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Division of Infectious and Immunological Diseases, British Columbia’s Children’s Hospital,
Canadian Bacterial Diseases Network, and Departments of
Paediatrics,
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Pathology, and
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Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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) in a serum-free environment is a crucial
first step in the pathogenesis of this facultative intracellular
pathogen. We present data demonstrating that freshly explanted alveolar
M do not efficiently bind M. tuberculosis in a
serum-free system, although a small subpopulation of these M
(10–15%) can bind mycobacteria. In contrast, almost 100% of a
peritoneal M population bind mycobacteria under the same conditions.
The poor binding of mycobacteria by alveolar M does not reflect a
general inability to associate with particles; binding and ingestion of
latex beads and zymosan particles were comparable with that seen with
peritoneal M. Resident alveolar M did not efficiently bind
mycobacteria in the presence of serum and expressed poorly several M
surface receptors, including CR3. Furthermore, we demonstrate that
bovine surfactant protein A does not enhance the association of
M. tuberculosis with alveolar M. Differentiation of
alveolar M in vitro resulted in increased expression of M surface
receptors and an increased capacity to bind mycobacteria in the
presence and absence of serum. Evidence is presented that opsonic
binding of M. tuberculosis by differentiated alveolar M
is mediated by complement and CR3, and that the poor binding by
resident alveolar M is due to their poor expression of CR3. The
receptor mediating nonopsonic binding of M. tuberculosis to
differentiated alveolar M was not unequivocally identified in this
study, but could also be CR3. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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3 and located primarily
in the apical regions of the lung. To better understand the interaction
of M. tuberculosis with its host cell, investigators have
developed models that utilize M obtained from humans and mice.
Presumably, the best model would be the human alveolar M.
Unfortunately, due to the constant exposure of human lungs to inhaled
particles and microbes, the normal healthy human lung will undoubtedly
have a mixed population of cells in the pulmonary lavage, including
resident alveolar M, elicited (monocyte-derived) M, and possibly
immune activated M. We have shown previously that the phenotype of a
M greatly affects its interaction with mycobacteria (1). Thus, to
fully understand the pathogenesis of mycobacteria in the different lung
M populations, it will be necessary to study the interaction of
mycobacteria with pure populations of M. Defined, pure, or enriched
M populations can be obtained readily from animal models following
experimental manipulations (e.g., the introduction of phlogistic agents
into the lung will induce a population of predominantly elicited M).
As the association of mycobacteria with human and murine M is known
to be mediated by similar mechanisms (1, 2), we consider the mouse
alveolar M to be a representative model for the human alveolar M.
Moreover, the mouse model facilitates the acquisition of populations of
M of a desired phenotype (resident, elicited, immune activated) and
also favors reproducibility between samples. However, it will still be
of importance to study the human pulmonary lavage M population as a
representation of the in situ situation probably encountered by an
inhaled mycobacterium.
Whereas opsonic phagocytosis of mycobacteria appears to be mediated by
complement receptors binding to complement components fixed to the
mycobacteria (3, 4, 5, 6), the process of nonopsonic binding of
mycobacteria is understood poorly. It has been suggested that
mycobacteria can bind to M nonopsonically via the vitronectin
receptor, CR1, CR3, Fc
R, transferrin receptor, mannose receptor, or
a glucan receptor (4, 5, 6, 7, 8). We have shown previously that the nonopsonic
binding of M. tuberculosis to mouse peritoneal M is
mediated predominantly by an epitope within CR3 distinct from that
which binds iC3b (1), possibly the glucan binding site of CR3 recently
described (9). These results have been confirmed using CHO cells
transfected with human CD11b/CD18 (2). In contrast, little is known
about the interaction of mycobacteria and alveolar M (8, 10, 11),
the cell with which the bacteria initially associate.
We have investigated the ability of murine alveolar M to bind
M. tuberculosis and have related those binding
characteristics to the expression of M surface receptors. We have
compared the alveolar M to the murine peritoneal M, as we have
previously characterized the latter cell’s ability to bind M.
tuberculosis (1). We describe a number of observations concerning
the interaction of alveolar M and M. tuberculosis: 1)
nonopsonic binding of M. tuberculosis and other mycobacteria
by resident alveolar M is extremely poor; 2) resident alveolar M
do not efficiently bind mycobacteria (or other particles) in the
presence of normal serum, presumably because they do not express
receptors for serum opsonins (12–17 and this study) or because there
are minimal amounts of serum in the lung (18), consequently creating a
redundancy for receptors on alveolar M that recognize serum
opsonins; 3) SP-A does not enhance binding of M.
tuberculosis to resident alveolar M; and 4) differentiation of
resident alveolar M results in an increase in their ability to bind
mycobacteria nonopsonically and opsonically, correlating with the
increased expression of a number of M surface receptors.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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M. tuberculosis, strain Erdman (Trudeau Mycobacterial Collection (TMC) No. 107; American Type Culture Collection (ATCC) No. 35801, Rockville, MD); M. tuberculosis, strain H37Rv (TMC 102, ATCC 27294); M. tuberculosis, strain H37Ra (TMC 201, ATCC 25177); and Mycobacterium bovis BCG, strain Pasteur (TMC 1011, ATCC 35734) were grown and stored as previously described (1).
Macrophages
Resident and thioglycollate-elicited murine peritoneal M from
6- to 8-wk-old female BALB/c mice were isolated and maintained as
previously described (1). To obtain murine alveolar M, 6- to
8-wk-old female BALB/c mice were injected with a lethal dose of
pentabarbitol, and the heart and lung were dissected out into cold PBS
and washed free of blood. A 22G catheter (Critikon, Tampa, FL) was
inserted into the trachea and tied off. While supporting the lungs in a
jig, they were lavaged with 10 ml of PBS, containing 0.1% EDTA, in 1-
to 2-ml aliquots (preliminary studies showed that the inclusion of EDTA
increased the yield of M, but did not affect subsequent M
functions). Pooled washings were pelleted and washed with supplemented
RPMI (RPMI 1640 medium (Life Technologies, Grand Island, NY) plus 10%
v/v FCS (Life Technologies), 10 mM L-glutamine, and 10
mM sodium pyruvate). The final pellet was resuspended in supplemented
RPMI at 5 x 105/ml, and 100-µl aliquots were placed
onto 13-mm coverslips. The cells were allowed to adhere for 1 h at
37°C in 5% CO2, washed, and used immediately in
experiments or, if being maintained in vitro, transferred to 24-well
plates containing 1 ml supplemented RPMI and reincubated at 37°C in
5% CO2 (if kept past day 4, the media were replenished on
that day by removing 500 µl supernatant and adding 500 µl
supplemented RPMI).
Particles for probing M receptors
The function of FcR was investigated using EIgG, and
complement receptors were identified using EIgMC', whereas zymosan
particles (prepared from bakers yeast, Sigma, St. Louis, MO) were used
to probe for lectin-like phagocytic receptors, all as described
previously (1).
In vitro assay for binding of particles to M
Adherent peritoneal and alveolar M were washed twice using
binding medium (19), 138 mM NaCl, 8.1 mM
Na2HPO4, 1.5 mM
KH2PO4, 2.7 mM KCl, 0.6 mM CaCl2, 1
mM MgCl2, and 5.5 mM D-glucose. A 500-µl
aliquot of binding medium was added to each well, and the cells were
acclimatized for 10 min at 37°C, 5% CO2. In some
experiments, the M were incubated for an additional 15 min in the
presence of mAb, bSPA (a gift from Dr. F. Possmeyer and K. Inchely,
University of Western Ontario, London, Ontario), or PMA (Sigma) in
binding medium. The overlay was then removed and replaced with 250 µl
of binding medium containing any material to be tested in the system
and 250 µl of binding medium containing particles, as previously
described (1). Particles and M were then gently rocked for 1 h
at 37°C, 5% CO2 (Nutator; Becton Dickinson, Mountain
View, CA), followed by an additional 2 h stationary at 37°C, 5%
CO2. The monolayers were then processed as previously
described (1). In experiments comparing the binding characteristics of
M maintained in vitro for different times (e.g., freshly explanted
alveolar M and alveolar M maintained in vitro for 4 days), the
M were set up on different days so that their binding
characteristics could be compared on the same day using the same
preparation of particles.
The binding of zymosan, EIgG, and EIgMC' was assessed by counting the
total number of particles associated with 100 M (Association Index),
whereas the association of mycobacteria with M was estimated by
counting the percentage of M with 0, 1 to 5, 6 to 10, or >10
associated bacteria, as previously described (1). The purpose of these
studies was to identify the binding mechanisms involved in
M/mycobacteria interactions, so no attempt was made to differentiate
attachment and ingestion.
In some experiments, the association of M. tuberculosis with
M in the presence of serum was assessed. Serum from BALB/c mice,
prepared as described previously (20), was added to M and
mycobacteria to give a final concentration of 1% serum.
Heat-inactivated serum was prepared by heating at 56°C for 30
min.
FACS analysis
The expression of surface Ags on alveolar M was investigated
using FACS analysis. Cells from five mice were pooled and incubated in
supplemented RPMI in a Teflon container at 37°C, 5%
CO2 for 4 days before processing. On day 4, the cells
were gently resuspended and pelleted by centrifugation. At the same
time, cells from three mice were pooled and processed immediately after
lavage. Both freshly isolated and day 4 cells were washed in wash
buffer (RPMI with 2% v/v FCS, 20 mM HEPES, and 20 mM azide),
resuspended in wash buffer, processed for FACS, as previously described
(1), and read on a Becton Dickinson FACScan.
The mAbs used in this study were, unless stated otherwise, prepared
from hybridomas obtained from ATCC and were as follows: M1/9.3.4.HL.2
(rat IgG2a, 
CD45 (21)); F4/80 (rat IgG2b, murine M marker
(22)); 2.4G2 (rat IgG2b, mouse FcRII (23)); M1/70.15.11.5.HL
(rat IgG2b, mouse CD11b, recognizing an epitope that binds iC3b
(21)); 5C6 (rat IgG2b, mouse CD11b, recognizing an epitope that is
involved in M attachment and spreading, but is distinct from that
which binds iC3b (24, 25)); M17/4.4.11.9 (rat IgG2a, mouse CD11a
(26), was obtained from Developmental Studies Hybridoma Bank);
M18/2.a.12.7 (rat IgG2a, mouse CD18 (26), was obtained from
Developmental Studies Hybridoma Bank); 8C12 (rat IgG from nude mouse
ascites, mouse CR1 (27), a generous gift from Dr. T. Kinoshita,
Osaka University, Osaka, Japan); 2F8 (rat IgG2b, murine scavenger
receptor (28), a generous gift from Dr. S. Gordon, University of
Oxford, Oxford, England); and N418 (hamster IgG, mouse CD11c (29)).
Controls for the mAb were as follows: SFR8-B6 (30) for the rat IgG and
1% normal hamster sera in wash buffer for N418. Following incubation
with the primary Ab, cells were incubated with donkey anti-rat IgG
FITC, or goat anti-hamster IgG FITC (Jackson ImmunoResearch, West
Grove, PA), as appropriate, and processed as previously described
(1).
Statistical analysis
Data are expressed as mean ± SEM. Student’s t
test for independent means was used to evaluate M binding;
p < 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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: a comparison with peritoneal M
Confirming earlier studies (1), we demonstrated that freshly
explanted murine, resident peritoneal M readily bind M.
tuberculosis (Fig. 1
). In contrast,
freshly explanted, resident alveolar M did not bind M.
tuberculosis efficiently (Fig. 1); the percentage of the cell
population binding mycobacteria was significantly less
(p < 0.001) than that seen with peritoneal
M. Assessment of the distribution of mycobacteria within the M
populations demonstrated that, in addition to very few freshly
explanted, resident alveolar M binding M. tuberculosis,
those M that did bind mycobacteria bound only one to five bacteria.
In contrast, the majority of freshly explanted resident peritoneal M
bound mycobacteria and, in addition, bound more bacteria (approximately
40% of the population bound more than five bacteria). Furthermore, the
binding seen with alveolar M was obtained using an MOI 10 times
higher than that used for peritoneal M. When mycobacteria were added
to resident peritoneal M at an equal MOI as had been added to
resident alveolar M (approximately 500:1), almost 100% of the
population bound >10 bacteria (data not shown).
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populations in their ability to bind latex
particles (Fig. 1
). This demonstrated that the poor binding of
mycobacteria by alveolar M was not the result of a global inability
to bind particles, and also demonstrated that the procedure for
obtaining the M had not damaged them.
The poor binding of M. tuberculosis, strain Erdman, by
resident alveolar M was not limited to that strain; binding of
M. tuberculosis, strains H37Rv and H37Ra, and of M.
bovis BCG to alveolar M was equally poor (data not shown).
Binding of M. tuberculosis, EIgG, EIgMC', and zymosan by
pulmonary and peritoneal M maintained in vitro for up to 7 days
During the in vitro maintenance of resident alveolar M, the
ability to bind M. tuberculosis was enhanced transiently,
peaking at about day 4 and then declining (Fig. 2A). Over the same time
period, resident peritoneal M were observed to efficiently bind
M. tuberculosis until day 4, whereupon this ability began to
decline (Fig. 2A).
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during their maintenance over 7 days. Freshly explanted resident
peritoneal M bound all of the particles efficiently, with more than
90% of the population binding each particle. Over a 7-day period,
peritoneal M lost their ability to bind EIgMC' (Fig. 2C), while their ability to bind EIgG (Fig. 2B) and zymosan (Fig. 2D) increased
initially and then returned to base level. Fresh alveolar M did not
bind EIgG (Fig. 2B) or EIgMC' (Fig. 2C) as efficiently as they did zymosan (Fig. 2D) with, respectively, approximately 40, 10, and
90% of the population binding each of these particles. Their ability
to bind zymosan and EIgG increased with time in culture, but their
ability to bind EIgMC' remained negligible over the 7 days. Over the
7-day period, no correlation was seen between the binding of M.
tuberculosis by alveolar M and their ability to bind control
particles.
Binding and ingestion of zymosan by alveolar M were particularly
efficient. More than 90% of freshly explanted alveolar M ingested
zymosan, and after 4 days maintenance in vitro, this had increased to
100%. The number of zymosan particles ingested by each alveolar M
at day 4 was between 20 and 30, resulting in the M appearing to be
completely full of zymosan. The binding and ingestion of zymosan by
alveolar M further demonstrated that the poor binding of
mycobacteria by alveolar M was not the result of a general inability
to bind particles.
Induction of particle binding by treatment of M with PMA
Elicited peritoneal M express CR3 in a nonfunctional state that
can be activated to become functional by treatment with PMA (1, 31). We
investigated the possibility that alveolar M could be stimulated
with PMA to induce functional binding of particles by CR3. In these
experiments, we used thioglycollate-elicited peritoneal M as a
positive control for the action of PMA on M. As previously reported
(1), treatment of thioglycollate-elicited peritoneal M with PMA
induced a significant (p < 0.05) increase in
EIgMC' binding, but not (p > 0.05) M.
tuberculosis binding (Table I). PMA
did not induce an increase in the binding of EIgMC' or of mycobacteria
by freshly explanted alveolar M (Table I). Day 4 alveolar M were
similarly unaffected by PMA (Table I). This experiment shows that
alveolar M do not express CR3 in a nonfunctional state.
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with bSP-A
There are reports describing how the binding of some bacteria by
alveolar M can be increased by treatment with SP-A (32, 33, 34). We
tested whether bSP-A could enhance the binding of M.
tuberculosis to murine, resident alveolar M (Table II). No increase in binding of M.
tuberculosis by alveolar M was observed after treatment with
bSP-A. Identical treatment of resident peritoneal M appeared to
induce an observable, albeit statistically insignificant, increase in
binding of mycobacteria (Table II). Binding of zymosan by both resident
alveolar and peritoneal M appeared to be increased by bSP-A
treatment (Table II), although this increase was not statistically
significant. Binding of EIgMC' by alveolar M was unaffected by
bSP-A, whereas peritoneal M demonstrated a decrease in their ability
to bind EIgMC' following bSP-A treatment, although this decrease was
not statistically significant.
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surface ligands with alveolar
M
FACS analysis of alveolar M (Table III) revealed that freshly explanted,
resident cells expressed high levels of CD45 (>20 times that of the
control background fluorescence) and intermediate levels of CD18,
CD11a, CD11c, and FcR (2–5 times that of the control fluorescence).
Expression of CD35, CD11b, the M marker F4/80, and the scavenger
receptor 2F8 by resident alveolar M was very low (less than
twice that of the control fluorescence). Interestingly, the expression
of the epitope within CD11b, which is recognized by 5C6, was expressed
strongly by a small subpopulation (approximately 10%) of freshly
explanted alveolar M (data not shown), whereas the epitope
recognized by M1/70 was not expressed at all.
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maintained in vitro for 4 days (Table III) showed that mAb-reactive receptor expression had increased during
the maintenance of the phagocytes. High levels of CD45 were still
expressed. Surprisingly, the expression of CD11a had declined. The
expression of CD11b (both epitopes) and CD11c had increased to more
than 5 times that of the control values. While all of the other ligands
investigated were expressed at higher levels than at day 0, expression
still remained relatively low (less than 5 times that of control
levels). More than 90% of the alveolar M expressed the 5C6-reactive
epitope after 4 days in culture (data not shown). It should be noted,
however, that at no time did the expression of surface receptors on
alveolar M approach the levels seen for resident peritoneal M
(Table III) (1).
Inhibition of binding of M. tuberculosis to day 4
alveolar M by mAb recognizing M receptors
The demonstration that day 4 alveolar M were better able to
bind mycobacteria (Fig. 2) than were freshly explanted alveolar M
and that this ability coincided with an increase in surface receptor
expression (Table III) suggested that the increased binding was
mediated by these receptors. To test this, mAb recognizing M
receptors were investigated for their ability to inhibit the enhanced
binding of mycobacteria by day 4 alveolar M. Binding was
significantly (p < 0.05) inhibited by six of
the eight mAb tested (recognizing CR3 (not the iC3b epitope), CR1, LFA,
CR4, FcR, and the scavenger receptor) and appeared to be slightly
suppressed by the remaining two mAb (recognizing CD18 and the iC3b
epitope of CR3), albeit at a statistically insignificant
(p > 0.05) level (Fig. 3). In contrast to these results, no
significant inhibition (p > 0.05) of
mycobacteria binding to freshly explanted alveolar M was observed in
the presence of the same mAb (data not shown).
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in the presence of serum
Binding of M. tuberculosis to freshly explanted
resident peritoneal M was significantly (p
< 0.001) enhanced in the presence of 1% normal murine serum. This
increase was dependent on a heat-labile component of serum, as
heat-inactivated serum did not significantly (p
> 0.05) enhance binding (Fig. 4). Normal
serum (1%) had no significant effect (p >
0.05) on the binding of M. tuberculosis to freshly
explanted, resident alveolar M (Fig. 4), whereas the binding of
M. tuberculosis by day 4 alveolar M was significantly
(p < 0.001) increased in the presence of 1%
whole, but not (p > 0.05) heat-inactivated,
serum (Fig. 4). Serum-mediated increases in binding were manifested as
an increase in the percentage of the M population binding
mycobacteria (Fig. 4) and as an increase in the number of mycobacteria
bound; the percentage of peritoneal M binding >10 mycobacteria
increased from 39 ± 6.8 in serum-free controls to 94.8 ±
1.4 in the presence of serum, and for day 4 alveolar M increased
from 4 ± 0.6 to 83.8 ± 3.5, each significant
(p < 0.001) increases. The increased binding
of M. tuberculosis by day 4 alveolar M in the presence of
1% serum was inhibited significantly (p <
0.001) by mAb recognizing CR3, but not those recognizing other
receptors (CR1, CR4, FcR) for serum opsonins (Fig. 5).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and that this event
initiates an infection, the outcome of which is then regulated by such
factors as the virulence of the mycobacteria and the inherent
microbicidal power of the alveolar M (35). Our data demonstrate that
the supposition that mycobacteria are ingested readily by the alveolar
M should be modified. We have shown previously that elicited and
activated peritoneal M are less able to bind and ingest mycobacteria
nonopsonically than are resident peritoneal M (1). In this study, we
extend those observations to show that resident alveolar M are
considerably less able to bind M. tuberculosis than are
resident peritoneal M. This inferior binding was evident for both
nonopsonic and opsonic binding of mycobacteria, but was not the result
of a generalized inability of alveolar M to bind and ingest
particles: their ability to ingest latex beads and zymosan was
comparable with that of peritoneal M.
Freshly explanted alveolar M were also poorly able to bind particles
coated with IgG or iC3b. This is in agreement with earlier reports
(12, 13, 14, 15, 16, 17) and suggests a lack of functional receptors for serum
opsonins. This failing appeared to be due to a lack of expression of
receptors rather than a lack of receptor function, as FACS analysis of
freshly explanted alveolar M showed minimal expression of receptors
for IgG (FcR) or C3b/iC3b (CR1/CR3/CR4). Furthermore, treatment of
alveolar M with PMA did not induce an increase in the binding of
EIgMC'; PMA has been shown to increase binding of EIgMC' by some M
populations due to the induction of functional binding of iC3b by
receptors expressed on the M surface in a previously nonfunctional
state (1, 31, and Table I). The nonopsonic binding of bacteria by
murine alveolar M has not been studied extensively, but appears to
be uniformly poor; they do not bind Listeria monocytogenes
(13), Escherichia coli, Pseudomonas aeruginosa,
Staphylococcus aureus (32), M. bovis BCG (36), or
M. tuberculosis (this study) very efficiently. In contrast
to their poor binding of bacteria, alveolar M avidly bind fungal
pathogens (37, 38), presumably by the mechanism with which they bind
zymosan.
Analysis of surface receptor expression of resident alveolar M from
humans and other primates shows low levels of CD11b and CD35 with
relatively higher expression of CD11a, CD11c, and CD18 (39, 40, 41, 42, 43, 44), just
as we demonstrate in this work for resident murine alveolar M. Human
alveolar M, like their murine counterpart, bind bacteria poorly,
both in the absence and presence of normal serum. Binding of P.
aeruginosa by human alveolar M was minimal and was not enhanced
by normal serum (40). Similarly, the binding of M.
tuberculosis to human alveolar M has been shown to be very
poor; using an infection ratio of 100 bacteria to 1 M in the
presence of 2% normal serum, only 20% of a M population ingested
an average of 3 mycobacteria each (10). In contrast, the association of
M. tuberculosis with human MDMs under similar conditions
(MOI of 100 to 1 in the presence of 1% serum) results in 95% of the
M binding in excess of 10 bacteria each (R. W. Stokes,
unpublished observations).
The maintenance of alveolar M in vitro resulted in an increase in
surface expression of a number of receptors (F4/80, FcR, CR3, CR4,
and scavenger receptor), as detected by FACS, which corresponded with
the increased binding of M. tuberculosis by these M. It
is possible that the day 4 alveolar M are representative of an in
vivo M population that has differentiated after recent ingress into
the lung. However, in the same way that there is uncertainty as to
whether the in vitro differentiated human MDM represents any population
of M found in vivo, we cannot be sure that the in vitro
differentiated alveolar M truly represent a possible M phenotype
found in vivo. This question could be addressed by characterizing the
phenotype of M obtained from the lungs of mice infected with
mycobacteria or treated with phlogistic agents and is currently under
investigation.
M. tuberculosis binds nonopsonically to resident peritoneal
M predominantly via an epitope within CR3 distinct from that which
binds iC3b (1, 2). If resident alveolar M use the same mechanism,
their poor binding of M. tuberculosis could be explained by
their low level of CR3 expression, and consequently, the increased
binding of M. tuberculosis by alveolar M maintained in
vitro for 4 days could be due to their increased expression of CR3.
However, inhibition of nonopsonic binding of mycobacteria to alveolar
M by mAb recognizing M receptors did not support the above
contention. Binding by freshly explanted alveolar M was not
inhibited significantly by any mAb, but as initial binding levels of
mycobacteria by resident alveolar M were so low, it is likely that
inhibition studies were unresolvable. With day 4 alveolar M, all of
the mAb tested appeared to inhibit binding to some degree, ranging from
25 to 50% of the control value. This may reflect a diverse array of
receptors mediating the nonopsonic binding of M.
tuberculosis to alveolar M, as has been previously suggested
(8). Alternatively, it may indicate nonspecific effects of the mAb
interacting with M (45), thus demonstrating the problem with relying
on mAb inhibition as the only indication of receptor involvement in the
binding of particles to M. We consider the latter explanation to be
the most likely, as inhibition of binding appeared to be equally
effective with mAb that recognized receptors that had been up-regulated
(e.g., CR3) or down-regulated (e.g., LFA) during in vitro maintenance.
It is hard to reconcile the importance of a receptor in binding
mycobacteria (as shown by the mAb inhibition) with the observation that
the receptor is down-regulated over the same period that binding of
mycobacteria is up-regulated. Thus, we consider that the receptor that
mediates the nonopsonic binding of mycobacteria to alveolar M has
yet to be determined.
Evidence that binding of M. tuberculosis to day 4 alveolar
M in the presence of serum is mediated by heat-labile complement
components that bind to CR3 was obtained. These results suggested that
complement fixes to the mycobacterial surface and mediates binding to
differentiated alveolar M CR3, as has been reported in other models
of serum-mediated binding of mycobacteria by M (3, 4, 5, 6). Other
receptors for complement components (CR1 and CR4) and the receptor for
IgG (FcR) did not appear to be involved in the serum
opsonin-mediated binding of M. tuberculosis to day 4
alveolar M. This is in contrast to a study on serum opsonin-mediated
binding of mycobacteria to human alveolar M in which CR4 was shown
to predominate, with CR1 and CR3 playing a minor role (10). However, in
another study (46), it was found that human alveolar M bind
complement-coated particles predominantly via CR3 with little
involvement of CR4. These contrasting results may reflect variation in
the constituent populations of M within the human alveolar lavages
of these two studies.
The observation that day 4 alveolar M bind only complement-coated
mycobacteria and not complement-coated SRBC, whereas peritoneal M
bind both (Figs. 4 and 2C) was a surprising
observation that we currently cannot explain. Our favored explanation
is that resident alveolar and peritoneal M express different
densities of CR3 on their surface (1 and Table III), which, coupled
with the difference in size of the SRBC and the mycobacteria, results
in the variation in binding. This possibility and other possibilities
are currently being investigated further. However, it must be
emphasized that the freshly explanted alveolar M binds neither SRBC
nor mycobacteria in the presence of serum.
It is possible that other, nonserum opsonins may mediate increased
binding of mycobacteria to resident alveolar M. One candidate, the
lung surfactant SP-A, has been suggested to increase the binding of
mycobacteria to human MDM and alveolar M (47) and to murine alveolar
M (34). SP-A is also reported to enhance the association of other
microorganisms with M in a species-dependent manner (32, 33, 38). We
found that bSP-A did not affect the binding of M.
tuberculosis to resident murine alveolar M, whereas binding to
peritoneal M was slightly increased (Table II). The difference
between our result and those of others may be due to the different
origins of the SP-A that was used. Alternatively, it may merely reflect
the way the data are presented; in one report, the binding of M.
tuberculosis by normal alveolar M was said to increase in the
presence of SP-A and was defined as an increase of 2% (from 10 to 12)
of the M-binding mycobacteria (34), whereas in another report,
binding of M. tuberculosis by alveolar M increased in the
presence of SP-A, but it was not clear how substantial was this
increase, as only percentage changes in binding were reported and not
the actual levels of binding (47). However, bSP-A was not without
effect on the binding characteristics of murine M. In agreement with
earlier studies using rat SP-A (16), we found that bSP-A inhibited
binding of iC3b-coated particles to peritoneal M. In addition, the
binding of zymosan by resident alveolar M (and peritoneal M) was
increased in the presence of bSP-A. We conclude from these experiments
that bSP-A has no affect on the binding of M. tuberculosis
to resident, murine alveolar M, but can increase binding of the
bacteria to other M populations. Thus, within the human alveolar
M population of mixed phenotypes, there may be a subpopulation of
M (comparable with MDM) that can bind mycobacteria opsonized
with SP-A.
Our observations have major implications for the pathogenesis of
M. tuberculosis in the lung. On deposition in the lung, an
inoculum of M. tuberculosis has been considered previously
to be avidly ingested by alveolar M (35). We now present evidence
that this is not so; a very small subpopulation of resident M will
ingest the mycobacteria nonopsonically. If the mycobacteria are able to
survive and replicate, they could induce the production of cytokines
from cells in the vicinity of the infection, which could result in the
differentiation of resident M and also in the deposition of elicited
M, along with accompanying serum at the site of infection.
Differentiated alveolar M (as represented by day 4 alveolar M in
this study) are better able to bind mycobacteria nonopsonically and
opsonically. Elicited M bind mycobacteria poorly in the absence of
serum opsonins (1), but strongly in the presence of normal serum (R. W.
Stokes, unpublished observations). Thus, in this study, we provide
further evidence for our contention (1) that the interaction of
mycobacteria with M is dependent on the phenotype of the M. This
interaction is impacted further by the presence or absence of opsonins.
Mycobacteria will therefore enter their host cell via a number of
possible receptor/ligand interactions, and the nature of this
receptor/ligand interaction will preferentially direct the bacteria to
certain M phenotypes. For example, if the bacterium is coated with
iC3b, it will direct it away from resident alveolar M, but toward
differentiated alveolar M and elicited M. We would suggest that
the survival and subsequent pathogenesis of a mycobacterium are
affected by the precise nature of the interaction of a bacterium with
its host cell and by the phenotype of the M that ingests that
bacterium.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Richard W. Stokes, Department of Pediatrics, The Research Institute, 950 West 28th Avenue, Vancouver, British Columbia, V5Z 4H4 Canada. E-mail address:
3 Abbreviations used in this paper: M, macrophage; BCG, bacille bilié de Calmette-Guérin; bSP-A, bovine surfactant protein A; EIgG, sheep red blood cells coated with immunoglobulin G; EIgMC', sheep red blood cells coated with immunoglobulin M and inactivated complement component C3b; iC3b, inactivated complement component C3b; MDM, monocyte-derived macrophage; MOI, multiplicity of infection; SP-A, surfactant protein A.
Received for publication August 22, 1997. Accepted for publication February 4, 1998.
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1
mycobacterium or 
) and peritoneal (•) M
