␤2 Integrins Are Involved in Cytokine Responses to Whole
Gram-Positive Bacteria1
Maria Cuzzola,* Giuseppe Mancuso,* Concetta Beninati,* Carmelo Biondo,*
Francesco Genovese,* Francesco Tomasello,* Trude H. Flo,† Terje Espevik,† and
Giuseppe Teti2*
Proinflammatory cytokines have an important pathophysiologic role in septic shock. CD14 is involved in cytokine responses to a
number of purified bacterial products, including LPS. However, little is known of monocyte receptors involved in cytokine
responses to whole bacteria. To identify these receptors, human monocytes were pretreated with different mAbs and TNF-␣ was
measured in culture supernatants after stimulation with whole heat-killed bacteria. Human serum and anti-CD14 Abs significantly
increased and decreased, respectively, TNF-␣ responses to the Gram-negative Escherichia coli. However, neither treatment influenced responses to any of the Gram-positive bacteria tested, including group A and B streptococci, Listeria monocytogenes, and
Staphylococcus aureus. Complement receptor type III (CR3 or CD18/CD11b) Abs prevented TNF-␣ release induced by heat-killed
group A or B streptococci. In contrast, the same Abs had no effects when monocytes were stimulated with L. monocytogenes or
S. aureus. Using either of the latter bacteria, significant inhibition of TNF-␣ release was produced by Abs to CD11c, one of the
subunits of CR4. To confirm these blocking Ab data, IL-6 release was measured in CR3-, CR4-, or CD14-transfected Chinese
hamster ovary cells after bacterial stimulation. Accordingly, streptococci triggered moderate IL-6 production (p < 0.05) in CR3
but not CD14 or CR4 transfectants. In contrast, L. monocytogenes and S. aureus induced IL-6 release in CR4 but not CR3 or CD14
transfectants. Collectively our data indicate that ␤2 integrins, such as CR3 and CR4, may be involved in cytokine responses to
Gram-positive bacteria. Moreover, CD14 may play a more important role in responses to whole Gram-negative bacteria relative
to Gram-positive ones. The Journal of Immunology, 2000, 164: 5871–5876.
T
he incidence of sepsis caused by Gram-positive bacteria
has been steadily increasing in recent years, while the
incidence of Gram-negative sepsis has remained fairly
constant (1, 2). As a result, septic shock caused by Gram-positive
bacteria is, in many centers, as frequent as, or more frequent than,
shock caused by Gram-negative organisms. Many of the manifestations of septic shock have been related to the exaggerated release
of proinflammatory cytokines upon interaction of host cells with
microbial products (3, 4). Knowledge of the cell activation mechanisms involved in sepsis would be useful to devise adjunctive
therapies aimed at decreasing cytokine production.
Less is known of cell activation mechanisms initiated by whole
bacteria relative to the many studies conducted with the LPS component of the Gram-negative cell wall. Monocyte activation triggered by LPS involves binding to CD14, a GPI-anchored protein
lacking an intracytoplasmic portion (5–7). Binding of LPS to
CD14 is greatly increased by serum LPS-binding protein, which
may account for the ability of serum to enhance LPS responses (6,
8). Host cell receptors capable of transducing signals originated by
*Istituto di Microbiologia, Facoltà di Medicina e Chirurgia, Università degli Studi di
Messina, Messina, Italy; and †Institute for Cancer Research, University of Trondheim,
Trondheim, Norway
Gram-positive bacteria, which are devoid of LPS, have been only
recently investigated. Similar to LPS, peptidoglycan, a component of
both Gram-positive and Gram-negative bacteria, and lipoteichoic acids, present in the membrane of Gram-positive bacteria, appear to
activate cells through CD14-dependent mechanisms (9 –13).
Complement receptor type III (CR3),3 a ␤2 integrin formed by
noncovalently linked CD18/CD11b complexes, can bind, in a
complement-independent fashion, a number of protozoal, bacterial, and fungal components (14 –18). CR3 can also bind to LBP
(19), and, in the presence of serum and LPS, associate transiently
with surface CD14 on the plane of the neutrophil membrane (20).
CR4, also a ␤2 integrin, is made up by CD18/CD11c and may be
involved in transducing LPS signals in the absence of serum (21).
Recent studies indicate that some human homologues of the Drosophila melanogaster Toll proteins, known as Toll-like receptor
(TLR) 2 and TLR4, can function as LPS signal transducers (22, 23).
The present study was undertaken to identify monocyte receptors involved in cytokine responses to whole bacterial cells, focusing particularly on different Gram-positive bacteria. Our data
indicate that ␤2 integrins may play a role in responses to whole
Gram-positive bacteria. Moreover, cytokine responses to the latter
may be less CD14 dependent than those to Gram-negative bacteria
or soluble products from both types of bacteria.
Received for publication June 29, 1999. Accepted for publication March 14, 2000.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
Materials and Methods
1
This work was supported by grants from the Consiglio Nazionale delle Ricerche
(“Progetto Finalizzato Biotecnologie”), Ministero dell’Universitá e della Ricerca Scientifica e Tecnologica (“Progetti di Rilevanza Nazionale” ex 40%), and Istituto Superiore di Sanitá (“Progetto AIDS” and “Progetto Nazionale Tubercolosi”) of Italy.
COH-1, an encapsulated type III Streptococcus agalactiae (group B streptococcus or GBS) strain, was kindly provided by Craig Rubens, University
2
Address correspondence and reprint requests to Dr. Giuseppe Teti, Istituto di Microbiologia, Torre Biologica (IIp.) Policlinico Universitario, Via Consolare Valeria 1,
98125 Messina, Italy. E-mail address: [email protected]
3
Abbreviations used in this paper: CR3, complement receptor type III; TLR, Toll-like
receptor; GBS, group B streptococcus; GAS, group A streptococcus; CHO, Chinese
hamster ovary.
Copyright © 2000 by The American Association of Immunologists
Heat-killed bacteria
0022-1767/00/$02.00
5872
CYTOKINE RESPONSE TO GRAM POSITIVES
of Washington, Seattle. Streptococcus pyogenes (group A streptococcus or
GAS), Staphylococcus aureus, Listeria monocytogenes, and Escherichia
coli were recent clinical isolates. All of these bacteria were grown to the
early stationary phase in Todd-Hewitt broth and harvested by centrifugation. Killed bacteria were prepared by heat-treatment (60°C for 45 min),
followed by extensive washing with distilled water and lyophilization.
Reagents
The following mAbs (all mouse IgG1) were purified by G protein (GammaBind G Sepharose; Pharmacia Biotech, Milan, Italy) affinity chromatography (24) from culture supernatants of the following hybridomas purchased from the American Type Culture Collection (Manassas, VA):
TS1/18 (anti-human CD18); LM-2/1.6 (anti-human CD11b); HB 247 (antihuman CD14). Anti-human CD11c mAb was purchased from Santa Cruz
Biotechnology (Santa Cruz, CA). Mouse IgG1, used as a control, LPS from
Salmonella enteriditis, and polymixin B were obtained from Sigma
Chimica (Milan, Italy).
Monocyte cultures
Mononuclear cells were obtained from the peripheral blood of healthy
adult donors by centrifugation on Ficoll-Hypaque (Pharmacia) (25). Cells
at the interface were extensively washed and resuspended to a concentration of 2.5 ⫻ 106/ml in RPMI 1640 supplemented with streptomycin (50
␮g/ml), benzylpenicillin (50 IU/ml), and 10 mM N-2-hydroxyethylpiperazine-N⬘-2-ethanesulfonic acid (Life Technologies, Milan, Italy). Then 200
␮l of the cell suspension were dispensed to the wells of microtiter plates
and incubated for 2 h in a 5% CO2 humidified atmosphere at 37°C. Thereafter, nonadherent cells were aspirated and adherent monocytes were
washed twice in RPMI 1640. Monolayers were incubated with mouse IgG1
or mAbs at the indicated concentration for 30 min at 37°C, before the
addition of the stimuli. After a 4-h incubation, culture supernatants were
collected and stored at ⫺70°C until assayed for TNF-␣ measurement. At
the end of the experiments, cell viability, as determined by trypan blue
exclusion, was always ⬎90%. To study the effects of serum components,
in some experiments monocytes were cultured with the stimuli in the presence of 10% heat-inactivated (56°C for 30 min) FCS (Fetalclone I, HyClone Laboratories distributed by Celbio, Milan, Italy) or human serum
obtained from healthy volunteers. Human sera were devoid of Abs against
the Gram-positive bacteria tested, as assessed by ELISA (26, 27). In the
latter test, 1 ⫻ 106 killed bacteria per well were used as sensitizing Ags.
Human sera producing absorbance values ⱕ0.2 at 405 nm using a 1:200
serum dilution were considered negative.
FIGURE 1. TNF-␣ production induced in human monocytes by E. coli
(upper panels) or LPS (lower panels) in the absence and in the presence of
heat-inactivated FCS. Human monocytes were pretreated with mAbs (100
␮g/ml) before stimulation with 50 ␮g/ml of E. coli (dry weight of lyophilized bacteria) or LPS (1 ␮g/ml). Columns and bars represent means ⫾
SDs of three independent observations each conducted in separate experiments. These were performed in duplicate. ⴱ, Significantly (p ⬍ 0.05)
different from controls.
TNF-␣ measurement
TNF-␣ measurement was performed with a cytotoxicity assay using TNF␣-sensitive WEHI 164 clone 13 cells (28, 29). Ten serial 2-fold dilutions
(from 1:4 to 1:2048) were tested in duplicate for each sample. The assay
was calibrated with human recombinant TNF-␣ (sp. act., 20 U/ng; Genzyme, Cinisello Balsamo, Italy) as a standard. TNF-␣ concentrations were
determined by comparing the cytotoxic activity of each sample with that of
the standard using a standard curve generated by linear regression analysis.
TNF-␣ activity in selected plasma samples was totally inhibited by a 1:100
dilution of anti-human TNF-␣ rabbit serum (Genzyme), but not normal
rabbit serum. Because bioassays, such as the one described, may underestimate the amounts of cytokine produced because of the possible coinduction of soluble receptors, TNF-␣ was also measured, in selected supernatants, using a commercial immunoassay with a sensitivity of 5 pg/ml
(Cytoscreen hTNF-␣ ELISA kit, BioSource International, distributed by
Celbio). For this purpose, 12 samples from four separate experiments were
tested. The immunoassay showed a strong correlation with the bioassay.
Transfected cells lines
Chinese hamster ovary (CHO) cells transfectants expressing CD14, CR3,
CR4, or CD14/CR3 and CD14/CR4 combinations and CHO/NEO control
cell (30 –32) were a kind gift of Dr. Golenbock (Boston University, Boston,
MA). Transfectants were maintained in RPMI 1640 medium supplemented
with 10% FCS and 1 mg/ml of G418 (Life Technologies) in a 5% CO2
humidified atmosphere at 37°C. CHO cells were incubated for 4 h with the
bacterial stimuli at the indicated concentrations. Culture supernatants were
then harvested and stored at ⫺70°C until assayed for IL-6 concentration.
None of the tranfectants used in the present study produced detectable IL-6
in the absence of stimuli.
IL-6 was measured using the IL-6-dependent 7TD1 cell proliferation
assay, as described (33). One unit of IL-6 was defined as the amount that
induced 50% maximal proliferation. The assay was calibrated using recombinant murine IL-6 (1 ⫻ 108 U/mg; Boehringer Mannheim, Milan,
Italy) as a standard. The detection limit of the assay was 1 U of IL-6. IL-6
concentrations were determined by comparing absorbance values of each
sample with those of the standard using a standard curve generated by
linear regression analysis.
Data expression statistical analysis
Cytokine levels were expressed as means ⫾ SDs of three independent
observations, each conducted on duplicate samples. Differences in cytokine
levels were assessed by one-way ANOVA and Student-Newman-Keuls
test. Differences were considered significant at values of p ⬍ 0.05.
Results
Stimulation with whole bacteria
In the absence of bacterial stimuli, TNF-␣ levels of monocyte culture supernatants were below the detection levels of either the
cytotoxicity or immune assays. To determine whether CD14, CR3
(CD18/CD11b), or CR4 (CD18/CD11c) are involved in TNF-␣
responses to whole bacteria, monocytes were pretreated with antiCD14, anti-CD18, anti-CD11b, or anti-CD11c mAbs before the
addition of killed bacteria. In preliminary experiments, spontaneous or bacteria-induced TNF-␣ production was not affected by
monocyte pretreatment with up to 100 ␮g/ml of control mouse
IgG1 (not shown).
Moreover, in the absence of bacteria, monocytes did not produce detectable TNF-␣ after the addition of anti-CD14, anti-CD18,
or anti-CD11c Abs at concentrations of up to 100 ␮g/ml (not
shown). Anti-CD11b at concentrations of 10 ␮g/ml or higher produced, in the absence of other stimuli, TNF-␣ elevations that never
The Journal of Immunology
5873
FIGURE 2. TNF-␣ production induced in human monocytes by killed
GAS (upper panels) or GBS (lower panels) in the absence or in the presence of heat-inactivated FCS. Human monocytes were pretreated with
mAbs (100 ␮g/ml) before stimulation with 50 ␮g/ml of GBS or GAS (dry
weight of lyophilized bacteria). Results represent means ⫾ SDs of three
independent observations each conducted in separate experiments. These
were performed in duplicate. ⴱ, Significantly (p ⬍ 0.05) different from
controls.
FIGURE 3. TNF-␣ production induced in human monocytes by S. aureus (upper panels) or L. monocytogenes (lower panels) in the absence and
in the presence of heat-inactivated FCS. Human monocytes were pretreated
with mAbs (100 ␮g/ml) before stimulation with 50 ␮g/ml of S. aureus or
L. monocytogenes (dry weight of lyophilized bacteria). Data represent
means ⫾ SDs of three independent observations each conducted in separate experiments. These were performed in duplicate. ⴱ, Significantly (p ⬍
0.05) different from controls.
exceeded 6% of maximal, LPS-induced stimulation (not shown).
These slight TNF-␣ responses were not inhibited by polymixin B
(15 ␮g/ml), ruling out endotoxin contamination of the Ab
preparation.
Figs. 1–3 show the effects of anti-receptor Abs on TNF-␣ responses to 50 ␮g/ml of killed bacteria (dry weight of lyophilized
cells). Experiments were conducted both in the absence and in the
presence of 10% heat-inactivated FCS. Fig. 1 shows that serum
markedly increased the TNF-␣ response to E. coli (upper panels).
Anti-CD14 markedly reduced ( p ⬍ 0.05) E. coli-induced stimulation both in the absence and in the presence of serum. Anti-CD18
or anti-CD11b produced moderate, but significant ( p ⬍ 0.05), inhibition in the presence, but not in the absence, of serum. Conversely, anti-CD11c significantly decreased TNF-␣ responses to E.
coli in serum-free conditions only.
Results obtained with 1 ␮g/ml of LPS as a stimulus (Fig. 1,
lower panels) generally paralleled those observed with E. coli. In
fact, anti-CD14, anti-CD18, or anti-CD11b Abs had similar inhibitory effects when using LPS or E. coli (Fig. 1). However, antiCD11c had no effects on LPS-induced cell activation either in the
presence or in the absence of serum. This was at variance with the
above described inhibition by anti-CD11c with E. coli in serumfree conditions.
Fig. 2 shows experiments using GAS (upper panels) and GBS
(lower panels) as stimuli. At variance with previous observations
with LPS or E. coli, serum, anti-CD14, or anti-CD11c did not
affect GAS- or GBS-induced TNF-␣ release. In contrast, antiCD18 or anti-CD11b produced marked inhibition both in the absence and in the presence of serum. However, these anti-CR3
mAbs did not affect TNF-␣ release by S. aureus and L. monocytogenes (Fig. 3). Anti-CD11c produced moderate, but significant
( p ⬍ 0.05), inhibition in S. aureus- or L. monocytogenes-induced
TNF-␣ responses. Neither anti-CD14 nor serum affected S. aureusor L. monocytogenes-induced stimulation (Fig. 3). Results obtained by measuring TNF-␣ with ELISA produced virtually identical results as to the inhibitory effects of the various Abs (not
shown). In further studies, the experiments presented in Figs. 2 and
3 were repeated using decreasing doses of anti-receptor Abs. The
minimal concentration of either anti-CD18 or anti-CD11b that
could still produce significant ( p ⬍ 0.05) inhibitory effects was 5
␮g/ml using either GBS or GAS as stimuli. With anti-CD11c Abs,
minimal inhibitory concentrations were 10 and 20 ␮g/ml, respectively, using listeria and staphylococci (not shown).
Release of IL-6 by CHO-transfected cells
To analyze the role of CD14, CR3, and CR4 in the absence of
competing receptors or small amounts of complement products
produced by phagocytes, we used transfected cell lines, i.e., CHO
cells expressing CD14, CR3, or CR4. Control CHO/NEO cells did
not produce detectable IL-6 in the presence of bacteria or LPS (not
shown). Fig. 4 shows that even in the absence of serum, moderate
production of IL-6 was observed in CD14 transfectants stimulated
with LPS (10 ␮g/ml) or E. coli ( p ⬍ 0.05). Similar results were
observed with lower LPS concentrations (1 and 0.1 ␮g/ml, not
shown). Serum increased IL-6 release induced by either E. coli or
LPS ( p ⬍ 0.05; Fig. 4). In contrast, no IL-6 production was detectable in CHO/CD14 cells using any of the Gram-positive bacteria as stimuli either in the absence or in the presence of serum
(Fig. 4).
Both GAS and GBS were capable of inducing moderate, but
significant, IL-6 production in CR3-transfected cells (Fig. 5). In
those cells, E. coli or LPS were also capable of triggering moderate
IL-6 release, but only in the presence of serum. Conversely, S.
aureus and L. monocytogenes were not capable of inducing IL-6
5874
CYTOKINE RESPONSE TO GRAM POSITIVES
FIGURE 4. IL-6 production induced in CHO/
CD14 cells by various doses of killed whole bacteria
(dry weight of lyophilized bacteria) or LPS in the
absence and in the presence of heat-inactivated FCS.
Points and bars represent means ⫾ SDs of three independent observations each conducted in separate
experiments. These were performed in duplicate.
release in CR3-transfected cells. However, these bacteria induced
moderate ( p ⬍ 0.05) IL-6 production in CR4 transfectants (Fig. 6).
Using these cells, either E. coli or LPS triggered moderate IL-6
release, and this effect was augmented by serum. Coexpression of
CD14 with either CR3 or CR4 did not increase the ability of these
cells to respond to any of the bacterial stimuli-relative cells expressing only one receptor (data not shown). Collectively, these
transfected cell data confirmed those previously obtained with
blocking Abs.
Discussion
Much less is known of receptors and signal transduction pathways
activated by Gram-positive bacteria relative to the many observations conducted with the LPS component of Gram-negative bacteria. Clinical manifestations are similar in septic shock caused by
Gram-positive and Gram-negative bacteria, suggesting common
pathways of cell activation. Accordingly, a recent study indicates
that, similar to LPS, S. aureus or Streptococcus pneumoniae can
initiate activation signals via TLR2 (34).
Data presented here suggest that, at least early in the signaling
pathway, at the receptor level, differences may exist in responses
to different bacterial species. First, CD14 appeared to play a more
important role in TNF induction by E. coli or LPS, relative to any
of the Gram-positive species tested. Second, different ␤2 integrins
may be involved in responses to streptococci on the one hand and
S. aureus or L. monocytogenes on the other.
The possibility that CD14 does not play an obligatory role in
monocyte responses to whole Gram-positive bacteria is indicated
by several lines of evidence. First, anti-CD14 Abs did not prevent
TNF release from monocytes upon stimulation with Gram-positive
bacteria, while almost completely blocking responses to E. coli.
Second, serum (or combinations of soluble CD14 and LPB, our
unpublished results) markedly increased TNF induction by E. coli,
but not Gram-positive bacteria. Third, the latter organisms were
unable to stimulate CD14 transfectants for increased IL-6 production. Our data are in general agreement with those of Tuomanen et
al. (35), who also found that anti-CD14 Abs do not inhibit responses to whole pneumococci. It was recently observed that coexpression of CD14 in CHO cells enhanced TLR-mediated translocation of NF-␬B in response to S. aureus or S. pneumoniae (34).
Moreover, recent data from our laboratories indicate that moderate
(30%) inhibition of L. monocytogenes-induced monocyte activation can be produced by the anti-CD14 mAb 3C10 (not shown).
In the present study, virtually no inhibition was observed with a
different anti-CD14 mAb (HB247). Therefore, it cannot be excluded that, under certain circumstances, CD14 may participate to
cell activation by Gram-positive bacteria. While not ruling out this
possibility, our data suggest that CD14 plays a more important role
in monocyte responses to Gram-negative bacteria relative to
Gram-positive ones.
This relative lack of involvement of CD14 may seem surprising,
because ubiquitous and quantitatively important Gram-positive
components such as peptidoglycan and acid lipoteichoic bind to
CD14 and initiate cell activation phenomena through CD14-dependent pathways (9 –13). The type-specific and group-specific
polysaccharides from GBS (our unpublished results), as well as
purified cell walls from GBS (30), S. aureus (36), and pneumococci (35), were also capable of inducing cytokines through CD14dependent mechanisms.
FIGURE 5. IL-6 release induced in CHO/CR3
cells after stimulation with various doses of killed
whole bacteria (dry weight of lyophilized bacteria) or
LPS in the absence and in the presence of heat-inactivated FCS. Points and bars represent means ⫾ SDs
of three independent observations each conducted in
separate experiments. These were performed in
duplicate.
The Journal of Immunology
5875
FIGURE 6. IL-6 release induced in CHO/CR4
cells by various doses of killed whole bacteria (dry
weight of lyophilized bacteria) or LPS in the absence
and in the presence of heat-inactivated FCS. Points
and bars represent means ⫾ SDs of three independent
observations each conducted in separate experiments.
These were performed in duplicate.
Why should receptors involved in cytokine response to whole
Gram-positive bacteria differ from those engaged by purified components? It is possible that some of these components may not be
expressed on the cell surface in a position or in sufficient quantities
to activate host cells. Alternatively, differences in size (i.e., soluble
vs particulate) of the stimuli may lead to the selection of different
receptors. For example, phagocytosis, albeit not absolutely necessary to induce TNF-␣ responses, may provide coactivation signals
that eventually obscure those initiated by single surface components. It is of interest that receptors mediating TNF-␣ responses to
whole bacteria (e.g., CD14 and CR3 for, respectively, E. coli and
GBS) can also mediate nonopsonic phagocytosis of the corresponding organism (37, 38). Interestingly, it was shown that soluble products, specifically mannuronic acid polymers, which are
capable of activating cells only through CD14, acquire CR3 dependency in their stimulating activity after conjugation to latex
particles (39).
In the present study, anti-CR3 Abs were highly effective in
blocking cytokine responses to GBS or GAS, but not staphylococci
or listeria. Conversely, anti-CR4 Abs inhibited TNF-␣ release by
monocytes stimulated with staphylococci or L. monocytogenes, but
not streptococci. Experiments with transfected cells were in general agreement with those using blocking Abs and confirmed that
CR3 and CR4 may be involved in responses to streptococci on the
one hand and S. aureus and L. monocytogenes on the other. Our
GBS data are in general agreement with previous studies showing
that CR3 may mediate GBS-induced nitrous oxide production in
mouse macrophages (40). In addition, macrophage cell lines expressing CR3, but not the WEHI 3 line that is devoid of CR3,
produced detectable TNF-␣ after GBS stimulation (41). To our
knowledge, the involvement of CR3 in cytokine induction by GAS
was not reported. This data may be of interest in view of the ability
of GAS to induce massive cytokine release and fulminant shock in
the course of invasive infections. Although the importance of superantigenic exotoxins in cytokine induction has been emphasized
(42, 43), the potential role of whole bacteria should not be overlooked (44).
It is perhaps too early to speculate on whether CR3 blockade can
be an effective strategy to decrease TNF production during streptococcal sepsis. Theoretically, anti-CR3 treatments may prevent
not only streptococci-induced TNF-␣ production, but also TNF-␣
toxicity. This is suggested by the ability of anti-CD18/CD11b Abs
to prevent tissue injury and cardiopulmonary changes induced by
recombinant TNF-␣ infusion (45).
However, anti-CR3 approaches should be considered with great
caution in view of the important role of these molecules in host
defenses. Patients with congenital defects in the CD18/CD11b
complex have impaired neutrophil functions (46, 47). In addition,
anti-CD18/CD11b Abs exacerbated infection in animals challenged with E. coli (48, 49), S. aureus (50) or L. monocytogenes
(51). These detrimental effects likely occurred, at least in part, as
a result of decreased neutrophil adhesion to vascular endothelium
and recruitment in infected sites. It will be of interest to determine
whether binding of streptococci to CR3 can be selectively blocked
without affecting the ability of this receptor to mediate cell to cell
interactions involved in host responses. This may be feasible if
bacterial- and host-derived CR3 ligands bind to different sites of
this molecule. Our data do not exclude that, in addition to ␤2
integrin, other receptors may be involved in such responses, especially in view of the fact that moderate responses only were observed in cells expressing single receptors. In this respect, the role
of TLR should be further investigated, in view of the ability of
TLR2 expression in CHO cells to confer responsiveness to staphylococci and streptococci. Studies are underway to address this
point.
In conclusion, our data indicate that CR3 is involved in TNF-␣
induction by streptococci. In addition, CR4 may be also involved
in responses to L. monocytogenes or S. aureus. Clearly other receptors, in addition to CR4, may play a significant role in listeriaor S. aureus-induced stimulation. In fact, using these bacteria, the
inhibitory effects of anti-CD11c Abs were more modest relative to
those induced, for example, by anti-CR3 Abs using GBS as a stimulus. Data presented here may be useful to devise alternative therapeutic strategies aimed at preventing mediator production. However, future studies are necessary to assess the clinical relevance of
these findings.
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