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. 1999 Jul;97(3):429–437. doi: 10.1046/j.1365-2567.1999.00788.x

Lipopolysaccharide enhances FcγR-dependent functions in vivo through CD11b/CD18 up-regulation

C Rubel 1, P Miliani De Marval 1, M Vermeulen 1, M A Isturiz 1, M S Palermo 1
PMCID: PMC2326862  PMID: 10447764

Abstract

Fc receptors for immunoglobulin G (IgG) (FcγR) mediate several defence mechanisms in the course of inflammatory and infectious diseases. In Gram-negative infections, cellular wall lipopolysaccharides (LPS) modulate different immune responses. We have recently demonstrated that murine LPS in vivo treatment significantly increases FcγR-dependent clearance of immune complexes (IC). In addition, we and others have reported the induction of adhesion molecules on macrophages and neutrophils by LPS in vivo and by tumour necrosis factor-α (TNF-α) in vitro. The aim of this paper was to investigate CD11b/CD18 participation in LPS enhancing effects on Fcγ-dependent functionality of tissue macrophages. Our results have demonstrated that LPS can enhance antibody-dependent cellular cytotoxicity (ADCC) and IC-triggered cytotoxicity (IC-Ctx), two reactions which involve the Fcγ-receptor but different lytic mechanisms. In vitro incubation of splenocytes from LPS-treated mice with anti-CD11b/CD18 abrogated ADCC and IC-Ctx enhancement, without affecting FcγR expression. Similar results were obtained with physiological concentrations of fibrinogen. In this way cytotoxic values of LPS-splenocytes decreased to the basal levels of control mice. Time and temperature requirements for such inhibition strongly suggested that anti-CD11b/CD18 could modulate intracellular signals leading to downregulation of FcγR functionality. Data presented herein support the hypothesis that functional and/or physical associations between integrins and FcγR could be critical for the modulation of effector functions during an inflammatory response.

INTRODUCTION

Fc receptors for immunoglobulin G (IgG) (FcγR) are involved in the clearance of immune complexes, phagocytosis of antibody-coated pathogens, enhancement of antigen presentation, generation of reactive oxygen intermediates (ROI) and antibody-dependent cellular cytotoxicity (ADCC). These receptors are particularly important in the immunological defence against pathogens during infectious diseases. Opsonization of pathogens with IgG and/or complement factors allows cells to recognize a wide variety of organisms using a limited number of receptors. It has been widely demonstrated that Gram-negative bacterial infection is a strong stimulus of mononuclear phagocyte functions and several reports have analysed the in vitro effects of lipopolysaccharides (LPS) on FcγR.5 However, the results obtained are partial and contradictory, depending on experimental conditions and the cell populations studied.5–8 We have recently demonstrated that in vivo LPS inoculation markedly enhanced Fcγ-dependent immune complex clearance.9 In addition, several authors have reported the induction of integrins on macrophages and neutrophils by LPS in vivo and by tumour necrosis factor-α (TNF-α) in vitro.

Integrins are a familiy of transmembrane glycoproteins that mediate cell–cell and cell–substrate interactions. The subfamily of β2 integrins comprises four homologous heterodimers: CD11a/CD18 (leucocyte-function associate molecule-1, LFA-1), CD11b/CD18 (complement receptor type 3: CR3, Mac-1), CD11c/CD18 (CR4, p150/95) and CD11d/CD18 (αd2).11 CD11b/CD18 recognizes a variety of exogenous and endogenous substances, including complement component iC3b, intracellular adhesion molecule-1 (ICAM-1), fibrinogen, factor X, zymosan, β glucans, Escherichia coli and Leishmania.12 In addition to the recognition phenomena, β2 integrins mediate transmembrane chemical signalling and transmit mechanical stress to the cytoskeleton. There is increasing evidence that ligation of integrins and Fc receptors is able to initiate complex transmembrane signalling pathways contributing to the activation of several effector functions of leucocytes such as the oxidative burst15 and degranulation,16 which are required for effective killing of the target. Moreover, it was suggested that although CD11b/CD18 does not induce target cell lysis by itself, it could mediate an enhanced phagocytosis via FcγR.

The aim of this paper was to investigate CD11b/CD18 participation in the enhancement of Fcγ-dependent functionality by LPS. We further discuss the relevance of functional and/or physical associations between integrins and FcγR for modulating effector functions during an inflammatory response.

MATERIALS AND METHODS

Animals and treatment

LPS from Escherichia coli O111:B4 was dissolved in 0·15 m NaCl in order to obtain a final concentration of 50 μg/ml. Eight to 10-week-old BALB/c mice were i.v. injected with 0·1 ml of this solution and 20 hr later were sacrificed and their spleens used for cytotoxic studies.

Reagents

LPS from Escherichia coli O111:B4, phorbol myristate acetate (PMA), zymosan A from Saccharomyces cerevisiae yeast (Zy), catalase (bovine liver, type V, 3090 U/mg protein) and bovine fibrinogen were purchased from Sigma Chemical Company (St Louis, MO)

Antibodies

Purified monoclonal antibodies to CD11b/CD18 (M1/70·15 clon, rat IgG2b anti CD11b) azida free, utilized in functional studies was a generous gift of Dr Françoise LePault from Unit 1461 CNRS URA. Hopital Necker, 161 Sevres 75743, France.19 The F(ab′)2 fragment of monoclonal antibody to CD11b/CD18 was prepared by pepsin treatment and protein A purification using a Immunopure kit (Pierce Chemical Co., Rockford, IL). Purity of F(ab′)2 was confirmed by reducing and non-reducing sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) with Coomassie staining. Isotype control of rat IgG2b (Immunotech, France) and rat monoclonal antibody against murine CD18 (Immunotech, France) used in functional studies were exaustively dialysed to remove azide. Cytometric studies were developed using rat monoclonal antibodies against murine FcγR (CD32/ CD16) (Pharmingen, San Diego, CA) and murine CD11b/ CD18 (Boehringer Mannheim, Buenos Aires, Argentina). Fluorescein isothiocyanate-conjugated goat antirat inmunoglobulins (FITC-goat antirat IgG) were purchased from Pharmingen. Fluorescein isothiocyanate-conjugated mouse monoclonal antibody to mouse RFcγII/III (CD32/CD16) and R-phycoerythrin-conjugated rat monoclonal antibody to mouse CD11b were purchased from Caltag Laboratories (Burlingame, CA), and utilized in double staining assays

Cytotoxic assays

Cytotoxic assays were performed by reacting splenocytes suspended in complete medium (RPMI-1640, 3% fetal calf serum, Gibco BRL, Gaithersburg, MD), with the appropiate stimulus. Neutrophil contamination of splenocyte suspension was always lower than 1%. Macrophage in vivo depletion with liposomes containing dichloromethylene biphosphonate (Clodronate) completely abrogated cytotoxic functions, ensuring that these reactions were carried out by the macrophage population (unpublished observation). After incubation for 18 hr at 37°, the culture plate was centrifugated and the radioactivity of supernatants and pellets was measured. The mean of 51Cr released was expressed as the percentage of total radioactivity. Spontaneous release was always less than 5% and was substracted to obtain specific cytotoxicity.

Antibody-dependent cellular cytotoxicity (ADCC)

ADCC was assayed by the chicken red blood cells (CRBC) anti-CRBC system as previously described.20 Unless otherwise stated, cytotoxic assays were performed by reacting 1 × 106 splenocytes with 2 × 10551Cr-labelled CRBC sensitized with subagglutinating amounts of specific rabbit IgG.

Non-specific immune-complex triggered cytotoxicity (IC-Ctx)

Precipitating immune complexes (IC) were prepared with rabbit IgG antiovalbumin (OA), isolated by affinity chromatography and chromatographically purified OA (Cappel Lab., Detroit, MI). Immune complexes were formed at the equivalent zone, previously determined by a precipitating curve. Antigen and antibody in a molar ratio Ag:Ab=5:1 were incubated for 1 hr at 37° and 18 hr at 4°. Then, IC were washed and resuspended. Unless otherwise stated, 20 μl of these IC were added to 1 × 106 splenocytes 5 min before the addition of 2 × 105 non-sensitized labelled erythrocytes (CRBC).

PMA-induced cytotoxicity (PMA-Ctx)

Splenocytes (1 × 106) were incubated with different concentrations of PMA to induce the respiratory burst of splenocytes, and 2 × 10551Cr-labelled non-sensitized erythrocytes (CRBC).

Opsonized zymosan-induced cytotoxicity (OpZy-Ctx)

Zymosan A was washed in saline solution and suspended in human serum to a concentration of 10 mg/ml. After 30 min of incubation at 37°, serum was removed by centrifugation and opsonized Zymosan (OpZy) was washed five times with saline solution. Finally, OpZy was resuspended in saline solution to a concentration of 10 mg/ml. Different concentrations of OpZy were added to 1 × 106 splenocytes 5 min before the addition of 2 × 10551Cr-labelled non-sensitized erythrocytes (CRBC).

Immunofluorescence flow cytometry

Measurement of the expression of FcγR and CD11b/CD18 on splenocytes was carried out by indirect immunofluorescence flow cytometry. The splenocyte suspension (20 μl; 25 × 106 cells/ml) obtained from saline and LPS-treated mice was washed with cool phosphate-buffered saline (PBS) and incubated with monoclonal antibodies (mAb) (20 μg/ml to 1 × 106 cells) for 30 min at 4°. Then, the cells were washed and stained with fluoresceinated antirat IgG for 30 min at 4°. After two washes with PBS, the cells were resuspended in 0·4 ml of ISOFLOW (International Link, S.A., Buenos Aires, Argentina) and the fluorescence was analysed by FACScan (Becton Dickinson, Mountain View, CA). Double staining assays (CD11b+/FcγR+) were carried out utilizing fluorescein and R-phycoerythrin-conjugated antibodies. The analysis was made on 20 000 events on each sample by using the Cell Quest Program (Becton Dickinson).

Statistical analysis

All data are presented as the mean±SEM. Comparisons between multiple groups were performed with one-way analysis of variance (anova) followed by Bonferroni t-tests.

RESULTS

LPS in vivo treatment up-regulates CD11b/CD18

It has been established that the process of integrin up-regulation involves translocation of presynthesized intracytoplasmic pools to the cell surface.21 However, depending on the stimulus and the cell target assayed this enhanced expression could be transient or sustained.22 In order to analyse integrin induction on effector cells by LPS in vivo injection, CD11b/CD18 expression was evaluated on splenocyte surface after LPS treatment. Doses as low as 5 μg/mouse (250 μg/kg, i.v.) of LPS induced a marked increase in splenocyte CD11b/CD18 fluorescence intensity 3 hr post LPS inoculation and, this effect persisted for at least 24 hr (Fig. 1).

Figure 1.

Figure 1

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Effect of LPS on CD11b/CD18 expression on splenocytes.At 24 hr post LPS injection, mice were killed and their spleens were excised. The cells were stained with specific mAb anti-CD11b/CD18 as described in Materials and Methods. (a) Representative histograms of CD11b/CD18 expression on splenocytes from saline and LPS-treated mice are shown. Controls of isotype were drawn in dotted lines. The ordinate and the abscissa represent the cell number and the fluorescence intensity, respectively. In (b), saline and LPS-treated splenocytes were excised at different times after LPS injection and stained with specific mAb anti-CD11b/CD18 as described in Materials and Methods. Each bar represents the mean±SEM of arithmetic mean of fluorescence of four mice. *P < 0·05 compared to saline treated group.

We had previously demonstrated that IC-clearance and ADCC were markedly enhanced by LPS treatment, without any alteration in FcγR expression.9 Then, LPS effects on the double possitive cells (CD11b+/FcγR+) were evaluated using direct immunofluorescence. Results shown in Fig. 2 demonstrated that the percentage of such cell population was increased after LPS treatment, due to an enhanced expression of CD11b.

Figure 2.

Figure 2

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Effect of LPS on double positive CD11b+/FcγR+ population. At 24 hr post-LPS injection, mice were killed and their spleens were excised. The cells were stained with specific mAbs anti-CD11b and anti-FcγR as described in Materials and Methods. (a) Representative dot plots of CD11b+/FcγR+ expression on splenocytes from saline and LPS-treated mice are shown. The ordinate (Fl2) corresponds to R-phycoerythrin-conjugated anti-CD11b and the abscissa (Fl1) corresponds to fluorescein conjugated anti-FcγR antibodies. In (b), data represent the mean±SEM of percentage of positive population and arithmetic mean of fluorescence of nine mice.*P < 0·0001 compared to saline treated group.

CD11b/CD18 involvement in Fcγ-mediated functions

Considering that integrins have been postulated as co-signalling molecules of several receptors,12 we investigated CD11b/CD18 involvement in the enhancement of Fcγ-mediated functions by LPS. Splenocytes from LPS or saline treated mice were incubated with anti-CD11b/CD18 monoclonal antibody simultaneously with target cells in order to assay ADCC, as described in Materials and Methods. Under these experimental conditions, anti-CD11b/CD18 antibody did not exert any modulatory effect on cytotoxicity. However, when splenocytes were preincubated for 1 hr with the monoclonal antibody the enhancement of ADCC induced by LPS was abrogated by anti-CD11b/CD18 at every effector:target ratio assayed (Fig. 3a). Serial dilutions of the antibody showed that total inhibition occurred at a concentration of 0·7 μg/ml (5 nm) (Fig. 3b). The inhibition of ADCC from LPS-treated mice was not due to non-specific blocking of FcγR since the F(ab′)2 preparation was also able to inhibit ADCC. Moreover, equivalent concentrations of isotype matched mAb rat IgG2b (isotype control) and anti-CD18 had no effect on cytotoxicity (Fig. 3b). The requirement of a preincubation time suggested the involvement of a metabolic process in anti-CD11b/CD18 inhibition of ADCC. In order to address this issue, splenocytes from LPS and saline treated mice were preincubated with anti-CD11b/CD18 monoclonal antibody for 60 min at 37° or 4°. In these experiments a final concentration of 4 μg/ml anti-CD11b/CD18 was used after which target cells were added and the reaction was carried out at 37°. When the temperature of preincubation with monoclonal antibody was 4°, there was no inhibition of ADCC from LPS-treated mice (Table 1). These results demonstrated that anti-CD11b/CD18 inhibition was time and temperature dependent, suggesting the involvement of an active mechanism.

Figure 3.

Figure 3

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(a) Effect of anti-CD11b/CD18 on ADCC from saline (□) and LPS (▪) treated mice. Splenocytes were incubated for 60 min at 37° with or without anti CD11b/CD18 at different effector:target ratios, using 0·5, 1 and 2 × 106 splenocytes for 2·5:1, 5:1 and 10:1 ratios, respectively. After 18 hr of incubation at 37°, the cytotoxicity was measured as described under Materials and Methods. Each point represents the mean±SEM of nine animals per group. (b) Splenocytes (1 × 106) were incubated for 60 min at 37° without or with different concentrations of anti-CD11b/CD18. Additional controls of the F(ab′)2 fragment of anti-CD11b/CD18 (▵), isotype matched antibody (○) and anti-CD18 antibody (•) are also shown.

Table 1.

Time and temperature-dependence in anti CD11b/CD18 inhibitory effect on ADCC

graphic file with name imm0097-0429-t1.jpg

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Splenocytes (1 × 106) from saline- or LPS-treated mice were incubated at 37° or 4° without mAb or with anti-CD11b/CD18 (4 μg/ml), added 60 min before (−60), simultaneously (0) or 60 min after (+60), the addition of 51Cr-CRBC. After 18 hr of incubation at 37°, the ADCC was measured as described under Materials and Methods. Each point represents the mean±SEM of n animals per groups. *P < 0·001 compared to LPS-treated splenocytes without mAb.

We investigated the possibility that FcγR splenocyte membrane expression was modulated by this anti-CD11b/CD18 monoclonal. Our results were consistent with previous data,23 where the binding of monoclonal anti-FcγR was not modified by preincubation with anti-CD11b/CD18 antibody (data not shown).

We then examined the effect of anti-CD11b/CD18 on other Fcγ-dependent function, such as non-specific immune-complex triggered cytotoxicity (IC-Ctx). ADCC and IC-Ctx are both strictly dependent on FcγR, although they involve different activation pathways.24 The results showed in Fig. 4(a) demonstrate that IC-Ctx is also enhanced upon LPS in vivo treatment. Moreover, anti-CD11b/CD18 preincubation significantly diminished this enhancement. In order to confirm the participation of different lytic mechanisms in both Fc-dependent cytotoxic assays, the reactions were carried out in the presence of 15 U/ml of catalase. This enzyme has been reported to impair H2O2-mediated lysis generated upon burst respiratory stimulation by IC, but it does not interfere with the ADCC lytic mechanism.24 The results of a representative experiment are shown in Table 2. We also confirmed previous evidence that ADCC does not involve postphagocytic events, since cytochalasin B was not able to inhibit the level of cytotoxicity (data not shown).

Figure 4.

Figure 4

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(a) Effect of anti CD11b/CD18 on IC-triggered cytotoxicity (IC-Ctx) from saline (□) and LPS (▪) treated mice. Splenocytes (1 × 106) were incubated for 60 min at 37° without mAb or with different concentrations of anti CD11b/CD18. After 18 hr of incubation at 37°, the lysis was measured as described in Materials and Methods. Additional controls of the F(ab′)2 fragment of anti-CD11b/CD18 (▴) and isotype matched antibody (○) are also shown. Each point represents the mean±SEM of seven animals per group. †P < 0·001 compared to IC-Ctx from saline treated mice.*P < 0·001 compared to IC-Ctx from LPS-treated mice without anti-CD11b/CD18. (b). Effect of the anti CD11b/CD18 on PMA-triggered cytotoxicity from saline (□) and LPS (▪) treated mice. Splenocytes (1 × 106) were incubated for 60 min at 37° without mAb (−) or with anti-CD11b/CD18 (+, 4 μg/ml). Different concentrations of PMA were added 5 min before the addition of non-sensitized 51Cr-CRBC. After 18 hr of incubation at 37°, the lysis was measured as described under Materials and Methods. Each point represents the mean±SEM of eight animals per group. †P < 0·01 (LPS versus saline). (c) Effect of anti-CD11b/CD18 on zymosan-triggered cytotoxicity from saline and LPS treated mice. Splenocytes (1 × 106) were incubated for 60 min at 37° without mAb (□ saline, ▪ LPS) or with anti-CD11b/CD18 (4 μg/ml, ○ saline, • LPS). Different concentrations of OpZy were added 5 min before the addition of non-sensitized 51Cr-CRBC. After 18 hr of incubation at 37°, the lysis was measured as described under Materials and Methods. Each point represents the mean±SEM of 11 animals per group. †P < 0·001 (LPS versus saline).

Table 2.

Effect of catalase on ADCC and IC-CTX from LPS and saline treated mice

graphic file with name imm0097-0429-t2.jpg

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Splenocytes (1 × 106) were incubated in presence or absence of 15 U/ml of catalase with 2 × 105 52Cr-CRBC sensitized, with subagglutinant amounts of specific rabbit IgG for the ADCC assay, or with IC (20 μg/ml) for the IC-Ctx. After 18 hr of incubation at 37°, the cytotoxicity was measured as described under Materials and Methods. Each point represents the mean±SEM of five animals per groups.

*P < 0·0001 compared to IC-Ctx in the absence of catalase.

CD11b/CD18 involvement in PMA-cytotoxicity

Considering that PMA is able to directly stimulate protein kinase C (PKC) without any membrane receptor participation, we incubated splenocytes from LPS- and saline-treated mice with different doses of PMA. Splenocytes from LPS-treated animals showed an increased PMA-cytotoxicity, although preincubation with anti-CD11b/CD18 was not able to inhibit this reaction in any of the doses evaluated (Fig. 4b). This was also observed for control mice.

CD11b/CD18 involvement in zymosan cytotoxicity

Zymosan opsonized with human serum (OpZy) represents yeast particles coated with complement fragments, predominantly iC3b, a ligand for CD11b/CD18 (CR3). When phagocyte CD11b/CD18 binds to iC3b on yeast, phagocytosis and degranulation are triggered because of simultaneous recognition of iC3b via a CD11b I-domain binding site and specific microbial polysaccharides via a lectin site located at the COOH-terminal I-domain.25 Taking into account that CD11b/CD18 was up-regulated by LPS, the lytic activity was assayed on unsensitized erythrocytes, triggered by OpZy. The possible inhibition by anti-CD11b/CD18 antibody was also analysed. As shown in Fig. 4(c), this function was significantly increased by in vivo treatment with LPS. However, anti-CD11b/CD18 antibody preincubation only slightly, but not significantly, suppressed this enhancement at the highest dose of OpZy assayed.

ADCC-modulation by fibrinogen, a CD11b/CD18 natural ligand

Considering that fibrinogen is the natural ligand of CD11b/CD18 on monocytes and neutrophils, the modulatory action of this ligand on ADCC was evaluated. Physiological concentrations of fibrinogen, added in solution to culture medium, were able to induce inhibition of ADCC carried out by splenocytes from LPS-treated mice (Fig. 5) at all effector:target ratio assayed. However, normal ADCC was only inhibited at the higher effector:target ratio.

Figure 5.

Figure 5

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Effect of fibrinogen (FG) on ADCC from LPS and saline treated mice splenocytes. Splenocytes were incubated for 60 min at 37° with or without FG (6 μm) at different effector:target ratios. After 18 hr of incubation at 37°, the cytotoxicity was measured as described under Materials and Methods. Each point represents the mean±SEM of 12 animals per group. *P < 0·01, compared with ADCC from LPS-treated mice without FG. †*P < 0·05, compared with ADCC from saline-treated mice without FG.

DISCUSSION

We have presented a pannel of cellular cytotoxic reactions mediated by splenocytes which are markedly enhanced after in vivo LPS-treatment. These included FcγR-dependent and -independent functions. The enhancement on FcγR-mediated reactions could not be associated with a higher expression of FcγR on cell membrane.9 On the other hand, a marked and persistent increase in β2-integrins has been observed, confirming previous information obtained in neutrophils. Considering that recent reports have postulated that β2- integrins might function in a cooperative manner with Fc-receptors,28 we investigated the involvement of CD11b/CD18 in LPS enhancing effects on FcγR-dependent functions.

While CD11b/CD18 is normally found in small amounts on the macrophage–monocyte cell surface, a larger quantity exists as a cytoplasmic pool, which becomes inserted into the plama membrane in response to the treatment of the cells with a number of cytokines and lipids. Enhanced surface expression is often accompanied by conformational changes in the integrin itself31 resulting in the expresion of activation epitope mAb2432 and the induction of a high avidity binding state of CD11b/CD18. Neutrophils and macrophages treated with LPS in vivo or TNF in vitro dramatically increase the level of cell-surface CD11b/CD18 expression.10 We consistently found that LPS in vivo treatment increased the level of this molecule not only in peripheral neutrophils but also in splenocytes. Moreover, FcγR+ cells which express a low density of CD11b molecules, significantly increase their expression after LPS treatment, resulting an enhancement in double high possitive cells (CD11b+/FcγR+). However, enhancement in CD11b/CD18 splenocyte expression was minor and happened later in comparison with polymorphonuclear cell (PMN) induction. Peripheral PMN showed maximal expression of CD11b/CD18 1 hr post-LPS treatment (250% compared to untreated cells), and splenocytes showed a 100% enhancement 24 hr post LPS injection. TNF-α has been implicated as the endogenous cytokine which mediates several LPS actions,33 and TNF-αin vitro induces CD11b/CD18 expression on PMN surface.10 However, after in vivo injection of murine TNF-α, we could not obtain any splenocyte modulation of this molecule nor Fcγ-dependent functions (data not shown). The difference in kinetics of CD11b up-regulation between PMN and splenic macrophages, as well as the differential effect of TNF-α on both cell types, could reflect differences in the underlying mechanism, i.e. translocation of an intracellular store (PMN) versus the de novo synthesis (splenocytes). This hypothesis remains to be tested.

CD11b/CD18 binding to its counter-receptors (ICAM-1) has been found to play an important role in leucocyte adhesion to endothelial cells34 and to modulate H2O2 production.35 Moreover, β2-integrins are phagocytic receptors that bind a variety of bacterial products, including LPS. This binding to integrins activates intracellular signalling pathways that co-ordinate and regulate a variety of cellular responses.36 Additionaly, CD11b/CD18 forms transmembrane complexes with glycosylphosphatidyl inositol-linked membrane glycoproteins such as FcγRIIIB (CD16), the uroquinase-plasminogen activator receptor (CD87), or the LPS receptor CD14, providing these surface-bound molecules with transmembrane signalling, and modulating their activities. In this regard, inhibition of neutrophil phagocytosis by anti-CD11b/CD18 has been extensively reported. On the other hand, little is known concerning anti-CD11b/CD18 effects on tissue macrophages carrying out other effector functions, specially under inflammatory conditions. Although phagocytosis, ADCC and IC-Ctx are all FcγR-dependent functions, the intracellular pathways and lytic mechanisms involved in each reaction are quite different.24 In addition, monocytes and tissue macrophages employ different molecular mechanisms from those used by PMN for the same activity.24 In this context, we report new and additional CD11b/CD18 modulatory action on effector immune functions. The data presented herein strongly suggest that LPS enhances FcγR-dependent functions: ADCC and IC-Ctx, through the up-regulation of CD11b/CD18. We demonstrated that anti-CD11b/CD18 antibody inhibits FcγR-dependent functions in a temperature and time dependent manner. Anti-CD11b/CD18 inhibits splenocyte activities specially when previously amplified by a systemic inflammatory process, such as LPS in vivo treatment, which leads to the up-regulation of CD11b/CD18 cell-surface expression. However, basal cytotoxicity in control mice was not significantly modified. In this regard, PMN from patients with leucocyte adhesion deficiency (LAD), expresing a β2 integrin deficiency, exhibit normal Fcγ-mediated phagocytic function in the absence of stimulation. However, they fail to amplify ingestion of either IgG- or C4b-opsonized targets in response to stimulation by phorbol esters, cytokines or Arg-Gly-Asp (RGD)-containing adhesive proteins. Both results, taken together, strongly suggest that during inflammatory situations enhancement of Fcγ receptor-dependent functions is associated with up-regulation of CD11b/CD18.

On the contrary, PMA-induced cytotoxicity, which is also increased by LPS treatment, was not affected by anti-CD11b/CD18 antibody. These results would indicate that the interaction of monoclonal antibody with the integrin act on the earliest steps before PKC activation. Anti-CD11b/CD18 also failed to inhibit OpZy-Ctx enhanced by in vivo LPS, indicating that anti-CD11b/CD18 monoclonal antibody did not disrupt the signalling pathway triggered by the simultaneous recognition of iC3b via a CD11b I-domain and the polysaccharides via a lectin site.39

The observed inhibition of LPS-enhanced Fcγ-dependent functions by anti-CD11b/CD18 antibody could potentially be explained by two different mechanisms. Firstly, antibody to CD11b/CD18, either whole IgG or F(ab′)2 fragments, could sterically interfere with FcγR on splenocytes or down regulate its expression upon co-capping. However, we could not obtain downregulation of FcγR after anti-CD11b/CD18 incubation either at 4° (steric interference) or at 37° (down-regulation upon co-capping), evaluated by cytometric assays. These results are in concordance with Annenkov et al.,40 who did not find any direct evidence of cross-blocking between CD11b and FcγR in the K562 cells. Second, β2 integrins could participate as active co-signalling molecules which would affect the cellular response to signals transmitted via Fc receptors. The results presented herein favour the interpretation that β2 integrins activated by inflammatory mediators could co-operate in transmitting intracellular signals triggered by FcγR. Moreover, the ligation of CD11b/CD18 receptor with its specific antibody, or with the physiological ligand fibrinogen, could interfere with a positive signal transduced by activated β2 integrins or trigger a negative signal initiated after binding. Recently, Fukushima et al.41 reported evidence that a similar mechanism is involved in the activation of Na+/H+ exchange upon FcγR-binding in neutrophils. Considerable progress has been made in the last few years in defining heterogeneity for IgG receptors through their molecular cloning. These studies make it apparent that FcγR share structurally related ligand binding domains, but differ in their transmembrane and intracellular domains.42 These structural differences account for different cellular signalling pathways. In this regard, our results lead us to speculate that overexpression and/or activation of CD11b/CD18 enable specific FcγR to transmit signals through integrin molecules. Overexpression of integrins on cellular membrane probably increases the proximity between these molecules, allowing for close physical associations.

To understand how integrins regulate these events, it is critical to identify and characterize the intracellular signalling pathways activated by integrin–ligand interactions. In this regard it has been described that ligand occupancy of β2 integrins lead to multiple intracellular events such as interaction with cytoskeleton43 and tyrosine phosphorylation.21

In conclusion, new and compelling evidence would indicate that multiple and common receptor-dependent intracellular pathways can synergize or antagonize in order to regulate cell function. A better understanding of these processes could contribute to future manipulation of immune functions. Although further in vivo investigation is necessary, it is interesting to speculate that the anti-inflammatory in vivo effects of the anti-β chain antibodies seen in various pathological conditions could be explained by the inhibition of cytotoxic reactions. Finally, the results obtained with fibrinogen have physiological implications, considering that it is an important acute phase protein normally present in plasma. The role of fibrinogen in regulating leucocyte functions during inflammation, remains an unexplored field of investigation.

Acknowledgments

The authors thank Nora Galassi, Norma Riera, Juan Portaluppi and Antonio Morales for their excellent technical assistance and Dr Jorge Geffner for reviewing the manuscript. We also thank Fundación de la Hemofilia and Academia Nacional de Medicina for the use of the FACScan flow cytometer. This work was supported by a grant from Consejo Nacional de Ivastigaciones Cientificas y Tecnológicas (CONICET) and Fundación Alberto J. Roemmers and An torchas.

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