INTRODUCTION

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The receptors for the Fc portion of immunoglobulin G (IgG) (FcRs) are widely distributed in cells of the immune system and have been considered a link between cellular and humoral immunity by serving as a bridge between antibody specificity and effector cell functions. They also enable monocytes/macrophages and neutrophils to exert regulatory functions, as well as to trigger a variety of cytotoxic mechanisms (8, 9).

Three different classes of FcRs, FcRI, FcRII, and FcRIII, have been identified on human leukocytes through the use of monoclonal antibodies (MAbs), functional analysis, and the molecular characterization of the primary structure. One of them, FcRI, is a high-affinity receptor capable of induction of phagocytosis, clearance of immune complexes, respiratory burst, antibody-dependent cell-mediated cytotoxicity (ADCC), enhancement of antigen presentation, and secretion of inflammatory cytokines (28). This receptor is expressed primarily on mononuclear phagocytes, and this expression is regulated by cytokines and hormones such as gamma interferon (IFN-) (24), IFN- (29), interleukin-10 (IL-10) (30), granulocyte colony-stimulating factor (5), and glucocorticoids (13).

The monocyte/macrophage activation by IFN- is characterized by a pronounced increase in FcRI expression that frequently results in a concomitant enhancement of several FcR-dependent functions (7, 14, 24). This effect has also been shown in vivo on circulating monocytes of cancer patients who received IFN- treatment (20). Surprisingly, a similar upregulation can be induced by IL-10 (30), a cytokine known by its anti-inflammatory activity. On the other hand, IL-1 and IFN- downregulate IFN--induced FcRI expression (3, 29), while corticoids can either enhance or inhibit this FcRI expression, depending on whether cells have been primed with IFN- (21).

We have recently demonstrated that the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP), a prototype of N-formyl peptides, is capable of inducing a time-dependent downregulation of both FcRIIIB and FcRII in human neutrophils, altering different FcR-dependent functions (1). These N-formyl peptides are released at the site of infection as a consequence of bacterial destruction by the immune system or by autolysis.

Considering the biological relevance of FcRI, the regulation of this receptor would be of fundamental importance in different immunological mechanisms. Therefore, taking into account our previous results and the pleiotropic and proinflammatory activities of the formyl peptides on the immune system (26, 27), we investigated the possible effect of FMLP on the induction of FcRI by both IFN- and IL-10 on human monocytes. In addition, we studied other important effects induced by IFN- such as the overexpression of molecules of the major histocompatibility complex (MHC) class II and the enhancement of lipopolysaccharide (LPS)-induced secretion of tumor necrosis factor alpha (TNF-).

	MATERIALS AND METHODS

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Reagents. FMLP, Ficoll 400, tissue culture medium (RPMI 1640 medium), LPS from Escherichia coli O111:B4, anti-sheep red blood cell (anti-SRBC) antibody, human recombinant IFN-, human recombinant IL-10, anti-IL-1 polyclonal antibody, and catalase were obtained from Sigma, St. Louis, Mo. Fluorescein isothiocyanate-labeled anti-FcRI (clone 10.1) and FcRIII (clone 3G8), phycoerythrin-labeled anti-FcRII (clone C1KM5) MAbs, and mouse IgG1 (clone MOPC21) isotype control antibody were obtained from CALTAG Laboratories, Burlingame, Calif. Phycoerythrin-labeled anti-CD14 (clone RMO52) and mouse IgG2a (clone U7.27) isotypes were from Immunotech, Marseille, France. Fluorescein isothiocyanate-labeled anti-HLA-DR (clone L243) was purchased from Becton Dickinson, San Jose, Calif.

Preparation of human mononuclear cells. Fresh human blood was obtained by venipuncture from healthy adult volunteers and was placed in citrate (84%)-dextrose (84%)-adenine (0.003%). Blood was diluted 1:2 with saline, layered on a Ficoll-Hypaque cushion, and centrifuged at 400 × g for 25 min as described previously (6). Peripheral blood mononuclear cells (PBMCs) were collected, washed, and resuspended in RPMI 1640 medium with 10% heat-inactivated (56°C, 30 min) fetal calf serum and 50 µg of gentamicin per ml. The viability of the mononuclear cells was always more than 98%, as measured by the trypan blue exclusion test.

FMLP treatment. FMLP was diluted in dimethyl sulfoxide at a concentration of 0.1 M. The subsequent dilutions of FMLP were made in saline. PBMCs were incubated with FMLP at a concentration of 1 µM in polypropylene tubes unless stated otherwise. After 1 h of incubation at 37°C in 5% CO₂, the cells were washed and incubated with either 240 U of IFN- per ml or 100 U of IL-10 per ml for 24 h. After this period the cells were used as effector cells in ADCC and phagocytosis assays or were used for flow cytometry studies.

ADCC assay. When total PBMCs at 4 × 10⁶ cells/ml (100 µl) were used as effector cells, ADCC assay was performed in 96-well polystyrene plates. The adherent PBMC population was obtained from 4 × 10⁶ PBMCs/ml that had been left to adhere in 96-well round-bottom plates for 1 h at 37°C. After that, the nonadherent cells were removed and ADCC assay was performed. In both cases the cells were incubated with 10^{5 51}Cr-labeled chicken red blood cells (⁵¹CRBCs) and a suboptimal concentration of rabbit IgG anti-CRBCs as described previously (12). After 18 h of incubation at 37°C in 5% CO₂, the culture plate was centrifuged and the percentage of cytotoxicity was calculated as follows: percent ADCC = (amount of ⁵¹Cr released into the supernatant × 100)/total amount of radioactivity. This value was corrected by subtracting the percentage of ⁵¹Cr released in the absence of antibody (spontaneous release). Quadruplicates were set up for each sample.

Phagocytosis assay. One hundred microliters of human mononuclear cells at a concentration of 2 × 10⁶ cells/ml were seeded onto slides and allowed to adhere for 1 h at 37°C. This procedure was repeated once under the same conditions. Then, the samples were rinsed with RPMI 1640 medium to remove nonadherent cells and incubated with 100 µl of a suspension of IgG-sensitized 1% SRBCs for 30 min at 37°C. After this period, nonphagocytosed SRBCs were removed by hypotonic lysis and the cells were stained with May-Grünwald and Giemsa stains. At least 100 cells per donor were counted. The phagocytic index represents the percentage of monocytes/macrophages containing erythrocytes (percent phagocytosis) (18). The ingestion of unsensitized SRBCs (control group) was less than 2% in all cases.

Measurement of TNF-. TNF--sensitive actinomycin D-treated murine L-929 fibroblasts were used to quantify TNF- activity by the method of Wang et al. (31). One milliliter of mononuclear cells at a concentration of 10⁶ cells/ml was incubated with different agonists and LPS (1 µg/ml) for 24 h at 37°C in 5% CO₂. After this period, the cells were centrifuged and supernatants were removed for TNF- evaluation. The amount of TNF- secreted by cells not treated with LPS was negligible. The results are expressed as 50% lytic units.

Flow cytometry. After different treatments, 10⁶ mononuclear cells were washed and incubated with the MAbs indicated above. The cells were washed and resuspended in ISOFLOW, and flow cytometry was performed on a fluorescence-activated cell sorter analyzer (Becton-Dickinson Immunocytometry Systems, San Jose, Calif.). The results were expressed as the percentage of the median fluorescence intensity (MFI) for CD14-positive cells (CD14⁺) compared with that for nontreated groups (controls). In some experiments permeabilization of PBMCs for staining of intracellular FcRI was performed. Briefly, PBMCs were treated with FMLP or IFN-, or both. After 24 h of incubation, the cells were fixed for 30 min with cold 0.5% paraformaldehyde. Aliquots of these cells were permeabilized in a buffer containing 0.04% saponin, 0.1% ovalbumin, phosphate-buffered saline, and glycine and were then stained with the MAbs. Parallel aliquots of fixed PBMCs were treated identically but without saponin for nonpermeabilized controls. Background fluorescence intensity was obtained by using mouse IgG1 isotype-matched control antibody.

Statistical analysis. All experiments were repeated at least five times, and the results of a representative experiment are presented unless stated otherwise. The statistical significance of the results was calculated by the nonparametric Mann-Whitney or Wilcoxon test (two tailed).

	RESULTS

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FMLP downregulates IFN--induced expression of FcRI. As described previously (24), FcRI, which is constitutively expressed on human monocytes, was significantly upregulated by 240 U of human recombinant IFN- per ml after 24 h of incubation. However, incubation of the cells with 1 µM FMLP for 1 h, which did not modify the basal level of expression of FcRI, exerted a drastic inhibition of the FcRI upregulation induced by IFN- (Fig. 1). The untreated cells (controls) were incubated overnight under the same conditions used for the experimental groups. The magnitude of the FcRI expression after FMLP treatment was different with PBMCs from various cell donors, as follows. After treatment with FMLP, IFN-, or both FMLP and IFN-, the magnitudes of FcRI expression were 96 ± 2, 238 ± 32 (significantly different [P < 0.0002] from that for the control), and 130 ± 12 (significantly different [P < 0.05] from those for the control and IFN- treatment), respectively (the data are percent MFI relative to the control ± standard errors for the CD14⁺ population of PBMCs [n = 12]). In this experiment PBMCs (10⁶/ml) were incubated with medium or 1 µM FMLP for 1 h. Then, the cells were washed and incubated with medium or IFN- (240 U/ml) for 24 h. After this period, the PBMCs were stained with anti-FcRI and anti-CD14 antibodies. Statistical significance was calculated by Mann-Whitney test (two tailed). This variability can be attributed to the susceptibilities of the individual donors to FMLP or IFN- and/or to the heterogeneity of monocyte subpopulations. These results were observed by using concentrations of FMLP as low as 0.01 µM (percent MFIs of that for the control were as follows: IFN-, 284%; 0.01 µM FMLP plus IFN-, 173%; 0.01 µM FMLP, 109% [the data are representative of four experiments]). Concentrations of 1 nM had no effect (data not shown). It is noteworthy that in these experiments PBMCs were exposed for 1 h to FMLP, and after this period the cells were washed and incubated with IFN- for 24 h in the absence of FMLP. Similar results were achieved by incubation of human mononuclear cells with 1 µM FMLP for 5 min instead of 1 h (n = 3). Dimethyl sulfoxide alone at the same concentration used for the dilution of FMLP (see Materials and Methods) had no effect. In addition, the FMLP-induced FcRI downregulation was observed when we used 500 or 1,000 U of IFN- per ml. These concentrations induce the maximal expression of FcRI, indicating that larger amounts of the cytokine did not overcome the effect of 1 µM FMLP (n = 2; data not shown). These results were also observed when PBMCs were incubated with FMLP plus IFN- for 48 h.

View larger version (29K): [in this window] [in a new window]   FIG. 1.   Effect of FMLP on IFN--induced FcRI expression. PBMCs (106 cells/ml) were incubated with medium or 1 µM FMLP for 1 h. Then, the cells were washed and incubated for 24 h with medium (a) or IFN- (240 U/ml) (b). After this period, the PBMCs were stained with anti-FcRI and anti-CD14 antibodies. The histograms represent the CD14+ population of PBMCs and correspond to a representative experiment of 12 experiments conducted. The background fluorescence intensity (filled peak) was obtained with control isotype antibodies. The x axis represents fluorescence intensity (arbitrary units); the y axis represents the cell number.

FMLP downregulates IL-10-induced expression of FcRI. It has previously been reported that IL-10 is also capable of inducing an increase in the level of expression of FcRI on human monocytes (30). As shown in Fig. 2 and below, preincubation of PBMCs with 1 µM FMLP for 1 h was also able to inhibit the FcRI upregulation induced by 100 U/ml of IL-10. After treatment with 1 µM FMLP, IL-10, 1 µM FMLP plus IL-10, 0.01 µM FMLP, and 0.01 µM FMLP plus IL-10, the magnitudes of FcRI expression were 107 ± 5 (n = 7), 190 ± 17 (n = 9), 142 ± 9 (n = 9), 116 ± 2 (n = 9), and 135 ± 8 (n = 9) (the data are percent MFI relative to the control ± standard errors for the indicated numbers of experiments). Data for the IL-10 treatment were significantly different (P < 0.0002) from those for the control. Data for the 1 µM FMLP plus IL-10 and 0.01 µM FMLP plus IL-10 treatments were significantly different (P < 0.05) from those for the control and were significantly different (P < 0.05) from those for the IFN- treatment. PBMCs (10⁶ cells/ml) were incubated with medium, 1 µM FMLP, or 0.01 µM FMLP for 1 h. Then, the cells were washed and incubated with medium or IL-10 (100 U/ml) for 24 h. After this period, PBMC were stained with anti-FcRI and anti-CD14 antibodies. Statistical significance was calculated by the Mann-Whitney test (two tailed). Similar results were obtained with 200 or 400 U of IL-10 per ml at either 24 or 48 h of incubation (data not shown). Although monocytes also express FcRII and FcRIII on their membranes, this expression was not affected by treatment with either FMLP, IL-10, or IFN- or any combination of them (data not shown).

View larger version (39K): [in this window] [in a new window]   FIG. 2.   Effect of FMLP on IL-10-induced FcRI expression. PBMCs (106 cells/ml) were incubated with medium or 1 µM FMLP for 1 h. Then, the cells were washed and incubated with IL-10 (100 U/ml) for 24 h. After this period, the PBMCs were stained with anti-FcRI and anti-CD14 antibodies. The histograms represent the CD14+ population of PBMCs and correspond to a representative experiment of eight experiments conducted. The background fluorescence intensity (filled peak) was obtained with control isotype antibodies. The x axis represents fluorescence intensity (arbitrary units); the y axis represents the cell number.

FMLP does not alter other immunoregulatory actions of IFN-. It is well known that IFN- also enhances the expression of MHC class II antigens and the release of TNF- from LPS-stimulated mononuclear phagocytes (10). With the purpose of investigating whether FMLP was also capable of modifying these IFN--induced effects, PBMCs were incubated with 1 µM FMLP for 1 h and were then washed and treated with LPS (1 µg/ml) plus 240 U of IFN- per ml for 24 h at 37°C. After that, the cells were centrifuged and the supernatants were collected for TNF- evaluation. On the other hand, after FMLP treatment, PBMCs were incubated with IFN- for 24 h and MHC class II antigen expression was analyzed by flow cytometry. As depicted in Table 1, neither the overexpression of MHC class II antigens nor the enhancement of TNF- secretion induced by IFN- was modulated by FMLP.

View this table: [in this window] [in a new window]   TABLE 1.   Effect of FMLP on the IFN--induced HLA-DR expression and enhancement of TNF- secretion

Effect of FMLP on ADCC and phagocytosis. The upregulation of FcRI induced by IFN- could correlate with FcR-dependent functions such as ADCC and phagocytosis (24). As shown in Fig. 3, the enhancement of ADCC by IFN- 240 U/ml was completely inhibited by 1 µM FMLP in total PBMCs. However, when ADCC was carried out with adherent cells, an enriched monocyte preparation, FMLP inhibited the enhancement of cytotoxicity only in the presence of catalase. This means that the adherence of monocytes to the plates activates these cells, which exert part of their cytotoxic effect by oxygen radicals, masking the conventional ADCC (essentially independent of oxygen radicals), as shown previously (17).

View larger version (36K): [in this window] [in a new window]   FIG. 3.   Effect of FMLP on ADCC enhancement induced by IFN- in total and adherent PBMCs. PBMCs (4 × 106 cells/ml) were incubated in polypropylene tubes with medium or 1 µM FMLP for 1 h. Then, the cells were washed and incubated with medium or IFN- (240 U/ml) for 24 h. After this period, ADCC assay was performed (see Materials and Methods). The adherent PBMC population was obtained as indicated in Materials and Methods and was treated with 1 µM FMLP and IFN- as indicated for PBMCs. After 24 h of incubation, ADCC assay was performed on the 96-well round-bottom plates in the presence or absence of catalase (5,650 U/ml). The figure shows the results of a representative experiment of six experiments conducted. Data are expressed as percent ADCC of that for the control ± standard deviation *, significance (P < 0.05) of difference for results of six experiments compared to the results for IFN--treated cells by the Wilcoxon test (two tailed).

View larger version (31K): [in this window] [in a new window]   FIG. 4.   Effect of FMLP on ADCC enhancement induced by IL-10 in adherent PBMCs. Adherent PBMCs were obtained as indicated in Materials and Methods and were incubated with medium or 1 µM FMLP for 1 h. Then, the cells were washed and incubated with medium or IL-10 (100 U/ml). After 24 h of incubation, ADCC assay was performed on the 96-well round-bottom plates in the presence or absence of catalase (5,650 U/ml). The figure shows the results of a representative experiment of six experiments conducted. Data are expressed as percent ADCC of that for the control ± standard deviation. *, significance (P < 0.05) of difference for results of six experiments compared to the results for IL-10-treated cells by the Wilcoxon test (two tailed).

View larger version (23K): [in this window] [in a new window]   FIG. 5.   Effect of FMLP on IFN--induced enhancement of phagocytosis. PBMCs (2 × 106 cells/ml) were incubated with medium or 1 µM FMLP for 1 h. Then, the cells were washed and incubated with medium or IFN- (240 U/ml) for 24 h. After this period, the level of phagocytosis was determined (see Materials and Methods) (n = 5), *, significantly different (P < 0.008) from the control by the Mann-Whitney test (two tailed).

	DISCUSSION

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When bacteria are destroyed either by autolysis or by cytotoxic mechanisms, formyl peptides (26, 27) are released into the milieu, establishing a close contact with neutrophils and monocytes, the most representative cells in acute and chronic inflammation, respectively (15).

The aim of the study described here was to examine the effect of FMLP, a prototype of N-formyl peptides, on the expression of the high-affinity Fc receptor (FcRI), which is a key effector molecule in monocyte/macrophage function. We were able to demonstrate that FMLP induced a significant and dose-dependent downregulation of IFN--induced FcRI expression. As shown in Fig. 1, although this formyl peptide strongly inhibited the IFN--dependent FcRI expression on human monocytes, the basal level of expression of this receptor was not altered by FMLP treatment.

The FMLP downregulatory effect was not observed when naive PBMCs were incubated with supernatants from FMLP-stimulated monocytes, suggesting that the mechanism responsible for FcRI downregulation in these cells is different from those described for FcRII and FcRIII in human neutrophils (1). In fact, we have previously demonstrated that FMLP induces the release of a serine protease(s) (2), which in turn downregulates the basal levels of FcRII and FcRIII expressed on these cells.

Our results demonstrate that FMLP induces a rather specific action on FcRI expression since the enhancement of LPS-dependent TNF- secretion and the MHC class II antigen upregulation induced by IFN- were not altered by FMLP treatment. These results suggest that the effect of FMLP would not be exerted at the first steps of the IFN- transduction signal cascade, in which the activation pathway is common for different signals (11, 29). Thus, central molecules from the IFN- signaling pathway such as the receptor for IFN-; JAK1 and JAK2 kinases, or the transcription factor STAT1 cannot be altered (4, 16, 19, 25). Whether FMLP acts on a specific step in the pathway of FcRI upregulation is not known since this is a complex mechanism that depends on the IFN- response region localized in the promoter of the FcRI gene and multiple transcription factors that have not been yet elucidated (22, 23). It has recently been reported that, similar to FMLP, IFN- has an antagonistic action on the IFN--induced FcRI expression (29). However, although this effect seems to be due to a posttranscriptional event, the mechanism of action is not known. In our experiments, the pool of FcRI was not modified by FMLP on IFN--treated cells, suggesting a posttranscriptional event.

The FMLP-induced FcRI downregulation is an early event since incubation of cells with FMLP for 5 min was sufficient for inhibition of IFN--induced FcRI upregulation. In addition, we observed that this effect was reversible, discarding any toxic effects, and that the presence of FMLP throughout the experiment was not necessary. Taking into account the fact that FMLP also inhibited the FcRI upregulation induced by IL-10 (Fig. 2), we can speculate that FMLP acts on a common step in the pathway of FcRI upregulation shared by IFN- and IL-10.

It has also been reported that LPS is capable of inducing FcRI downregulation through the secretion of IL-1 (3). However, in our experiments, the effect of FMLP can not be ascribed to this cytokine since the incubation with the formyl peptide in the presence of anti-IL-1 antibody did not modify the FMLP downregulatory effect.

To investigate whether the observed differences in FcRI expression could have any physiological relevance, FcR-dependent cytotoxic mechanisms such as phagocytosis and ADCC were assayed. The formyl peptide inhibited the IFN- enhancement of ADCC carried out by total and adherent PBMCs. Moreover, the inhibition of IFN--dependent enhancement of phagocytosis by FMLP confirms the monocyte/macrophage lineage of the cells and indicates that the upregulation of FcRI and FcR-dependent functions were tightly linked. Since the basal levels of phagocytosis and ADCC were not modified by FMLP treatment, it indicates that FMLP acts only on the overexpression of FcRI, without modifying the functional ability of this receptor.

Meanwhile, although FMLP completely inhibited the IFN-- and IL-10-induced enhancement of ADCC and phagocytosis, the overexpression of FcRI was only partially inhibited. This indicates that even when the level of expression of FcRI does not return to basal levels upon FMLP treatment, the remaining receptors are not enough to increase effector functions significantly.

As far as we know, this is the first description of the inhibitory effect exerted by FMLP on either IFN-- or IL-10-induced FcRI upregulation. This is a paradox in which a prototype proinflammatory molecule (FMLP) exerts a dramatic anti-inflammatory effect. Another paradox has been observed with glucocorticoids, a typical anti-inflammatory drug, which can nevertheless behave as a proinflammatory agent (32). In the pathophysiology of bacterial infections, in which bacteria multiply and die at the site of infection and phagocytic cells could be exposed to high concentrations of N-formyl peptides, we can speculate that the inhibitory effect of FMLP on IFN-- and IL-10-induced FcRI upregulation can exert an important regulatory and/or anti-inflammatory effect during the evolution of bacterial infections.