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transforming growth factor-fl-induced expression of FcyRIII (CD16) on human monocytes ..... CD16 expression may imply that GM-CSF does not support.

Immunology 1996 87 162-167

Granulocyte-macrophage colony-stimulating factor antagonizes the transforming growth factor-fl-induced expression of FcyRIII (CD16) on human monocytes M. KRUGER,*t L. COOREVITSt T. P. M. DE WIT,t M. CASTEELS-VAN DAELEt J. G. J. VAN DE WINKELt & J. L. CEUPPENSt tThe Laboratory of Clinical and Experimental Immunology, Departments of Pathophysiology and Paediatrics, the Catholic University of Leuven, Belgium, and

tThe Department of Immunology, University Hospital, Utrecht, the Netherlands

SUMMARY Fc-y receptor III (FcyRIII, CD 16) type A is expressed on natural killer cells, on a small subset of peripheral blood monocytes and on mature macrophages. Along with differentiation into macrophages, monocytes will express FcyRI1I when cultured with transforming growth factor-,B (TGF-f,). In view of the involvement of granulocyte-macrophage colony-stimulating factor (GMCSF) in myeloid cell differentiation, we investigated the effect of this cytokine on FcyRIII expression in cultures of peripheral blood monocytes. GM-CSF antagonized TGF-fl-induced expression of FcyRIII on monocytes in vitro in a dose-dependent way. The effect of GM-CSF persisted in cultures until at least day 7. The suppression was at the mRNA level, as shown by Northern analyses with a CD1 6 specific probe, and the signalling pathway involved tyrosine kinase activity. Interferon-y and interleukin-2 had no effect on the induced expression of FcyRIII by TGF-f,, while interleukin-4, similar to GM-CSF, antagonized this induction. Our findings suggest that regulatory cytokine networks can drive monocytes into different effector functions and differentiation pathways.

differentiation of peripheral blood monocytes into macrophages, we have selected FcyRIII expression as a differentiating characteristic of macrophages.7'8 FcyRIII is one out of three different classes of IgG-Fc receptors on human phagocytic cells. All three receptor classes are members of the immunoglobulin superfamily.9 FcyRI (CD64) binds monomeric IgG with high affinity, while FcyRII (CD32) and FcyRIII (CD16) mainly interact with aggregated or immune complexed IgG. These receptors mediate phagocytosis of antibody-coated micro-organisms or cells, antibody-dependent cellular cytotoxicity (ADCC), clearance of immune complexes from the circulation and stimulate the generation of superoxide.9 Two topographic forms of FcyRIII exist: a transmembrane receptor, known as FcyRIIIA, on macrophages and natural killer cells, and a glycosyl-phophatidylinositol-linked molecule, known as FcyRIIIB on neutrophils.'0 The molecular weight of the class III receptor is between 50 and 80 and the extracellular region consists of 190 amino acids.9 FcyRIIIA, in addition, contains a 25 amino acid cytoplasmic region. Although most tissue macrophages express FcyRII1A expression, only about 10% of circulating peripheral monocytes are positive for this receptor class." Monocytes, kept in culture, acquire FcyRIII after 3 to 7 days, in parallel with morphological differentiation into macrophages, and the expression reaches a plateau after 14 days.7 FcyRIII expression has been shown to be induced by transforming growth factor-,B (TGF-fl).'2

INTRODUCTION

Granulocyte-macrophage colony-stimulating factor (GMCSF), a glycoprotein of 14000 to 35000MW, is a cytokine that induces proliferation and differentiation of myeloid precursors.' Activated T cells, fibroblasts, endothelial cells, macrophages and mast cells can all produce GM-CSF upon activation.2-5 Recombinant (r) GM-CSF has proven efficacy for treatment of neutropenic cancer patients, and stimulates myeloid progenitor cells, with resulting leucocytosis.6 In order to ascertain whether GM-CSF plays a role in the Received 8 March 1995; revised 26 June 1995; accepted 28 June 1995.

Abbreviations: BCS, bovine calf serum; FCS, fetal calf serum; FcyRIII, Fc-y receptor III; FITC, fluorescein isothiocyanate; GM-CSF, granulocyte-macrophage colony-stimulating factor; HIV, human immunodeficiency virus; IFN-y, interferon-y; IL, interleukin; mAb, monoclonal antibody; PBMC, peripheral blood mononuclear cells; PE, phycoerythrin; PMA, phorbol myristate acetate; TGF-,B, transforming growth factor-,B. *Present address: Department of Paediatrics, Kalafong Hospital and University of Pretoria, Private bag X396, Pretoria, Republic of South Africa 0001. Correspondence: Jan Ceuppens, Laboratory of Experimental Immunology, Gasthuisberg-Onderwijs en Navorsing, 49 Herestraat, 3000 Leuven, Belgium.

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GM-CSF inhibits CDJ6 expression on monocytes The studies described here, surprisingly, indicate that GMCSF antagonizes the TGF-fl-induced expression of FcyRIII on peripheral blood monocytes. MATERIALS AND METHODS

Cytokines The cytokines used were purified recombinant human gene products. GM-CSF for in vivo use was donated by ScheringPlough (Brussels, Belgium). It contained less than 0-06 ng endotoxin/mg GM-CSF. A second GM-CSF preparation, interleukin-2 (IL-2), interferon-y (IFN-y), and TGF-,B were purchased from Janssen Chimica (Beerse, Belgium). Interleukin-4 (IL-4) and tumor necrosis factor-at (TNF-a) were donated by Dr A. Van de Voorde, Innogenetics (Ghent, Belgium).

Monoclonal antibodies The monoclonal antibodies (mAbs) for the immunomagnetic monocyte separation method (see later) were as follows: UCHT-l (anti-CD3), was donated by Dr P. Beverley (University Hospital and Middlesex School of Medicine, London, UK); CLB-19 (anti-CD19) was obtained from the Central Laboratory of the Netherlands Redcross Blood Transfusion Service (CLB, Amsterdam, the Netherlands), and 3G8 (antiCD16) was from Medarex (Annandale, NJ). FcyRIII expression was detected by phycoerythrin (PE)-conjugated mAb LeulIc (mIgG1) (Becton-Dickinson, San Jose, CA) or fluorescein isothiocyanate (FITC)-conjugated 3G8 (mIgGl) (Medarex). The control mIgGl and mIgG2a, anti-CD45, anti-HLA-DR (IgG2a), Leu 15 (anti-CD llb, IgG2a), Leu 54 (anti-CD54, IgG2b) and Leu M3 (anti-CD14, IgG2b) were purchased from Becton-Dickinson. The mAb used against FcyRI and FcyRII were 22(IgGl) and IV.3(IgG2b), respectively, and were from Medarex.

Other reagents Phorbol myristate acetate (PMA), a protein kinase C activator, was from Sigma Chemical Co. (St Louis, MO). Herbimycin A, a tyrosine kinase inhibitor, was purchased from Gibco (Paisley, UK). Actinomycin D, an inhibitor of mRNA transcription, was obtained from Merck, Sharp and Dohme Research Laboratories (Harlow, Essex, UK). Anti-TGF-f, (polyclonal rabbit IgG) was from British Biotechnology Ltd. (Oxon, UK).

Cell isolation Heparinized blood (60 ml) was obtained from healthy volunteers (20 to 50 yr). Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Hypaque sedimentation (density 1 077). Monocytes were obtained by cold agglutination.'3 For certain experiments, as indicated in the results section, monocytes were further purified by an immunomagnetic method. The cells were incubated with anti-CD3 (UCHT-1), anti-CD16 (3G8) and anti-CD 19 (CLB-19) mAb for 30 min at 4°. Thereafter, cells were washed and incubated with goat-anti-mouse-coated Dynabeads (Dynal, Oslo, Norway) for a further 30 min at 40 (bead to cell ratio: 20:1). The suspension of cells and beads was brought into a magnetic field for 15 min and the monocytes were obtained by negative selection. The purity (as evaluated by CD14 expression) was between 70% and 80% and viability exceeded 95%. The monocytes, used for Northern blotting, were prepared by Dr R. van Schie (University of Nijmegen, the © 1996 Blackwell Science Ltd, Immunology, 87, 162-167

Netherlands) by counterflow centrifugal purity was 88% (viability > 95%).

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Cell culture Cells at a concentration of 0-5 x 106 cells/ml were cultured in RPMI-1640 medium (Gibco, Paisley, UK) without phenol red, supplemented with 2mm L-glutamine, penicillin (1OOU/ml), streptomycin (100 pg/ml) and 10% fetal calf serum (FCS) (unless otherwise indicated). The FCS was obtained from Gibco (Paisley, UK) and contained less than 0 03 ng/ml endotoxin. The final concentration of TGF-fl in the medium RPMI-1640/10% FCS was 2 ng/ml. The cells were cultured either in 12 x 75 mm polystyrene or polyethylene tubes (Falcon, Becton Dickinson Labware, NJ). There were no differences in the results obtained with the different types of tubes. A serumfree medium, macrophage-SFM medium (Gibco), was used in some experiments as indicated in the results section.

Immunofluorescence analysis The cells were incubated with different mAb at 40 for 30 min in phosphate buffered saline (PBS), supplemented with 10% normal human serum. The latter was used to block non-specific binding of mAb via FcR. The cells were washed twice and fixed in 1 ml of 2% Paraformaldehyde. Fluorescence intensity was analysed on a FACScan (Becton-Dickinson), using the Consort 30 program. Electronic gating, based on scatter characteriztics served to analyse the monocytes. The data were expressed as the percentage of positive cells or as the mean fluorescence intensity of 5000 cells (arbitrary units).

Northern blot analysis Total cellular RNA was isolated from monocytes after 12, 24, 48 and 72 hr culture with/without TGF-fl, or TGF-fi in combination with GM-CSF or IL-4, using the RNA zol B method (Cinna Biotecx, Friendswood, TX).8 Ten pg of RNA was fractionated by electrophoresis in 1 % agarose/formaldehyde gels, and transferred to nitrocellulose (BA85, Schleicher and Schuell, Dassel, Germany). Hybridization with a 900 basepair (bp) HdIII/EcoRI fragment of FcyRIIIb,10 labelled with (cx-32P) dcTP, using a random primer labelling kit (BRL, Bethesda, MD), was carried out overnight in 50% formamide, 3 x saline sodium citrate (SSC), 0-1% sodium dodecyl sulphate (SDS), 10 x Denhardt's solution, supplemented with 50 ug/ml denaturated Salmon sperm DNA at 420. Filters were washed in 0-2 x SSC/0 1% SDS at 420 for 30 min, and exposed to Kodak XAR films (Hemel Hempstead, UK). RESULTS TGF-,B induces the expression of CD16 on human monocytes During culture of PBMC or enriched monocytes, there was a progressive increase in the expression of CD16 on monocytes. Figure 1 shows the results of one representative experiment (out of 10) with an enriched monocyte suspension, illustrating the spontaneous induction of CD 16 expression in FCScontaining medium. It has to be noted that the phenomenon of CD16 induction was very consistent, but that the level of CD 16 expression, expressed in arbitrary units of fluorescence intensity, was quite variable between individuals and/or monocyte preparations. This induction has previously been

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Figure 1. CD16 expression on fresh and cultured1 monocytes. (a) Freshly isolated peripheral blood monocytes. (b) Mc)nocytes cultured for 48 hr in RPMI-1640/10% FCS. Cells were stainedI with LeulIc-PE (anti-CD16) (dotted line) or isotype control mIgG-P]E (solid line) and analysed on a FACSscan.

shown to be dependent on the presence of TGIF-fl in media.'2 The final TGF-j3 concentration in the mediuim, used in our experiments, was 2 ng/ml. Similar results of C'D16 induction were also obtained when monocytes were incubzated for 48 hr in dlemi-nfi-i with serum-free macrophage-SFM medium, suppYIUMULIMU WIL11' recombinant TGF-f in a final concentration c f 2 ng/ml (data not shown). Moreover, anti-TGF-f3-antiserum blockedindMItion of CD16 expression on monocytes, culti 1640/10% FCS (Fig. 2a). Actinomycin D, an inhibitor of mRN[A translation, proving antagonized the expression of CD16 (Fig. 2b), 1 therebproving its dependence on de novo protein synthesis. PM of protein kinase C, also completely dow nregulated the expression of CD16 (Fig. 2c). Surprisingly, C:D16 expression was also low or absent on monocytes cultured in the presence of GM-CSF (Fig. 2d).

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Figure 3. (a) The inhibitory effect of GM-CSF on CD16 expression is dose-dependent. Monocytes were cultured for 48 hr in medium alone and with increasing doses of GM-CSF. (b) Kinetics of CD16 expression in the presence and absence of GM-CSF (50 U/ml). Monocytes were cultured in RPMI-1640/10% FCS and stained with anti-Leul Ic-PE or isotype control mIgG-PE. The mean intensity of cells stained with isotype control mIgG was subtracted from the mean intensity of cells, stained with anti-CD16-PE.

Further analysis of the effect of GM-CSF on CD16 expression The antagonism of GM-CSF on the expression of CD16 was obvious for both the percentage, and for the mean fluorescence intensity of the cells (P < 0-01, n = 10). The effect of GM-CSF was dose dependent, with a maximal inhibitory effect at concentrations higher than 25 U/ml (Fig. 3a). In kinetic studies, the antagonistic effect of GM-CSF on CD 16 expression was already seen at 24 hr (Fig. 3b), and continued when the cells

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with two different mAbs, 3G8 and Leu 1 lc (B73- 1), to different epitopes of CDl615"16 yielded the same results. Monocytes were also cultured in serum-free medium (macrophage-SFM medium), supplemented with rTGF-# (2 ng/ml), with/without GM-CSF. The GM-CSF was added at different points in time before and after TGF-fl. Maximal inhibition of CD1 6 induction was seen when GM-CSF and TGF-f were added simultaneously at initiation of the cultures (data not shown). Previous reports showed GM-CSF to upregulate FcyRII expression on monocytes, and Fcc-receptor II (CD23) expression on eosinophils and monocytes, accompanied by increased phagocytosis through these receptors. 17,i8 In our experiments GM-CSF increased FcyRII expression as evidenced by increasing mean fluorescence intensities (data not shown). GM-CSF decreased the expression of FcyRI slightly, albeit that

downregulation was not statistically significant (data not shown). In accordance with a previous report we could confirm that IL-4 also antagonized the induction of CD16 on monocytes.'9 Two other T-cell-derived cytokines (interferon-y and IL-2) had no significant influence on the expression of CD 16, neither alone, nor in combination with TGF-3 (data not shown).

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Figure 2. Inhibition of CD16 expression on cultured monocytes. Monocytes were cultured for 48 hr in RPMI-1640/10% FCS and then labelled with Leu-llc-PE (dotted line and broken lines) or isotype control mIgG-PE (solid line). CD 16 expression is shown after culture in medium alone (dotted line) or with (broken lines): (a) anti-TGF-13 (10 jug/ml) (one representative experiment out of five), (b) actinomycin D (lIg/ml) (one experiment representative of two), (c) PMA (10 ng/ml), (d) GM-CSF (50 U/ml). (a), (b), (c) and (d) represent separate experiments of CD 16 induction using monocytes from different donors.

Tyrosine kinase inhibitors antagonize the effect of GM-CSF on CD16 expression GM-CSF receptors lack tyrosine kinase domains, but evidence has been obtained that signalling by GM-CSF involves cytoplasmic tyrosine kinases.20'21 We incubated monocytes with the combination of a tyrosine kinase inhibitor, herbimycin A, with or without GM-CSF, in RPMI-1640/10% FCS (Fig. 4). Herbimycin A had no effect on TGF-fl-induced © 1996 Blackwell Science Ltd, Immunology, 87, 162-167

GM-CSF inhibits CD16 expression on monocytes

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Figure 4. Tyrosine-kinase-dependence of the GM-CSF effect on CD16 expression. Monocytes were incubated for 48hr in RPMI-1640/10% FCS with/without GM-CSF (50 U/ml) and/or herbimycin A (0-5 pM), a tyrosine-kinase inhibitor. The cells were labelled with Leu 1ic-PE to determine the expression of CD16 on the cell membrane (data of two experiments). The mean intensity of cells stained with isotype control mIgG was subtracted from the mean intensity of cells staining positive for CDl6. O, experiment 1; *, experiment 2.

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CD16 expression, but it overcame the effect of GM-CSF. This suggests that the signalling pathway of GM-CSF, leading to

inhibition of the TGF-f3-induced expression of CD 16, involves tyrosine kinase activity. The effect of GM-CSF is on the transcriptional level We isolated RNA from monocytes, cultured in medium with/ without 10% FCS, and/or GM-CSF, to determine whether the suppressive effect of GM-CSF was at the mRNA level. Northern blots were probed with a 32P-labelled CD16-cDNA probe at 24 and 48 hr (Fig. 5). In the sample incubated in serum-free medium (RPMI-1640 alone), there was little detectable transcript for CD 16. An increase in CD16 transcript was observed in the samples incubated in RPMI-1640/10% FCS medium for 24 and 48 hr. However, the monocytes incubated in RPMI-1640/10% FCS and GM-CSF (50U/ml), had little detectable CD16 mRNA. We conclude that the antagonism by GM-CSF is either on the transcriptional or post-transcriptional level. Similarly low transcript levels for CD16 were obtained when monocytes were incubated in medium, containing 10% FCS and IL-4, confirming the results of Wahl et al. (data not shown).'9 DISCUSSION In this study CD16 is used as a marker for differentiation of monocytes into macrophages. The majority of peripheral blood monocytes lack CD16 expression, whereas most tissue macrophages express CD 16.8,9 TGF-f induces FcyRIII expression on circulating monocytes in vitro.12 We confirm increased expression of CD16 (FcyRIII) when peripheral blood monocytes are cultured in the presence of TGF-f3. This induced CD16 expression was because of de novo synthesis, since the CD16 transcripts were increased, and expression was antagonized by actinomycin D (a transcription inhibitor). We confirm that IL-4 antagonizes the TGF-P-induced increase in expression of CD16.19 Moreover, we have shown that GM-CSF similarly suppresses the expression of FcyRIII, induced by TGF-/3, on 1996 Blackwell Science Ltd, Immunology, 87, 162-167

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Figure 5. RNA blot analysis of FcyRIII transcripts. Total cellular RNA was isolated from monocytes cultured for 24hr (lanes 1-3) or 48 hr (lanes 4-6) in medium without FCS (lanes 1,4), medium with FCS (lanes 2,5), or medium with FCS and GM-CSF (lanes 3,6). (a) RNA was separated on an agarose gel, and blotted to nitrocellulose. Blots were probed with 32P-labelled FcyRIII cDNA. (b) Ethidium bromide staining patterns for the same samples as in (a). Positions of the 28-S and 18-S ribosomal RNAs are indicated in (a).

human monocytes in vitro. Although GM-CSF induces differentiation of early myeloid cells, the antagonism on CD16 expression may imply that GM-CSF does not support further differentiation of monocytes into macrophages. The effect of GM-CSF was dose dependent and seems to be on the transcriptional or post-transcriptional level, as concluded from Northern blotting in which a faint CD16 transcript was observed in monocytes exposed to TGF-# and GM-CSF simultaneously, versus a strong 22 kb transcript seen with TGF-,B alone. The effect of GM-CSF on CD16 involved tyrosine kinase activity, as supported by the inhibitory effect of herbimycin A, a tyrosine kinase inhibitor. It is interesting to note that many effects of GM-CSF are very similar to the effects of IL-13 on monocytes. Like GM-CSF, IL-13 induces

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the expression of CD1ib, CD18, HLA-DR, HLA-DP and decreases the expression of CD 14, CD16 and CD64, although the last molecule is not significantly downregulated by GM-

CSF.22 TGF-fl is one of the first molecules released by platelets at sites of injury, and represents a potent monocyte activator.23 TGF-fl recruits monocytes and increases monocyte mRNA for IL-6, platelet-derived growth factor and, further, induces its own production.23 TGF-fl primes macrophages to express inflammatory gene products in response to certain stimuli.24'25 Enhanced expression of FcyRIII by TGF-fi may also be important in inflammatory processes. FcyRIIIA is indeed capable of mediating ADCC, clearance of immune complexes26'27 and phagocytosis of antibody-coated micro-organisms.28 FcyRIIIA triggering stimulates generation of superoxide.29 On the other hand, TGF-fl induces the synthesis of an IL-1-antagonist protein30 and it inhibits the expression of major histocompatibility complex (MHC) class II molecules.3' These data suggest that TGF-f stimulates the pro-inflammatory functions, but antagonizes antigen-presenting functions of monocytes, which can have important implications for certain clinical situations. For example, elevated TGF-# levels in serum are found concommitantly with FcyRIII expression on monocytes in HIV-infected patients.32 Synovial monocytes from inflamed joints also exhibit increased expression of FcyRIII, paralleled by increased concentrations of TGF-,B in the synovial fluid.33 In contrast, GM-CSF enhances a different type of effector functions of peripheral monocytes, e.g. it increases the expression of HLA-DR, HLA-DP, and CDl lb.34'35 GM-CSF primes monocytes for the production of IL-lB and TNF-a, although the cells need a second stimulus to be able to secrete these cytokines.36 The upregulated expression of these surface molecules and of cytokine secretion is especially important in accessory cell functions of monocytes. The effect of GM-CSF on the expression of some surface molecules, important in monocyte effector functions, was also investigated by us (unpublished results). First, we could demonstrate that GMCSF enhances CD54 (ICAM-1) expression. We also found increased expression of HLA-DR and of CDl lb as a result of GM-CSF activity, in agreement with other reports.34'35,37 These stimulatory effects were accompanied by a decrease in CD16 expression, clearly indicating a differential regulation. Several data from the litterature, together with our findings, thus suggest that TGF-# and GM-CSF exert antagonistic effects not only on monocyte differentiation, but also on monocyte function. At the level of mature monocytes, GMCSF and TGF-/3 apparently drive the monocytes into functionally different directions, with GM-CSF mainly supporting antigen-presenting functions, and TGF-,B favouring macrophage inflammatory functions. GM-CSF may, thus, be involved in the negative feedback control of certain TGF-f effects. In conclusion, although GM-CSF can enhance certain effector functions by upregulating the monocyte antigenpresenting ability34'37 it antagonizes CD16 expression, which may be important for downregulation of inflammatory responses involving CD16. TGF-,B antagonism by GM-CSF may have implications for in vivo treatment. GM-CSF treatment may indeed be useful to antagonize the immunosuppressive effects of TGF-,B produced in chronic inflammatory lesions.

ACKNOWLEDGMENTS We thank Lizette Meurs and Frieda Van Vaeck for excellent technical assistence. We also thank P. Beverley (Imperial Research Cancer Fund, London, UK), R. Van Schie (Nijmegen, the Netherlands), A. Van de Voorde (Innogenetics, Ghent, Belgium), and the company ScheringPlough (Brussels, Belgium) for kindly providing reagents or cells used in this study. This research was supported by a grant from Kom op tegen Kanker, by a grant for cancer research from the Algemene Spaar en Lijfrente Kas (ASLK), Belgium, by grant G3037.89 of the National Fund for Scientific Research (NFWO), Brussels, Belgium and by a grant from the Onderzoeksfonds of the Catholic University of Leuven, Leuven, Belgium.

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