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TGF- 1 Suppresses Myeloid Fc  Receptor Function by Regulating the Expression and Function of the Common  -Subunit

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TGF- 1 Suppresses Myeloid Fc  Receptor Function by Regulating the Expression and Function of the Common  -Subunit
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  TGF-  1 Suppresses Myeloid Fc    Receptor Function byRegulating the Expression and Function of the Common   -Subunit 1 Susheela Tridandapani, 2 * † Richard Wardrop,* Christopher P. Baran,* Yijie Wang,*Judy M. Opalek,* Michael A. Caligiuri, † and Clay B. Marsh* We have previously reported that Fc   R-mediated function in myeloid cells is a tightly regulated event that is influenced by thecytokines present in the milieu. TGF-  1 is an immunosuppressive cytokine with pleiotropic effects on immune responses; however,the molecular mechanism by which TGF-   suppresses immune responses is poorly understood. In this study, we have analyzedthe effect of TGF-   on Fc   R-mediated activation of myeloid cells. We report that TGF-  1-treated THP-1 human myeloid cellsdisplayed reduced ability to phagocytose IgG-coated particles. Because Fc   R expression is modulated by cytokines, we analyzedexpression levels of Fc   RI, Fc   RIIa, Fc   RIIb, and Fc   RIIIa in cells cultured with or without TGF-  1 and found while totalprotein levels of the Fc   R were not reduced, surface expression of Fc   RI and Fc   RIII was lower in cells cultured with TGF-  1.Concomitantly, there was a dose-dependent reduction in the expression of the Fc   R-associated    -subunit. This suppressive effectof TGF-   was likewise observed in bone marrow-derived murine myeloid cells and human monocytes. Importantly, TGF-  1 alsosignificantly reduced the production of monocyte chemoattractant protein-1 induced by immobilized IgG, which would furtherreduce monocyte recruitment to the site of inflammation. In contrast, human alveolar macrophages were refractory to this effect,expressing low levels of TGF-   type II receptors compared with peripheral blood monocytes from the same donor. These dataprovide insight into the regulation of immune responses by TGF-  1 and demonstrate the selectivity of these effects.  The Journal  of Immunology,  2003, 170: 4572–4577. T ransforming growth factor-   is an immunosuppressivecytokine with pleiotropic effects (reviewed in Ref. 1). TheTGF-   family consists of three members: TGF-  1, TGF-  2, and TGF-  3. In hemopoietic cells, TGF-  1 is the most plen-tiful isoform. TGF-   is well recognized to reduce host response toa variety of inflammatory stimuli and has been noted to reduceinflammation induced by IgG or immune complexes. DepositedIgG or immune complexes containing IgG stimulate host re-sponses by promoting the clustering of surface Fc   R. There arethree classes of Fc   R on the surface of human monocytes andmacrophages: Fc   RI, Fc   RII, and Fc   RIII (reviewed in Ref. 2).Fc   RI has the highest affinity for monomeric IgG. Fc   RII exists astwo isoforms: Fc   RIIa and Fc   RIIb. Fc   RIIa is an activating re-ceptor that contains a tyrosine-based activation motif (ITAM) 3 inits cytoplasmic domain (3). In contrast, Fc   RIIb has an immuno-receptor tyrosine-based inhibition motif in its cytoplasmic domainthat serves to recruit phosphatases to the receptor to reduce cellularactivation (4, 5). Fc   RIIb reduces Ig production by lymphocytes(6) and IgG-mediated phagocytosis by macrophages (7), and isthought to account for the suppressive effects of i.v. IgG (8), givenas treatment for patients with immune complex-mediated inflam-matory diseases. Fc   RIIIa is an activating receptor, which is ex-pressed at higher levels in macrophages than in monocytes.In addition to differences in surface expression and function,Fc   RI, Fc   RII, and Fc   RIII also differ in signaling elements. Al-though Fc   RIIa contains an ITAM sequence in its cytoplasmic tail,Fc   RI and Fc   RIIIa rely on the accessory    -subunit to supply theITAM (9, 10). Importantly, the surface expression of Fc   RI andFc   RIIIa depends on the presence of the accessory    -subunit, sug-gesting that processes suppressing expression of the    -subunit mayaffect signaling through these receptors. Consistent with this no-tion, IgG-mediated inflammation is dramatically reduced in    -sub-unit-deficient animals (11).In animal models of human diseases, TGF-   has been shown tomodify Fc   R-mediated inflammation. Perhaps this is most clearlyshown in models of allograft transplantation in which TGF-  1promotes acceptance of solid organ transplants (12). Likewise,graft acceptance is also promoted by the loss of IgG as seen in Bcell-deficient transgenic animals (13), and by reduced macrophagepopulations seen in M-CSF-deficient mice (14). Thus, the presenceof TGF-  1 or the loss of IgG production or reduced populations of macrophages can all prevent transplant rejection. These observa-tions led us to hypothesize that TGF-  1 may reduce inflammationby modifying Fc   R-mediated activation in monocytes and macro-phages and reduce cytokine or chemokine production.In this study, we report that THP-1 human monocytic cells cul-tured in the presence of TGF-  1 show decreased phagocytosis of IgG-coated SRBC. Second, while the overall protein expression of the Fc   R is unaffected by TGF-  1, the expression of the common *Department of Internal Medicine and Dorothy M. Davis Heart and Lung ResearchInstitute, and  † The James Cancer Hospital and Comprehensive Cancer Center, OhioState University, Columbus, OH 43210Received for publication September 13, 2002. Accepted for publication March5, 2003.The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked  advertisement   in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by P30 CA16058 and P01 CA095426 to S.T., and R01HL63800, R01 HL 66108, and P01 HL70294 to C.B.M. S.T. is a Fellow of theLeukemia and Lymphoma Society. 2 Address correspondence and reprint requests to Dr. Susheela Tridandapani, Room405B HLRI, 473 W. 12th Avenue, Columbus, OH 43210. E-mail address:tridandapani.2@osu.edu 3 Abbreviations used in this paper: ITAM, immunoreceptor tyrosine-based activationmotif; hIgG, human IgG; MCP-1, monocyte chemoattractant protein-1; MFI, meanfluorescence intensity; PBM, peripheral blood monocyte; WCL, whole cell lysate. The Journal of Immunology Copyright © 2003 by The American Association of Immunologists, Inc. 0022-1767/03/$02.00    -subunit is suppressed in a dose-dependent manner. Third, thesuppressive effect of TGF-   on the    -subunit and the signalingproperties of the associated Fc   R are suppressed both in murinebone marrow-derived myeloid cells and in human peripheral bloodmonocytes (PBM). TGF-  1 also substantially reduced monocytechemoattractant protein-1 (MCP-1) production from monocytesstimulated with immobilized IgG, suggesting a mechanism used byTGF-  1 to reduce leukocyte recruitment to the lung. Finally, thesuppressive effect of TGF-  1 was not seen in human alveolar mac-rophages derived from the same donors. Further analysis of thisdichotomy in TGF-  1 effect revealed that alveolar macrophagesexpress very low levels of TGF-   type II receptors in comparisonwith PBM from the same donor, indicating that the effect of TGF-   is specific, based on cellular context. Materials and Methods Cells, Abs, and reagents THP-1 cells were obtained from American Type Culture Collection (Ma-nassas, VA) and maintained in RPMI supplemented with 10% FBS. Mu-rine bone marrow-derived myeloid cells immortalized by retroviral infec-tion were a kind gift from B. G. Neel (Harvard Medical School, Boston,MA) (15). Murine myeloid cells were maintained in DMEM supplementedwith 10% FBS and 20% L cell-conditioned medium. F(ab  ) 2  of anti-Fc   RIAbs 32.2, anti-Fc   RIIa Ab IV.3, and anti-Fc   RIII Ab 3G8 were obtainedfrom Medarex (Annandale, NJ). Rabbit polyclonal Abs specific for Fc   RI,Fc   RIIa, and Fc   RIIb have been described elsewhere (16, 17). The anti-   -subunit mAb 4D8 was a generous gift from J. Kochan (Hoffmann-La-Roche, Nutley, NJ). The rabbit polyclonal anti-   -subunit Ab and the anti-TGF-   type II receptor Ab were obtained from Upstate Biotechnology(Lake Placid, NY). Anti-phosphotyrosine and anti-phosphoAkt Abs wereobtained from Cell Signaling Technology (Beverly, MA). Anti-  -actin Abwas from Sigma-Aldrich (St. Louis, MO).  Isolation of PBM  Monocytes were isolated from heparinized blood of volunteers, as previ-ously described (18). Polymyxin B (2 ng/ml) was used in PBS and RPMIunless specified to protect against LPS contamination. Briefly, monocyteswere enriched by diluting buffy coats 1/1 with sterile PBS and then layeringonto 15 ml of Histopaque. Cells were centrifuged at 18°C for 20 minwithout braking at   2000 rpm (800    g ). Mononuclear (interface layer)cells were pooled into larger tubes, washed twice with fresh endotoxin-freeRPMI, and centrifuged at 4°C at 1400 rpm (400    g ) for 10 min. Cellswere then resuspended in RPMI containing 10% FBS at a density of 5  10 7 cells/ml in polypropylene tubes. Tubes were gently rocked at 4°C for1 h to promote clumping of monocytes. After 1 h, cells were carefullylayered 1/1 over 100% FBS for 15 min on ice, allowing the monocytes tosink to the bottom of the tubes. Cells were collected from the bottom of thetubes washed in FBS-free RPMI. Using light microscopy and Diff-Quick (Dade Diagnostics, Aquada, Puerto Rico) staining, there was an average of 67% monocytes, 32% lymphocytes, and  1% neutrophils in the prepara-tions. Cell viability was  98%, as determined by trypan blue exclusion.  Isolation of CD14-positive PBM  For analysis of TGF-   type II receptor expression, CD14-positive mono-cytes were used. PBMCs were first isolated by density-gradient centrifu-gation over Histopaque (Sigma-Aldrich). Monocytes were then purifiedfrom the PBMCs by negative selection using the MACS Monocyte Isola-tion Kit (Miltenyi Biotec, Auburn, CA). Briefly, PBMCs were first treatedwith FcR blocking reagent (human IgG (hIgG)), followed by a hapten-Abcocktail (cocktail of hapten-conjugated CD3, CD7, CD19, CD45RA,CD56, and anti-IgE mAbs). The labeled cells were further treated withMACS anti-hapten magnetic microbeads that were conjugated to a anti-hapten mAb. The cells were then passed over a MACS column, and theeffluent was collected as the negative fraction representing enriched mono-cytes. The monocytes thus purified were subsequently analyzed for purityby double labeling with CD14 PE and CD45 FITC Abs, followed by flowcytometry. Data from 10,000 cells indicated that the isolated monocyteswere  97% CD14 positive. Cell viability was greater than 98%, as deter-mined by trypan blue exclusion. Preparation of human alveolar macrophages Macrophages were obtained from healthy donors by bronchoalveolar la-vage after signing an Institutional Review Board-approved consent. Cellswere washed twice with PBS and counted and analyzed by Diff-Quick staining for purity. Cell preparations were   95% positive for macro-phages. The cells were resuspended in RPMI 1640 medium with 5% FBSat 5    10 7 cells/condition. Cell viability was greater than 98%, as deter-mined by trypan blue exclusion. Cell lysis, immunoprecipitation, and immunoblotting For immunoprecipitations, 10 7 cells/sample were lysed (25 mM HEPES,pH 7.5, 20 mM Na 4 P 2 O 4 , 100 mM NaF, 4 mM EDTA, 1% Triton X-100,3 mM Na 3 VO 4 , 10   g/ml aprotinin and leupeptin, 2 mM PMSF) and in-cubated overnight with Ab of interest and protein G-agarose beads orSepharose beads coupled to F(ab  ) 2  of goat anti-mouse IgG Abs for Fc   Rimmunoprecipitations with F(ab  ) 2  Abs. The immune complexes were sep-arated by SDS-PAGE, transferred to nitrocellulose membrane, and immu-noblotted with the Ab of interest and developed by ECL. For whole celllysates (WCL), protein content was normalized between samples, 15   g of protein was loaded in each lane, and immunoblotting was performed, asdescribed above. The ECL signal was quantitated using a scanner and adensitometry program (Scion Image; Scion, Frederick, MD), as describedpreviously (19). To quantitate the phosphotyrosine signal in the activatedsamples, we first subtracted background, normalized the signal to theamount of precipitated protein, and plotted the values obtained as foldincrease of phosphotyrosine signal in activated samples over restingsamples. Preparation of IgG-coated SRBCs SRBCs (Colorado Serum, Denver, CO) were washed in PBS and labeledovernight with PKH-26 Red, as per the manufacturer’s protocol, washed inPBS, and incubated with a subagglutinating dose of rabbit anti-SRBC IgG(Diamedix, Miami, FL) at 37°C for 1 h. Unbound IgG was removed bywashing the cells with PBS. Phagocytosis assay Phagocytosis assays were performed, as previously described (20). Briefly,THP-1 cells or PBM cultured overnight with or without TGF-   were in-cubated with the SRBC described above. To measure phagocytic index (thenumber of SRBC ingested by 100 monocytic cells), the cells were incu-bated for 1 h at 37°C; externally bound SRBC were lysed by hypotoniclysis and fixed in 1% paraformaldehyde; and the ingested SRBC wereviewed and counted under a fluorescence microscope. To assess rosettingactivity (percentage of monocytes that bound three or more SRBC), sam-ples were incubated for 1 h at 4°C. In parallel, monocytic cells were alsoincubated with SRBC that had not been opsonized with IgG. No phago-cytosis or rosetting activity was seen in the latter samples. Flow cytometry analysis To analyze surface expression of Fc   R, cells cultured with or withoutTGF-   were labeled with F(ab  ) 2 of mAb 32.2 that specifically recognizesFc   RI, F(ab  ) 2  of mAb 3G8 that recognizes Fc   RIII, or Fab of the anti-Fc   RIIa mAb IV.3, followed by a FITC-conjugated secondary Ab. Recep-tor levels were analyzed by flow cytometry and expressed graphically aspercentage of mean fluorescence intensity (MFI) of the TGF-  -treated cellsin comparison with untreated cells.For experiments analyzing TGF-   type II receptor surface expression,cells were labeled with either TGF-   type II receptor Ab or normal rabbitIgG, followed by FITC-labeled secondary Ab. To ensure specific bindingof anti-receptor Ab, FcR on the cells were blocked with 100   g/ml hIgG.  ELISA determination of human MCP-1 production fromstimulated PBM  PBM isolated as described above were counted, washed, and resuspendedto 5    10 6 cells/ml for all cytokine experiments. Before cell culture inflat-bottom plates, cells were treated with or without 5 ng/ml active TGF-  for 2 h at 37°C before culture on flat-bottom plates, as described below. Atotal of 1.25  10 6 cells in 250-  l replicates was cultured on sterile flat-bottom Immulon IV plates (Fisher, Pittsburgh, PA) coated with either PBSalone, endotoxin-free intact hIg at 100   g/ml, or F(ab  ) 2  of hIgG at 100  g/ml. After 18 h in culture, supernatants of replicates were harvested ina sterile fashion, centrifuged to remove dead cells, and analyzed by ELISAusing a human MCP-1 OPT-EIA detection kit (BD PharMingen, San Di-ego, CA). Supernatants were thawed, and dilutions were made in assaydiluent (BD PharMingen) in accordance with recommendations of theOPT-EIA ELISA system. Immulon IV flat-bottom ELISA plates were pre-coated with specific capture Ab against human MCP-1 using recommendeddilutions. Human rMCP-1 was titrated on each plate for standardization 4573The Journal of Immunology  following kit instructions. Subsequent washing, detection, and developingsteps were accomplished following specific kit instructions. After 20 minof developing in the dark and stopping with 50   l 2 N H 2 SO 4 , plates wereread at 450 nm for quantification of MCP-1 production. Results TGF-  1 reduces phagocytic efficiency of THP-1 cells To assess the influence of TGF-  1 on Fc   R-mediated phagocyto-sis, THP-1 cells were cultured overnight with or without 5 ng/mlTGF-  1. The cells were then incubated for 1 h with IgG-coatedSRBC either at 4°C to assess binding ability, or at 37°C to measurephagocytosis. A total of 200 cells was analyzed in each experi-ment. We found that the phagocytic ability of cells cultured withTGF-  1 was significantly diminished (Fig. 1  A ) in comparison withthe phagocytic ability of cells that were cultured in the absence of TGF-  1. Interestingly, THP-1 cells cultured in the presence orabsence of TGF-   were similar in their ability to bind IgG-coatedSRBC (Fig. 1  B ). These findings suggest that the Fc   R-mediatedsignaling ability of THP-1 cells is diminished by TGF-  1. TGF-  1 does not influence Fc    R protein expression in THP-1 cells We and others have demonstrated that anti-inflammatory cytokinessuch as IL-4 diminished phagocytosis of IgG-containing immunecomplexes by altering the expression levels of Fc   R (19, 21). Spe-cifically, the expression of the inhibitory receptor Fc   RIIb wassignificantly increased in monocytes cultured with IL-4. Based onthese observations, we next asked whether the suppressive effect of TGF-  1 on phagocytic efficiency could be explained by alteredexpression of Fc   R. Our prediction was that TGF-  -treated cellswould either express enhanced levels of the inhibitory Fc   RIIb, ordecreased levels of activating Fc   R. Thus, we analyzed by West-ern blotting the protein expression of Fc   RIIb, Fc   RIIa, Fc   RI,and Fc   RIII in THP-1 cells cultured with or without TGF-  . Re-sults indicated that total cellular protein levels of the inhibitoryFc   RIIb were not significantly altered by the presence of TGF-  1(Fig. 2  A ). Likewise, there was no decrease in the expression levelsof the activating Fc   R, Fc   RI (Fig. 2 C  ), Fc   RIIa (Fig. 2  B ), orFc   RIIIa (Fig. 2  D ). All of these receptors are glycosylated molecules,which accounts for the diffuse nature of the bands seen in the figure. TGF-  1 suppresses the common    -subunit expression inTHP-1 cells To test whether the Fc   R signaling components were intact inTGF-  1-treated cells, we then analyzed expression levels of thecommon    -subunit, which is critical for Fc   RI and Fc   RIIIa sig-naling. Thus, we analyzed, by Western blotting, the protein ex-pression of the    -subunit in THP-1 cells cultured overnight with orwithout TGF-  1. As seen in Fig. 3  A ,    -subunit expression wasdecreased by TGF-  1 in a dose-dependent manner. We next askedwhether the decreased expression of     -subunit was accompaniedby a decrease in the surface expression of Fc   R because earlierreports indicated that the    -subunit is critical for surface expres-sion of both Fc   RI and Fc   RIIIa (11). For these experiments,THP-1 cells cultured with or without TGF-   were labeled withF(ab  ) 2  of mAb 32.2 that specifically recognizes Fc   RI, mAb 3G8that recognized Fc   RIII, or the anti-Fc   RIIa mAb IV.3, followedby a FITC-conjugated secondary Ab. Receptor levels were ana-lyzed by flow cytometry and expressed graphically as percentageof MFI of the TGF-  1-treated cells in comparison with untreatedcells. Results shown in Fig. 3  B  indicate that surface expression of Fc   RIIa was not significantly influenced by TGF-  1. However,there was a significant decrease in the surface expression of Fc   RIand Fc   RIII in THP-1 cells cultured in the presence of TGF-  1.These results suggest that the suppressive effect on TGF-  1 onFc   R-mediated phagocytosis is due to a decrease in the expressionof the    -subunit. FIGURE 1.  TGF-  1 down-regulates Fc   R-mediated phagocytosis inTHP-1 cells. THP-1 cells were cultured overnight with or without 5 ng/mlTGF-  1.  A , Cells were then incubated with PKH-26 Red-labeled, IgG-coated SRBC for 1 h at 37°C to allow phagocytosis to proceed. Externallybound SRBC were lysed by hypotonic lysis. Internalized SRBC wereviewed under a fluorescence microscope and counted. A total of 200THP-1 cells was assessed in each assay. The number of internalized par-ticles was expressed as a phagocytic index (total number of SRBC ingestedby 100 THP-1 cells).  B , In parallel, cells were incubated with SRBC for 1 hat 4°C to assess rosetting activity (percentage of THP-1 cells that boundthree or more SRBC). The graph represents the mean and SD of threeindependent experiments. The  p  values for phagocytosis and rosetting ac-tivity were 0.0007 and 0.46, untreated vs TGF-   treated, respectively. Sta-tistical analysis was performed with a paired, two-tailed Student’s  t   test. FIGURE 2.  Protein expression of Fc   R is not reduced by TGF-  1.THP-1 cells were cultured overnight with or without 5 ng/ml TGF-  1.  A ,Fc   RIIb were immunoprecipitated with the Fc   RIIb-specific rabbit poly-clonal Ab 163, and immunoblotted with the same Ab.  B , Fc   RIIa wereimmunoprecipitated with Fab of mAb IV.3, and immunoblotted with therabbit polyclonal Ab 260.  C  , Fc   RI were immunoprecipitated with F(ab  ) 2 of mAb 32.2, followed by immunoblotting with a rabbit polyclonal anti-Fc   RI Ab.  D , Fc   RIII were immunoprecipitated with F(ab  ) 2  of mAb 3G8,followed by immunoblotting with the mAb Gran1. These results are rep-resentative of three independent experiments. 4574 TGF-  1 DOWN-REGULATES THE COMMON    -SUBUNIT IN MONOCYTES  TGF-  1 suppresses    -subunit expression and Fc    R-mediated signaling events in murine myeloid cells To test whether the suppressive effect of TGF-  1 on the    -subunitexpression in human myeloid cell line could be extended to murinecells, we assessed TGF-  1 effects in murine bone marrow-derivedmyeloid cell line. In this study, the murine myeloid cells werecultured overnight with or without TGF-  1, and the followinganalyses were performed. First, we analyzed the influence of TGF-   on    -subunit expression by Western blotting. As indicatedin Fig. 4  A , murine myeloid cells, much like THP-1 cells, displayeddecreased levels of     -subunit upon TGF-   treatment. Second, weanalyzed the signaling events initiated by clustering Fc   R with themAb 2.4G2 that clusters both Fc   RIII and Fc   RII. Results indi-cated that concomitant with a decrease in    -subunit expression,cellular signaling events were also decreased in cells cultured withTGF-  . Specifically, overall tyrosine phosphorylation induced byFc   R clustering was diminished in TGF-  -treated cells (Fig. 4  B ).Likewise, phosphorylation of the survival factor Akt was also di-minished in cells cultured with TGF-  1 (Fig. 4 C  ). These results sug-gest that TGF-   suppresses Fc   R-mediated signaling events by sup-pressing the expression of the    -subunit in murine myeloid cells. TGF-  1 suppresses    -subunit expression in human PBM, but not in human alveolar macrophages To ensure that the effect of TGF-  1 on the expression of     -subunitwas not limited to transformed cell lines, similar experiments wereperformed in primary cells. Thus, human PBM and human alveolarmacrophages obtained from the same volunteers were culturedovernight with or without TGF-  1. Expression level of the    -sub-unit was assessed by Western blotting WCL from the above cellsnormalized for protein content. As seen in Fig. 5  A  ( upper panel ),PBM cultured in the presence of TGF-  1 displayed decreased ex-pression of the    -subunit. The  lower panel  (Fig. 5  A ) is a Westernblot of the same membrane with Ab to   -actin to demonstrateequal loading of protein in both lanes. Surprisingly, TGF-  1 didnot have a suppressive effect on    -subunit expression in humanalveolar macrophages (Fig. 5  B ,  upper panel ). Likewise, there wasno change in the surface expression of the Fc   R (data not shown).Further analysis by Western blotting using anti-TGF-   type II re-ceptor Abs revealed that alveolar macrophages, unlike PBM fromthe same donor, express very little TGF-   type II receptors (Fig.5 C  ). Analyzing surface-expressed TGF-   type II receptors by flowcytometry, we were not able to detect any expression on alveolarmacrophages (Fig. 5  D ). These results suggest that the effects of TGF-  1 are cell specific. TGF-   suppresses Fc    R-mediated MCP-1 production in humanPBM  We next assessed the functional capacity of human PBM culturedwith or without TGF-  1. First, we compared Fc   R-mediated pro-duction of MCP-1 in PBM cultured overnight on hIgG-coatedplates either in the presence or absence of 5 ng/ml TGF-  1. Ascontrols, PBM were also cultured in plates coated with F(ab  ) 2  of hIgG or in noncoated plates. Cell supernatants were collected 24 hlater and analyzed by ELISA for the amounts of MCP-1 produced.PBM cultured in IgG-coated plates, but not those cultured in non-coated plates or plates coated with F(ab  ) 2  of hIgG, producedMCP-1, which was significantly reduced by TGF-  1 (60–80%reduction;  p  value 0.01) (Fig. 6). Likewise, PBM cultured in thepresence of TGF-   displayed reduced phagocytic ability (25–30%)in comparison with PBM cultured in the absence of TGF-   (datanot shown). These results indicate that TGF-   suppresses PBMresponses to immune complexes and can reduce the production of chemokines that may recruit additional monocytes. Discussion In this study, we have analyzed the influence of TGF-  1 on ex-pression and function of Fc   R in human monocytes. Contrary toour initial hypothesis that the suppressive effect of TGF-  1 onFc   R-mediated functions would be due to altered Fc   R expres-sion, we found that TGF-  1 reduced protein expression of thecommon    -subunit. This finding has potentially far-reaching im-plications because the    -subunit is required for the proper surfaceexpression and functioning of a number of immune receptors, in-cluding the IgE receptor Fc  RI (22, 23), TCR (24–27), Fc  R (28),and the platelet receptor gpVI (29). Although this suppressive ef-fect of TGF-  1 on the    -subunit provides an explanation for thepleiotropic effects of TGF-  , surprisingly, we found that alveolarmacrophages could remain refractory to TGF-  1 effects by ex-pressing low levels of TGF-   type II receptors. These results in-dicate that the effects of TGF-   are specific and depend on cellularcontext. Indeed, Wahl and colleague (30) previously reported thatin vitro differentiation of PBM with    -IFN or LPS results in thegradual loss of expression of TGF-   type II receptors.The data presented in this study demonstrate that the suppres-sive effect of     -subunit can be extended to murine myeloid cells aswell. Preliminary experiments to identify similar effects in humanNK cells, which are reported to display reduced Ab-dependentcellular cytotoxicity in response to TGF-  1, revealed that whilethe suppression of     -subunit could be observed in one of the threedonor samples tested, surface expression of Fc   RIII remained un-changed (data not shown), perhaps due to the presence of the FIGURE 3.  TGF-  1 suppresses    -subunit expression with a concomi-tant decrease in Fc   RI and Fc   RIII surface expression.  A , THP-1 cellswere cultured overnight with the indicated concentrations of TGF-  1.   -subunit expression was analyzed by immunoprecipitating the    -subunitwith mAb 4D8, followed by immunoblotting with rabbit polyclonal anti-   -subunit Ab. The numbers below the panel indicate band intensity.  B ,THP-1 cells cultured with or without 5 ng/ml TGF-  1 were labeled withF(ab  ) 2  of anti-Fc   RI mAb 32.2, anti-Fc   RIIa mAb IV.3, or anti-Fc   RIIImAb 3G8, followed by F(ab  ) 2  of FITC-conjugated goat anti-mouse IgG sec-ondary Ab. Receptor expression was analyzed by flow cytometry and ex-pressed as MFI. MFI of untreated cells was set as 100%, and results are rep-resented graphically. The graph represents the mean and SD of threeindependent experiments. The  p  values for Fc   RI, IIa, and III were 0.002,0.089, and 0.04, untreated vs TGF-   treated, respectively. Statistical analysiswas performed with a paired, two-tailed Student’s  t   test. 4575The Journal of Immunology    -chain that is additionally associated with Fc   RIII in NK cells.Further investigation is needed to resolve these issues. Likewise,the exact mechanism by which TGF-   suppresses    -subunit ex-pression is not clear. Studies are underway in our laboratory todetermine the latter.The significance of the    -subunit is perhaps most effectivelydemonstrated in genetically altered mice that lack expression of the    -subunit. Ravetch and colleagues (11) elegantly demonstrated FIGURE 4.  TGF-   suppresses    -subunit expression and function in murine myeloid cells. Murine myeloid were cultured overnight with or without 20ng/ml of TGF-  1.  A ,    -subunit expression was assessed by immunoblotting protein-matched WCL with rabbit polyclonal anti-   -subunit Ab. The numbersbelow the panel indicate band intensity.  B , WCL from resting (R) cells and cells activated for 5  by clustering Fc   RIII/II with mAb 2.4G2 and mouse anti-ratIgG secondary Ab were analyzed for overall tyrosine phosphorylation by immunoblotting with anti-phosphotyrosine Ab.  C  , WCL from resting and activatedcells, as described above, were analyzed for Akt activation by immunoblotting with anti-phosphoAkt Ab ( upper panel ). The same membrane was reprobedwith anti-Akt Ab ( middle panel ).  Lower panel , Graphical representation of Akt phosphorylation, as determined by densitometry analysis of band intensitiesin the Western blots above. To quantitate phosphotyrosine signal in the activated samples, we first subtracted background, normalized the signal ( upper  panel ) to the amount of precipitated protein ( lower panel ), and plotted the values obtained as fold increase of phosphotyrosine signal in activated samplesover resting samples. These results are representative of three independent experiments. FIGURE 5.  TGF-  1 suppresses    -subunit expression in human PBM,but not in alveolar macrophages from the same donor. PBM (  A ) and al-veolar macrophages (  B ) were cultured overnight with or without 5 ng/mlTGF-  1. Protein-matched lysates were analyzed by immunoblotting for theexpression of the    -subunit ( upper panels ) and   -actin ( lower panels ).  C  ,Protein-matched lysates of PBM and alveolar macrophages were analyzedby Western blotting for TGF-   type II receptors ( upper panel ) and   -actin( lower panel ).  D , PBM and alveolar macrophages were analyzed for TGF-  type II receptor expression by flow cytometry. Shaded histograms representlabeling with control Ab and clear histograms with TGF-  type II receptor Ab.These results are representative of three independent experiments. FIGURE 6.  TGF-  1 down-regulates Fc   R-mediated production of MCP-1. Human PBM were cultured with or without 5 ng/ml TGF-  1 inplates that were either not coated or plates coated with F(ab  ) 2  of hIgG orintact hIgG. Cell supernatants were analyzed by ELISA for MCP-1. Theamount of MCP-1 produced by untreated PBM cultured on IgG-coatedplates was set as 100%. The graph represents the mean and SD of threeindependent experiments. The  p  value for percentage of MCP-1 productionby untreated vs TGF-  -treated PBM, plated on intact hIgG, was 0.01.Statistical analysis was performed with a paired, two-tailed Student’s  t   test. 4576 TGF-  1 DOWN-REGULATES THE COMMON    -SUBUNIT IN MONOCYTES
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