.................................................................................................................................................................................................. Downregulation of Mac-1 Expression in Monocytes by Surface-Bound IgG P. X. FernaÂndez-Calotti, G. Salamone, R. Gamberale, A. Trevani, M. Vermeulen, J. Geffner &M. Giordano Abstract Laboratorio de ImmunologõÂa, IIHEMA, Academia Physical and functional association between the b2-integrin Mac-1 (CD11b/ Nacional de Medicina, Buenos Aires, Argentina CD18) and receptors of immunoglobulin G(IgG)(Fc gRs) has been previously reported. In this study, we examined the modulation of Mac-1 expression by Received 26 June 2002; Accepted in revised form 4 September 2002 IgGin different leucocyte populations. Our data show that human monocytes, but not neutrophils, macrophages, dendritic or natural killer cells, downregulate Correspondence to: Dr M. Giordano, Laboratorio de the expression of Mac-1 after overnight exposure to surface-bound IgG. This ImmunologõÂa, Academia Nacional de Medicina, effect, which requires at least 6 h of incubation, is not associated with a general IIHEMA, Pacheco de Melo 3081, Buenos Aires 1425, downmodulation of membrane antigens, and is selectively induced by immobilized Argentina. E-mail: mirtagiordano@imaginaria. com.ar IgG(iIgG),as the stimulation of monocytes with N-formyl-methionyl-leucyl- phenylalanine, lipopolysaccharide, tumour necrosis factor-a (TNF-a)orsoluble IgGdid not modify the Mac-1 expression after 18 h in culture. The loss of Mac-1 was completely prevented by blocking antibodies (Abs) directed to FcgRII (CD32) or CD18. On the other hand, the serine protease inhibitor, phenyl methyl sulphonyl fluoride, but not inhibitors of cysteine proteases or neutral endopeptidases, partially prevented the downregulation of Mac-1 by iIgG. Monocytes cultured overnight on iIgG exhibited a dramatic decrease in their capacity to ingest zymosan particles that could be attributed to the reduced expression of Mac-1. However, there was no inhibition of TNF-a production induced by zymosan, suggesting that Mac-1-dependent responses require different levels of its expression to be fully activated. Introduction via FcgR, as was shown by its impairment with anti- Mac-1 (CD11b/CD18 or CR3) is a member of the Mac-1 Fab fragments and by the fact that patients lacking b2-integrin family which also includes LFA-1 (CD11a/ b2-integrins exhibit depressed immunoglobulin G(IgG)- CD18) and gp150/95 (CD11c/CD18 or CR4). These dependent effector activity [8±10]. receptors share a common b-subunit that is noncovalently Given the central role of Mac-1 in leucocyte biology, associated with unique but closely related a-chains [1, 2]. considerable effort has been devoted to elucidate the Mac-1, which is expressed by neutrophils, monocytes, mechanisms that regulate its surface expression. Our macrophages, dendritic and natural killer (NK) cells, interest has focussed on the modulation of Mac-1 by IgG plays an important role in cell adhesion through interaction in leucocytes that coexpress FcgRs. Previous studies of with intercellular adhesion molecule-1 (ICAM-1) (CD54) neutrophils demonstrated that Mac-1 interacts both [3, 4]. In addition, Mac-1 is a receptor of several well- functionally and physically with FcgRIIIB. Direct physical defined ligands, such as fibrinogen, complement fragment association was first suggested by experiments in which C3bi, coagulation factor X and heparin [1, 4]. On activation capping of one receptor resulted in the co-capping of the by ligand binding, Mac-1 is able to initiate signalling, other [11]. Further studies confirmed structural association which results in cellular responses, such as phagocytosis, between a lectin-like binding site present on Mac-1 and a respiratory burst and degranulation [1, 5, 6]. On the other putative carbohydrate moiety on FcgRIIIB [12, 13]. hand, Mac-1 has been recognized as a receptor that In contrast to what was observed in neutrophils, physical cooperates with FcgRs in promoting effector functions [7]. linkage between FcgR and Mac-1 involving lectin-like Thus, Mac-1 is required for the optimal phagocytosis interactions has not been found in mononuclear # 2003 Blackwell Publishing Ltd. Scandinavian Journal of Immunology 57, 35±44 35 36 Modulation of Mac-1 by Surface-Bound IgG P. X. FernaÂndez-Calotti et al. .................................................................................................................................................................................................. phagocytes [14]. Nevertheless, the treatment of monocytes were obtained from PeproTech (Rocky Hill, NJ, USA). with aggregated IgGconcomitantly reduces the expression of Human recombinant tumour necrosis factor-a (TNF-a) both receptors [15], while this effect could not be observed in was purchased from Sigma. murine macrophages treated with surface-bound IgG[16]. The IgGaggregates (aIgG)were prepared by heating The aim of the present study was to examine the human IgGat a concentration of 5 mg/ml for 12 min at modulation of Mac-1 expression by IgGin different leuco- 63 C. Then, aIgGwas centrifuged at 10,000 Â g for cyte populations. The data presented show that monocytes, 5 min and the precipitate discarded. but not macrophages, dendritic cells, NK cells or neutro- To prepare Zy-fluorescein isothiocyanate (Zy-FITC) phils, downregulate the expression of Mac-1 after prolonged particles, Zy A from Saccharomyces cerevisiae (Sigma) was exposure to immobilized IgG(iIgG). This effect, which suspended at a concentration of 10 mg/ml in phosphate- requires the interaction of both FcgRII and Mac-1 with buffered saline (PBS). FITC at 1 mg/ml was dissolved IgG-coated surface, is dependent on microfilament assembly in carbonate buffer, pH 9.8, and incubated with Zy and the activation of serine proteases. suspension at a concentration of 100 mg/ml, pH 7.2, for 2 h at room temperature on a rotating shaker. Unbound FITC was removed by extensive washings. Cell purification. Peripheral blood mononuclear cells Materials and methods (PBMCs) and neutrophils were obtained from heparinized Antibodies and reagents. The following monoclonal anti- samples of healthy donors. PBMCs were isolated by bodies (MoAbs) were obtained from Coulter-Immunotech centrifugation on Ficoll-Hypaque, washed twice and (Marseille, France): anti-CD1a (clone BL6, IgG1), anti- resuspended in RPMI-1640, supplemented with 10% CD11a (clone 25.3, IgG1), anti-CD11b (clone Bear 1, FCS and antibiotics (complete medium). Cell viability IgG1), anti-CD11c (clone BU15, IgG1), anti-CD18 was >98%, as determined by Trypan blue exclusion. (clone 7E4, IgG1), anti-ICAM-1 (clone 84H10, IgG1), Monocytes were purified by centrifugation on a dis- anti-very late activation antigen-4 (anti-VLA-4) (clone continuous Percoll gradient, with modifications of a HP2/1), anti-CD14 (clone RM052, IgG2a) and anti- previously described method [19]. Briefly, PBMCs were CD56 (clone C218, IgG1). The following MoAbs were suspended in Ca,Mg-free Tyrode's solution supple- 0 purchased from Medarex (Annandale, NJ, USA): F(ab )2 mented with 0.2% EDTA and incubated for 30 min fragments of anti-FcgRI (clone 197, IgG2a) and Fab at 37 C. During this incubation, the osmolarity of the fragments of anti-FcgRII (clone IV.3, IgG2b). Anti-CD18 medium was gradually increased from 290 to 360 mosmol (clone TS 1/18) was obtained from the American Type by the addition of 9% NaCl. Three different Percoll Culture Collection (Rockville, MD, USA). Rabbit IgG fractions were layered in polypropylene tubes: 50% at the anti-ovalbumin (anti-OVA) was prepared as previously bottom, followed by 46 and 40%. PBMCs (5±10 Â 106/ml) 0 described [17]. F(ab )2 fragments of anti-CD18 (TS 1/18) were layered at the top, and they were centrifuged at and anti-OVA IgGwere prepared in our laboratory by pepsin 400 Â g for 20 min at 4 C. Monocytes were recovered at digestion and further purified by Protein A Sepharose, 50±46% interface. The purity was checked by FACS according to standard techniques [18]. For blocking studies, analysis using anti-CD14 MoAbs and was found to be monocytes were preincubated with the corresponding MoAb >90%. Viability was consistently >85%. for 15 min at 4 C. Concentrations of MoAb three- to five- Neutrophils were isolated by centrifugation on Ficoll- fold higher than those needed to saturate all binding sites Hypaque and Dextran sedimentation, as previously described (1±10 mg/ml), as determined by fluorescence-activated cell [20]. Contaminating erythrocytes were removed by hypotonic sorter (FACS) analysis, were employed in these studies. lysis. After washing, the cells (>92% neutrophils on May± Ficoll, Dextran T-500 and Percoll were obtained from GruÈnwald±Giemsa-stainedCytopreps,>95%viablecells)were Pharmacia Fine Chemicals (Uppsala, Sweden); Hypaque resuspended at desired concentration in complete medium. was obtained from Winthrop Products (Buenos Aires, To induce the in vitro differentiation to macrophages, Argentina). Culture medium RPMI-1640 was purchased monocytes were cultured for 6 days at a cell density of from HyClone Logan, UT, USA). Fetal calf serum (FCS), 1 Â 106 cells/ml in complete medium supplemented with penicillin/streptomycin and Trypsin±EDTA (ethylenedia- 40 ng/ml of GM-CSF. At the end of the incubation, minetetraacetic acid) were from Life Technologies (Grand macrophages were gently scraped off with a rubber Island, NY, USA). OVA, herbimycin A, N-formyl-methio- policeman and