activity exhibited by untreated BAM or BAM treated with
unencapsulated M. dispar. From these results it can be concluded that the
presence of capsule, as a purified substance or in association with M. dispar cell
surfaces was linked with a diminished bactericidal activity of the BAM.
A previous report showed that M. dispar exerts a immunosuppressive effect
when injected in combination with other antigens (16). It also was reported that
M. dispar exerts an inhibitory effect on the mixed lymphocyte response to
mitogens. It also was suggested that this suppressive effect coukj be related to
the avidity with which this mycoplasma species adheres to the host cell
membrane, resulting in alteration in the membrane /receptor structures on the
host cell and the production of a suppressive factor (11,16). In the present
report we demonstrate that encapsulated M. dispar or purified capsule did not
induce TNF or IL-1 production by BAM, in contrast to what was seen when
unencapsulated M. dispar v/as used. In addition, encapsulated mycoplasma or
103
purified capsule did not Induce glucose consumption by BAM, indicating that
these cells were not activated. In contrast, activation of macrophages was
observed when macrophages were incubated with unencapsulated M. dispar or
with endotoxin.
It is possible that the lack of activation of BAM treated with purified capsule,
only reflects the inability of this material to interact with macrophages, but the
reduced bactericidal activity observed in BAM cultured with purified capsule,
indicates that a mechanism of inhibition was operative. It was observed that
encapsulated M. d/spar induced slightly higher levels of TNF production and
glucose consumption compared with that of purified capsule. These differences
can be explained by the small percentage of unencapsulated mycoplasmas that
are detected when expression of mycoplasma capsule was induced by
coculture (3). It also was shown that IIL-1 production by BAM stimulated with
heat-killed encapsulated mycoplasmas was significantly higher than that
observed with live encapsulated M. dispar. It is possible that during the process
of heat-killing, capsule material was lost from the surface of the mycoplasma
unmasking regions of the membrane that couki react with the IL-1 receptor in
the macrophage cell surface. It also was possible that fractions of disrupted
membrane, released during heat killing, could react with BAM causing IL-1
production. These differences were not detected with TNF production, which
indicated that TNF and IL-1 induction in BAM could be dissociated.
The inhibitory effect of encapsulated mycoplasmas or capsular material on the
induction of TNF and IL-1 production or glucose consumption, indicated that
this material was blocking the activation of the macrophages. It was previously
shown with other mycoplasma species that induction of TNF production resulted
r_.
104
from the interaction of a mycoplasmal membrane lipoprotein with the surface of
the macrophage (25). It is possible therefore, that the capsule material of M.
d/spar could mask a similar lipoprotein or its receptor in the surface of the
macrophage, abrogating their activation.
It was previously shown that Mycoplasma mycoides subsp. mycoides (LC)
induced TNF production, and that injection of TNF in goats reproduced the
disease caused by this mycoplasma species (23). It also was postulated that
Mycoplasma bovis may induce production of cytokines and the effect of these
could explain in part the necrotic lesions and lymphocyte accumulations
detected in the lung of naturally infected calves (11). In the present report we
show that encapsulated M. dispar diô not induce production of IL-1 and TNF or
glucose consumption by BAM. The lack of macrophage activation coukJ explain
the poor immunogenic reactivity induced by M. dispar and also the
characteristic interstitial alveolitis without peribronchial lymphocyte
accumulations caused by this mycoplasma species (13,16).
The inhibitory effect exerted by the capsular material or encapsulated M. dispar on
killing capacity of the BAM to secondary bacteria and the diminished activation and
secretion of cytokines, defines the important influence that the infection by M.
dispar may have on the development of severe pneumonic processes. We are
aware of the fact that these results describe the interaction of M. d/spar with BAM
under in vitro conditions, and these may not completely reflect the in vivo situation.
However, the immunosuppressive effects previously reported (16) and the results
presented here, can be taken together to help define the role that M. dispar has on
the development of pneumonic conditions in cattle.
105
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7. Cassen, G. H., and J. G. Woodward. 1989. Induction of class II MHC
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12. Goidstein, E., and H. C. Bartiema. 1977. Role of the alveolar
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107
13. Gourlay, R. N., C. J. Howard, L. H. Thomas, and S. G. Wyld.
1979. Pathogenicity of some Mycoplasmas and Ureaplasma species in the
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14. Henson, P. M., G. I. Larson, J. E. Henson, S. L. Newman, R. A.
Musson, and C. S. Leslie. 1984. Resolution of pulmonary
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15. Horwitz, M. 1982. Phagocytosis of microorganisms. Rev. Infect. Dis.
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16. Howard, C. J., and R. N. Gourlay. 1983. Immune response of calves
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Microbiol. 8:45-56
17. Howard, C. J., and G Taylor. 1985. Humoral and cell-mediated
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Exp. Med. 133:231-259.
108
19. Knudtson, W. U., D. E. Reed, and G. Daniels. 1985.
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Microbiol. 11:79-91.
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^
22. Rosenbusch, R. F., 1983. Influence of mycoplasma infection on the
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109
25. Sher, T., S. Rottem, and R. Gailily. 1990. Mycoplasma capricolum
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110
GENERAL CONCLUSIONS
The main objective of the present work was to elucidate the effect that the
infection with Mycoplasma cf/spar infection exerts on the activity of two main
components of the defense mechanism of the bovine respiratory system (e.g.,
tracheobronchial clearance and alveolar macrophage). In the first manuscript,
the focus was to determine the effect that M. dispar has on the mucociliary
activity of calves. The approach used was to compare the tracheobronchial
elimination of a bacterial marker {Serratia marcescens) before and after the
infection with M. dispar on the same calves. In order to assure that inoculation
with the bacterial marker does not in itself influence their rate of elimination, the
tracheobronchial elimination of the bacterial marker in calves non-infected with
M. dispar was determined using the same sampling routine and route
frequency of inoculation.
It was found that before the infection with M. dispar \he elimination
showed a peak around 2 hours after inoculation with the marker, and by 4 hours
only a few colonies were detected. In contrast, after infection with M. dispar, the
rate of elimination was steady and by 4 hours after inoculation with the marker,
the titer of the bacterial marker was 4 logs higher than that detected in the preinfection status calves. In addition, the bacterial marker was isolated in 83% of
the lungs from to the Md/spar infected group, compared with no isolation in the
non-mycoplasma infected calves. This can be taken as indication that the
clearance of the marker was incomplete 24 hours later in the M. dispar
inoculated group of calves, which was in contrast with that observed in the nonmycoplasma inoculated group of calves.
I'
111
The study was also extended to include histopathology and scanning
electron microscopic examination of the lung tissues. The main lesion
observed centred in the small and medium size airways . In these, the ciliated
ephitelium presented sign of degenration with shinkage of the cytoplasm,
pyknosis and sloughing of the cell. The lumen on'these airways contained
PMN's and mononuclear cells, and a moderate infiltration of PMN's and
macrophages were seen in the submucosa..
The SEM observation of these airways showed loss of ciliated epithelial
cells with patching distribution and presence of inflammatory cells along the
mucosa of small airways. No lesions of the respiratory epithelium was detected
in the large airways and no lesions were detected in the lungs of the calves not
exposed to M. dispar
The results obtained in this part of the project indicated that the infection
with M. dispar was associated with a reduction in the tracheal elimination of the
bacterial marker. This reduction was also associated with damage to the
ciliated epithelium of small airways, which was observed only in the M. dispar
exposed calves.
The second part of the project was to assess the effect that M. dispar
infection exerts on bovine alveolar macrophages (BAM). The approach used
was the evaluation of phagocytic functions, secretory functions, and activation
capacity of BAM infected with encapsulated, unencapsulated M. dispar and
purified capsule. It was found that phagocytosis did occur when
unencapsulated or encapsulated M. dispar was exposed to BAM in absence of
specific antibodies. The same results were observed when encapsulated M.
dispar was exposed to BAM in presence of antiserum against unencapsulated
112
M.dispar, or when unencapsulated mycoplasmas were exposed to BAM In
presence of antl-capsule antibodies. In contrast, reduction in the titer of the
mycoplasma was detected when unencapsulated mycoplasmas were
incubated with BAM in presence of specific antiserum, but complete reduction in
the titer was detected when encapsulated M. dispar was incubated with
macrophages in presence of specific anti-capsule antibodies. These results
indicated that the capsule exerts an anti-phagocytic effect, and that this effect
can be overcome by the addition of specific anti-capsule antibodies. The anti­
phagocytic effect of microbial capsules is not a novel discovery, since the same
effect was described long ago in many pathogenic microorganisms, but what
was interesting in the present work, was the finding that the encapsulated
mycoplasma or the purified capsular material also inhibited the killing of other
microorganisms added to the system. In effect, when macrophages were pretreated with encapsulated M. dispar or purified capsule and subsequently
exposed to Staphyloccocus aureus or Serratia marcescens in presence of
specific antibodies, a significant reduction of the killing activity of the
macrophages was detected, compared with that observed in non-mycoplasma
or capsule treated BAM. This effect was not detected when BAM were pretreated with unencapsulated M dispar.
It was also shown that encapsulated M. dispar or purified capsular
material did not emhance glucose consumption, production of TNF, or IL-1. In
contrast, when BAM were treated with unencapsulated M. dispar, increase in
glucose consumption, and production of TNF as well as IL-1 was observed.
However, some differences were observed when these results were compared
with that of heat-inactivated encapsulated or non-encapsulated M. dispar. For
113
instance, IL-1 production by BAM stimulated with heat-inactivated encapsulated
mycoplasmas was significant higher than that observed with live encapsulated
M. dispar. A possible explanation is that the heat-inactivation caused the
release of the capsule material from the surface of the mycoplasma leaving
exposed regions of the membrane that could react with the macrophage cell
surface. It is also possible that fractions of disrupted membrane, released
during this process, could react with BAM causing IL-1 production. These
differences were not detected with TNF production or glucose consumption,
which indicated that the inhibition of these functions operates by a different
mechanism .
It was previously shown with other mycoplasma species that induction of
TNF resulted from the interaction of a mycoplasmal membrane lipoprotein with
the surface of the macrophage (Sher et al, 1990). It is possible therefore, that
the capsule material of M. cf/spar could mask a similar lipoprotein or its receptor
in the surface of the macrophage, avoiding their activation. However, this can
not explain the inhibitory effect exerted by the capsular material to the killing
activity of BAM to a second microorganism such as S. marcescens or S. aureus
or the inhibitory effect exerted by the capsule on the activation of the
macrophages. This indicates that the action of the capsular material is not
simply mechanical by masking the surface of the mycoplasmas, but also by
inhibiting macrophage functions.
In conclusion, the work presented here indicates that M. d/spar exerts
negative effects on two major defensive elements of the bovine respiratory
system, and even when these effects can be limited by the relatively slow grow
of the mycoplasma in the lungs, they are important enough to reevaluate the
114
role of M. dispar'm the initiation and development of mixed respiratory infections
in cattle under severe stress.
115
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ACKNOWLEDGEMENTS
I wish to express my gratitude to my major professor Dr. Ricardo
F.Rosenbusch and members of my PhD committee for their patience, guidance,
support, and contribution during the execution of the present thesis project.
I extend my thanks to the people in our laboratory for their support and
technical assistance.
A very special appreciation is directed to my wife Eugenia Graciela and
our four kids, Camila Otilia, Antonio Mariano, Soledad, and Juan Manuel for
their continuous sacrifices, trust, and support given through this thesis wori<.