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Modulation of human mononuclear phagocyte FcγRII mRNA and protein
CELLULAR
IMMUNOLOGY
124,292-307( 1989)
Modulation
of Human Mononuclear Phagocyte FcyRll mRNA and Protein’
PAULG.COMBER,*MILTON D. ROSSMAN,*ERIC F. RAPPAPORT,PPAULCHIEN,* P. MARK HOGARTH,* AND ALAN D. SCHRPIBER* *University of Pennsylvania School ofMedicine and TChildren ‘s Hospital, Philadelphia, PennsyIvania 19104, and University ofMeIbourne,$ Melbourne, Australia Received July 10,1989; acceptedAugust 9.1989 Human monocytes and macrophages express three different classesof cell surface receptors for the Fc portion of IgG, Fc+yRI(CD64), FcyRII (CD32), and FcyRIII (CD16). We utilized a cDNA probe for FcyRII to examine the modulation of FcyRII mRNA by dexamethasone, a synthetic glucocorticoid, and interferon-y. We also determined the changesin the expression of both FcyRI and FcyRII protein following treatment with these agents by flow cytometry. In studies performed with the monocyte-like cell line, U937, Northern blot analysis revealed that cells treated with interferon-y showed a 2.5-fold increase in FcyRII mRNA levels that was maximal at 14 hr and declined to 1.4-fold over baseline by 48 hr of incubation. Treatment of U937 cells with dexamethasone did not significantly change the level of FcrRII transcripts, but was able to inhibit by up to 50% the increase seen following interferon-y treatment. The expression of FcyRII protein on U937 cells was increased 56-722 after 16-24 hr of interferon-y treatment, but was only 18%over baseline after 48 hr ofincubation. Treatment with dexamethasone caused a small, but significant, decreasein FcrRII protein, and inhibited by 20-60% the induction of FcyRII by interferon-y. The modulation by dexamethasone and interferon-y of FcyRI protein expression on U937 cells was markedly different from that of FcrRII in both magnitude and kinetics. Interferon-y treatment increased FcyRI expression by 240% at 16 hr, and FcyRI remained elevated through 48 hr. Treatment with dexamethasone decreasedFcyRI expression by 39%, and also inhibited by 40% the increase induced by interferon-y. In contrast to the findings with U937 cells, dexamethasone and/or interferon-y treatment had no significant effect on FcrRII mRNA levels or protein expression in monocytes. However, interferon-y treatment increased FcyRI expression on monocytes, and this increase was further augmented by treatment with dexamethasone. These data indicate that the modulation of FcyRII on U937 cells is at least in part due to changes in steady state levels of FcyRII mRNA. The difference between the magnitude of the changes in FcyRII mRNA and protein suggeststhat some translational or post-translational control is involved in regulating the expression of FcyRII. The differences between FcyRI and FcyRII, as well as between monocytes and U937 cells, in the extent to which they are modulated by dexamethasone and interferon-y indicate that modulation of Fey receptors by these two agents is both cell- and receptor-specific. o 1989 Academic press, IIIC.
INTRODUCTION Surface receptors for the Fc portion of IgG (FcrR) are found on many different human cells. On mononuclear phagocytes (blood monocytes and tissue macro’ This work was partially supported by National Institutes of Health Grant AI/HL-22 193. P.G.C. is the recipient of a National Institutes of Health Medical Scientist Training Program fellowship at the University of Pennsylvania. 292 0008-8749189 $3.00 Copyright 0 1989 by Academic Press,Inc. All rights of reproduction in any form reserved.
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phages), these receptors mediate important effector functions: phagocytosis of opsonized cells (l), clearance of immune complexes (2), and antibody-dependent cell cytotoxicity (3). The stimulation of FcyR on monocytes and macrophages also results in production and extracellular releaseof lysosomal enzymes, neutral proteases,prostaglandins, and superoxide anion. Three different classes of Fcr receptors have been identified on human cells (FcrRI, FcyRII, and FcrRIII) (reviewed in Refs. (4-7)). These receptors have been differentiated on the basis of their size, binding alfmities for various classesof murine IgG, and recognition by monoclonal antibodies. FcyRI (CD64) is a 72-kDa protein, recognized by monoclonal antibody mAb32.2 (8), that is found only on mononuclear phagocytes. It is the only Fey receptor that binds monomeric IgG (Kd = 1O-*- 1O-’ M). FcyRII, which has been designated CD32 (9), is a 40-kDa protein that is recognized by mAbIV.3 ( 10). In addition to being on monocytes and macrophages, FcyRII is also found on platelets, granulocytes, and B-lymphocytes. FcyRIII, a SO-to 70kDa protein formerly called Fc-yR,,, is not present on monocytes but is found on macrophages, granulocytes, natural killer cells, and some T-lymphocytes. FcyRIII is also the leu-1 1 antigen (11) and has been designated CD-16 (9). Recently, cDNA probes encoding for FcyRI ( 12) FcyRII ( 13- 15) and FcyRIII ( 16, 17) have been isolated and cloned. The availability of these clones enables the examination of the expression and regulation of mRNA for these different Fcr receptors. Mononuclear phagocyte Fey receptors have been implicated in the pathogenesis of several hematologic and immunologic diseases,and therefore the modulation of these receptors is of importance. Utilizing an in viva animal model, we observed that glucocorticoids inhibit the clearance of antibody-coated cells from the circulation (an Fey receptor-mediated event) ( 18-20). This finding was confirmed with in vitro studies, which demonstrated that glucocorticoids can inhibit Fey receptor expression and function on mononuclear phagocytes (2 1) and human cell lines (22,23). Interferon-y is a cytokine produced by T-lymphocytes during immune and inflammatory responsesthat has a wide range of effects, including stimulation of macrophage and monocyte oxidative metabolism (24) and differentiation (25). Several in vitro studies have demonstrated that interferon-y treatment of mononuclear phagocytes (26-29) as well as cell lines (30,3 l), markedly increases the expression of the FcrRI protein. We recently reported on the effects of both interferon-y and dexamethasone, a synthetic glucocorticoid, on FcrRI and FcyRII protein expression, and on ligand binding (32). However, the kinetics of these effects on Fey receptor expression have not been studied. Similarly, the modulation of Fcr receptor mRNA has not yet been examined. In this paper, we utilized one of the recently isolated full length FcrRII cDNA probes, HFc3.0 ( 13), to examine the changes in FcyRII mRNA levels following treatment with interferon-y and/or dexamethasone. We also examined the kinetics of the change in protein expression of FcrRI and FcrRII following treatment with these agents. These studies were performed with both fresh blood monocytes and U937 cells (33), a histiocytic lymphoma cell line which has characteristics of immature monocytes and, like monocytes, expressesthe Fcr receptors FcyRI and FcyRII. MATERIALS AND METHODS
Cell Culture U937 cells were obtained from the ATCC (American Type Culture Collection, Rockville, MD). These cells were maintained in continuous culture in RPM1 1640
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(GIBCO Laboratories, Grand Island, NY) culture medium supplemented with 10% heat-inactivated fetal calf serum (FCS), penicillin ( 100 U/ml), streptomycin ( 100 pg/ ml), and glutamine (25 mg/ml). This cell line was originally established from the pleural effision of a patient with a generalized, diffise histiocytic lymphoma (33). Human monocytes were isolated by removing peripheral blood from normal donors, which was anticoagulated with heparin (0.2 U/ml). Mononuclear cells were isolated by layering the blood on Lymphocyte Separation Media (Organon Teknika Corp., Durham, NC) followed by centrifugation at 900g. Cells were collected from the interface and adhered for 45 min at 37°C to T-75 tissue culture flasks that had been precoated with FCS. Nonadherent cells were removed by rinsing five to six times with Hanks’ balanced salt solution (HBSS). The monocytes were then cultured in RPM1 1640 medium containing 10% FCS, penicillin, streptomycin, and glutamine (as for the U937 cells). Monocytes were harvested by striking the flasks against a soft surface to dislodge the cells, followed by rinsing with HBSS (without Ca” or Me) containing 5 mM EDTA, 0.1% gelatin, and 0.1% sodium azide. We have previously shown that this isolation results in a cell population that is 90-95% monocytes as determined by nonspecific esterase staining and phagocytosis of latex particles (34,35). Dexamethasone and Interferon-y Preparations The synthetic glucocorticoid dexamethasone was purchased from Sigma Chemical Co. (St. Louis, MO). A stock solution of 0. I mM concentration was prepared by dissolving lyophilized dexamethasone in water. This stock was prepared fresh prior to each experiment and diluted to a final concentration of 400 n&J. Recombinant human interferon-y was the gift of Genentech, Inc. (South San Francisco, CA). A stock solution of 1.25 X 1O6U/ml was prepared and then diluted to a final concentration of 500 U/ml. All experiments were performed utilizing these dosage levels of 400 ti dexamethasone and 500 U/ml interferon-y for treatment. These doses of dexamethasone and interferon-y utilized in these experiments are the lowest levels at which our laboratory (unpublished observations) and others (36) have seen maximal modulation of FcyRI protein expression in U937 cells and monocytes. Additionally, these dosagesare sufficient to saturate the interferon-y (37) and glucocorticoid (38) receptors on monocytes in vitro. The dose of dexamethasone (400 nM) is well within the range of therapeutic concentrations achievable by pharmacologic administration of glucocorticoids (39,40). cDNA Probes We employed a recently isolated full length cDNA clone for human FcyRII [HFc3.0] (13). HFc3.0 is a full length human FcyRII cDNA clone which encodes a leader sequence,two immunoglobulin-like extracellular domains, a single transmembrane region, and an intracytoplasmic domain. This 1.4-kb clone contains the entire coding sequence for the 28 1 amino acid FcyRII protein, as well as some of the 3’untranslated region. Sequence analysis of overlapping clones has revealed that there is a polyadenylation site just 3’ to the sequence encoded by the HFc3.0 clone, as well as a second polyadenylation site approximately 1 kb further downstream (13). We also utilized a cDNA probe to the constitutively produced protein human y-actin (pHFyA- 1, gift of Larry Kedes) (4 1). The cDNA inserts were isolated by electroelu-
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tion after restriction enzyme digestion. The inserts were radiolabeled to high specific activity (>2 X lo* cpm/pg) utilizing random primer extension by the Klenow fmgment of DNA polymerase I (Random Primed DNA Labeling Kit, Boehringer Mannheim Biochemicals, Indianapolis, IN) in the presence of [32P]CTP (42). Unincorporated radioactivity was removed by spun column chromatography (43) on a prepacked Sephadex G-50 column (Quick Spin, Boehringer Mannheim Biochemicals).
Northern Blot Analysis Total cellular RNA was isolated by lysing the cells in a 4 M guanidine isothiocyanate buffer and pelleting through a 5.7 M cesium chloride gradient, purified by butanol/chloroform extraction, and isolated by ethanol precipitation (44). Equal amounts ( 10 pg), as determined by optical density at 260 nM and ethidium bromide staining, of the RNA samples were electrophoretically separated on a 1% (w/v) agarose/6% formaldehyde gel in a 3% formaldehyde/MOPS buffer solution (42, 45). The RNA was then transferred by capillary blotting in a solution of 1OX standard saline citrate (SSC) to a nylon hybridization membrane (GeneScreen Plus, NEN DuPont, Boston, MA). The blots were rinsed in a solution of 2X SSC and baked at 80°C for 2 hr. The blots were prehybridized for l-4 hr at 42°C in a solution of 50% deionized formamide, 1%sodium dodecyl sulfate (SDS), 1 A4 sodium chloride, and 10%dextran sulfate (46). 32P-labeledFcyRII cDNA was then added to the blots at a concentration of approximately 1 X lo6 cpm/ml and hybridized to the blots overnight at 42°C. The blots were washed twice in 2X SSC for 5 min at 20°C twice in 2X SSC/ 1.O%SDS for 30 min at 65°C and twice in 0.1 X SSC for 30 min at 20°C and then autoradiographed. Probe was stripped from the blots by incubation in a solution of 0.2% SDS/ 0.0 1 mM Tris (pH 7.4) for 60 min at 80°C. The blots were also hybridized with 32Plabeled y-actin cDNA in a similar manner in order to assure that equal amounts of monocyte or U937 RNA were loaded. The autoradiographs were analyzed with a densitometer (UltraScan XL, LKB Products, Bromma, Sweden) to determine the relative amounts FcyRII and y-actin mRNA present in each sample. The measurement of the relative amounts of FcyRII was adjusted for any variation in amount of total RNA loaded as detected by variation in the y-actin signal.
Flow Cytometry Monoclonal antibodies directed against FcyRII [mAbIV.3] (10) and FcyRI [mAb32.2] (8) were utilized in indirect immunofluorescence binding studies to assess protein expression of these receptors. The cells were incubated with the antibodies for 30 min at 4°C and washed twice with phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin and 0.02% sodium azide. Bound antibody was labeled by incubation with an FITC-labeled goat anti-mouse antibody (TAGO, Inc., Burlingame, CA) for 30 min at 4°C. The cells were again washed twice and then fixed with 4% paraformaldehyde until analyzed by flow cytometry. Fluorescence was measured by a FACStar cytometer with Consort-30 software (Becton-Dickinson, Mountain View, CA). For all samples, 10,000 events were recorded on a logarithmic fluorescence scale, and the actual mean fluorescence intensity (MFI) for each sample was determined using the Consort-30 software. In order to correct for autofluorescence, the MFI of a nonreactive murine IgG, antibody [P3 X 631 was subtracted from the
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MFI of the anti-FcyRII- and anti-FcyRI-stained cells. Percentage change in fluorescence intensity was calculated by % change (anti-FcyR MFI-treated cells - P3 X 63 MFI-treated cells) _ 1 x 100 = (anti-FcyR MFI-untreated cells - P3 X 63 MFI-untreated cells) ’
1
Two murine IgGZbantibodies were purchased from Becton-Dickinson and used as isotype controls for anti-FcyRII binding to monocytes [anti-Leud] (47) and U937 cells [anti&u-M31 (48).
Ligand Binding Assays Ligand binding studies were performed in order to determine the number of FcyRI and FcrRII receptor sites. The binding of ‘251-labeledmonomeric IgG and antiFcyRII [mAbIV.3] was utilized to measure FcyRI and FcyRII, respectively. Monomeric IgG was isolated from normal human serum by affinity chromatography utilizing a Sepharosecolumn of murine anti-human IgG antibody, as previously described (35). Both monomeric IgG and anti-FcyRII were radiolabeled with “‘1 by the chloramine-T method (49). Cells were collected, pelleted, resuspended in a serum-free buffer, and incubated for 30 min at 37°C to remove any nascent IgG. The cells were then pelleted and resuspended in HBSS (without Ca2+ or Mg2+) containing 5 mM EDTA, 0.1% gelatin, and 0.1% sodium azide (to prevent receptor endocytosis). The cells were then incubated with increasing amounts of the radiolabeled ligand, with or without an excessof unlabeled ligand, at 37°C for 45 min. We have previously shown that, under these conditions, binding equilibrium is reached within 20-30 min and 96-99% of the ligand is surface bound and not internalized (28, 32,36). Cell-bound ligand was separated from free ligand by centrifugation through silicon oil and quantitated by counting the radioactivity in the cell pellet. Specific binding was determined by subtracting the nonspecific binding (radiolabeled ligand with excessof unlabeled ligand) from total binding (radiolabeled ligand alone). Receptor number and affinity were determined by Scatchard analysis (50).
Statistical Analysis Two-way factorial (3 X 6) analysis of variance (ANOVA) with two groups (time and treatment) was utilized to analyze the results of the flow cytometry of U937 cells. Post hoc analysis was performed with the Scheffe test (51) to assessthe individual time and treatment effects. The Sche& test was chosen becauseit is the most conservative of the post hoc analyses. Since flow cytometry of monocytes was only performed at two time points, the Student t test was used to analyze these results. A paired Student t test was also used to assessany differences between treatments in the ligand binding assay.The ANOVA and ScheG test were performed using the Statistical Analysis System (SAS Institute, Inc., Raleigh, NC) on a Compaq 386/20 computer. The Student t tests were performed using the StatWorks program (Cricket Software, Inc., Philadelphia, PA) on an Apple Macintosh computer.
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A _
DMS
IFN
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FcrRII
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7 -Actin
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: FcyRII
B _
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FIG. 1. Northern blot analysis of U937 cells and monocytes: effect of dexamethasone and interferon~. U937 cells (A) and fresh human blood monocytes (B) were treated with medium alone (-), 400 nMdexamethasone (DMS), 500 U/ml interferon-r (IFN), or both dexamethasone and interferon-y (D + I) for 14 (U937 cells) or 48 (monocytes) hr. Total cellular RNA was isolated and Northern blots were prepared, hybridized with 3zP-labeledcDNA probes to human FcyRII and y-a&n, and autoradiographed. The positions of the 28 S and I8 S ribosomal RNA bands are indicated.
RESULTS
Modulation of FcyRII mRNA Total cellular RNA was isolated from U937 cells and human blood monocytes after incubation with normal medium (control), 400 nM dexamethasone, 500 U/ml interferon-y, and both dexamethasone and interferon-y. Northern blots were prepared and probed with 32P-labeledFcyRII cDNA. Approximately equal amounts of RNA were loaded in each lane of each blot. The blots were also hybridized with a probe for the constitutively produced protein y-actin. Representative autoradiographs of Northern blots from U937 cells and monocytes treated with dexamethasone and/or interferon-y are shown in Fig. 1. These autoradiographs indicate the presence of two transcripts for FcyRII, 2.5 and 1.5 kb in length, which are the same size in both monocytes and U937 cells. This is in agreement with the transcript sizes observed by the laboratories that originally cloned this gene (13- 15). All of the autoradiographs were subjected to densitometric analysis to determine the relative amounts of mRNA present in each sample. As shown in Fig. 1A, treatment with interferon-y
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FcyRII
Y-Actin
-
18 S
FIG. 2. Northern blot analysis of U937 cells: time course of interferon-y effect. U937 cells were treated with either medium alone (-) or 500 U/ml interferon-y (+) for 8 to 48 hr. Total cellular RNA was isolated and Northern blots were prepared, hybridized with ‘*P-labeled cDNA probes to human Fc-yRII and y-actin, and autoradiographed. The positions of the 28 S and 18 S ribosomal RNA bands are indicated.
increased the levels of steady state FcyRII mRNA in U937 cells, and this increase was partially inhibited by cotreatment with dexamethasone. In all of the experiments, both FcrRII transcripts were modulated to the same extent. There was no increase in FcyRII transcripts observed with any of the treatments in monocytes (Fig. 1B). In order to be certain that the autoradiographs were within the linear portion of the response curve of the densitometer, we performed a titration in which a Northern blot was prepared containing samples of from 3 to 75 rg of untreated U937 RNA. This blot was also hybridized with 32P-labeledy-actin and FcyRII cDNA and autoradiographed for an equivalent amount of time as the experimental blots. The densitometric readings for both y-actin and FcyRII increased linearly over this range (data not shown). Thus, the densitometric measurements were linear for changes from 0.3to 7.5-fold as compared to baseline. For the analysis of U937 cells, two complete time course experiments were performed and five separate Northern blots were prepared from each experiment. The results of the densitometric analyses for all of the blots from a particular experiment were compiled and averaged. The data presented represent the means of these compilations. For the monocyte analysis, only one Northern blot was prepared per donor for monocytes incubated for 24 (n = 2) or 48 (n = 4) hr. The analysis of U937 cells indicated that interferon-y treatment increased total FcyRII mRNA an average of 2.5fold (range 2.1-3.0) with a peak effect between 8 and 24 hr of incubation (Fig. 2). By 48 hr, the mRNA levels decreased to l.Cfold (range 1.l- 1.6) greater than untreated cells. The time course of the effect of interferon-y, dexamethasone, and both agents on FcrRII mRNA levels is shown in Fig. 3. Dexamethasone alone did not appear to have an effect on FcyRII mRNA, but did inhibit by up to 50% the increase seen with interferon-y treatment. Interferon-y and dexamethasone altered the levels of the 1.5- and 2.5-kb transcripts to approximately the same degree. In contrast, monocytes demonstrated no significant change in the levels of FcyRII transcript after 24 or 48 hr of incubation with any of the treatments.
MODULATION
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Time (hours) FIG. 3. Modulation of FcrRII mRNA levels in U937 cells. U937 cells were treated with medium alone, 400 n&f dexamethasone (open squares), 500 U/ml interferon-y (solid squares), or both dexamethasone and interferon--y (solid triangles), for 4 to 48 hr. Total cellular RNA was isolated and Northern blots were prepared, hybridized with ‘*P-labeled cDNA probes to human FcrRII and y-actin, and autoradiographed. The relative amount of FcyRII mRNA present at each time point was determined by densitometric analysis. The mean changes (from two experiments, except at 36 hr which is a single experiment) in FcyRII mRNA levels are plotted as the amount of mRNA (fold increase) in treated cells, as compared to untreated cells, at each time point.
Modulation of Fc-yRII and FcyRI Protein Expression U937 cells and monocytes were incubated with medium control, 400 nM dexamethasone, 500 U/ml interferon-y, or both dexamethasone and interferon-y. At various time points, cells were isolated and stained with either anti-FcyRII or anti-FcyRI and analyzed by flow cytometry. In all cases,only single fluorescent peaks were observed, suggesting that treatment with dexamethasone and/or interferon-y was not affecting a specific subpopulation of cells, but rather that the agents were modulating Fey receptors on all of the cells. Additionally, isotype control irrelevant antibodies for both FcrRII and FcrRI bound lessthan 5% of the cells in all treatment groups. The results of staining with anti-FcyRII are summarized in Fig. 4. In U937 cells, there was an overall significant difference between the treatment groups (P < 0.00 1), as well as a significant difference due to incubation time (P < 0.001). Additionally, there was a significant interaction of the effects of time and treatment (P < 0.02), indicating that the magnitude of the differences between the treatment groups varied with time. Therefore, we analyzed the effects of time and treatment separately. This analysis revealed that dexamethasone induced a significant change in FcrRII protein expression over time (P = 0.001) with a significant decreasebetween 4 and 16 hr (P < 0.05) to a level 15 + 3% below baseline. There was also a significant change in FcyRII expression with interferon-y-treated cells over time (P < 0.00 1). The expression of FcrRII protein increased significantly between 4 and 24 hr (P < 0.05), to a
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Dexanlethasone Interferon Dexamethasone + Interferon
-50. 24
46
Time (hours)
FIG. 4. Kinetics of the modulation of FcyRII on U937 cells and monocytes. U937 cells (A) and fresh human blood monocytes (B) were incubated with medium alone, 400 nM dexamethasone (open bars), 500 U/ml interferon-y (solid bars), or both dexamethasone and interferon-y (hatched bars) for 4-48 hr (U937 cells) or 24 and 48 hr (monocytes). The amount of FcyRII protein on these cells was then determined by flow cytometry utilizing an anti-FcyRII monoclonal antibody. The changes in FcyRII protein expression are plotted as the mean percentage change in the mean fluorescence intensity in treated cells, as compared to untreated cells, at each time point. The error bars represent the SEM for each point (n = 3-5).
maximum of 72 + 16% as compared to control cells. FcyRII expression then decreasedto only 18 + 4% above baseline after 48 hr of incubation. At each time point from 16 to 48 hr dexamethasone consistently (4/4 experiments) inhibited the interferon-y-induced increase in FcyRII expression, but this difference was not statistically significant. In contrast, when we examined the modulation of FcyRII expression on blood monocytes (Fig. 4B), we did not detect any significant changes in FcyRII expression.
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FIG. 5. Kinetics of the modulation of FcyRI on U937 cells and monocytes. U937 cells (A) and fresh human blood monocytes (B) were incubated with medium alone, 400 n&f dexamethasone (open bars), 500 U/ml interferon-y (solid bars), or both dexamethasone and interferon-y (hatched bars) for 4-48 hr (U937 cells) or 24 and 48 hr (monocytes). The amount of FcrRI protein on these cellswas then determined by flow cytometry utilizing an anti-FcyRI monoclonal antibody. The changesin FcyRI protein expression are plotted as the mean percentage change in the mean fluorescence intensity in treated cells, as compared to untreated cells, at each time point. The error bars represent the SEM for each point (n = 3-5).
The changes in FcyRI protein induced by treatment with dexamethasone and/or interferon-y are shown in Fig. 5. The modulation of FcyRl was markedly different from that of FqRII in both magnitude and kinetics, with much greater changes in protein expression being observed with FcyRI. As with the analysis of FcyRII, there was a significant overall effect of treatment (P < 0.00 1) and time (P c 0.00 1), as well as a significant interaction of these effects(P -C0.00 1). There was a significant change in FcyRI expression over time in U937 cells (Fig. 5A) treated with dexamethasone (P = 0.001) or interferon-y (P = 0.001). The significant change with dexamethasone
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treatment occurred between 4 and 16 hr (P < 0.05), when a 39 f 10% decrease in FcyRI expression was observed. Dexamethasone-treated U937 cells maintained this decreasedlevel of receptor expression from 16 to 48 hr of incubation. FcyRI expression on interferon-y-treated U937 cells increased by 240 + 25% from 0 to 16 hr (P < 0.05), and then remained at this elevated level for the remainder of the incubation period. This increase in FcyRI was inhibited by 40% by cotreatment with dexamethasone and required at least 16 hr of incubation. In contrast to the results with U937 cells, dexamethasone alone did not significantly alter FcyRI expression on monocytes (Fig. 5B). However, interferon-y increased the levels of monocyte FcyRI protein by 295 f 82% after 24 hr of incubation (P < 0.05). This increase is of a magnitude similar to that observed with U937 cells. However, in contrast to the effect on U937, cotreatment with dexamethasone resulted in a 155% further increase (P c 0.05) in FcyRI expression as compared to the increase induced by interferon-y alone. This effect was seenafter both 24 and 48 hr of incubation. Induction of FcyRII mRNA and Protein Expression by Interferon-y The above data indicate that there are differences in both the kinetics and the extent of modulation by dexamethasone and/or interferon-y between (a) the two receptors, FcyRI and FcyRII, and (b) the two cell types, U937 cells and monocytes. A comparison of particular interest is that between FcyRII mRNA levels and protein expression in U937 cells incubated with interferon-y. A direct comparison of the modulation of the Fcr receptor transcripts and protein is shown in Fig. 6. At all time points, the increase in mRNA levels was greater than the increase in protein expression. Additionally, the peak increase in FcrRII mRNA levels occurred prior to the increase in protein expression. In order to be certain that the changes observved in anti-Fey receptor immunofluorescence discussed above correlate with changes in actual receptor number, we directly determined the number of FcrRI and FcyRII receptors on U937 cells. U937 were incubated with or without 500 U/ml interferon-y for 24 hr (the time point of maximal increase in fluorescence), and then examined in ligand binding assays.The number of FcyRII receptors was determined by measuring the binding of ‘251-labeled monomeric human IgG to the U937 cells. The number of FcyRII sites on the U937 cells was enumerated by the binding of ‘251-labeledanti-FcyRII monoclonal antibody. There was no significant change following interferon-y treatment in the dissociation constants (&) for either monomeric IgG (3.03 f 0.36 n/M) or anti-FcyRII (0.68 f 0.13 nM). However, treatment with interferon-y increased the average number of FcyRI sites from 22,200 to 82,600, and the FcyRII sites from 45,900 to 104,000 (Table 1). This represents a 283 f 54% increase in FcyRI (P < 0.01) and a 128 & 7% increase in FcyRII (P < 0.05). FACS analysis indicated a 26 1 + 49% increase in antiFcyRI and a 72 f 16% in anti-FcyRII fluorescence after 24 hr of incubation with interferon-y. Thus the interferon-y-induced changes in FcyRI and FcyRII as determined by ligand binding correlate with, and are of a magnitude similar to, those seen by FACS analysis. Even though ligand binding indicated a slightly larger increase in FcyRII expression than did FACS analysis, this difference was not sufficient to explain the discrepancy between the early increase in FcyRII mRNA relative to the rise of protein expression induced by interferon-y.
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Time (hours)
FIG. 6. Effect of interferon-y on FcyRII protein and mRNA levels. U937 cells were incubated with either media alone or 500 U/ml interferon-y for 4 to 48 hr. FcyRII protein expression was determined by flow cytometry with anti-FcyRII, and FcyRII mRNA levels were determined by probing Northern blots with a “P-labeled cDNA probe to human FcyRII. The changes in FcrRII protein expression (open squares) are plotted as the mean percentage change (GEM) in the mean fluorescence intensity in treated cells, as compared to untreated cells, at each time point. The changes in FcrRII mRNA levels (solid squares) are plotted as the mean percentage change (from two experiments) in the relative amount of mRNA in treated cells, as compared to untreated cells, at each time point.
DISCUSSION Fey receptors are important in host defense and autoimmune diseaseand, thus, a thorough understanding of their modulation by both physiologic and pharmacologic signals is important. We studied the effect of two biologic signals on Fey receptors. We chose dexamethasone, a synthetic glucocorticoid, as a model signal because we have observed that Fey receptors are modulated by steroid hormones, such as glucocorticoids (l&21,52). We chose interferon-y because of its known stimulator-y action on Fey receptor functions (26-3 1, 53). We had previously studied the modulation by these agents of FcyRI and FcyRII on U937 cells at a single time point (48 hr) (32). Our current studies extend this work by determining the kinetics of the modulation of human Fcr receptor protein expression. Additionally, we utilized one of the recently isolated cDNA clones for human FcyRII to examine the modulation of this receptor at the mRNA level. Our primary goal was to examine the effects of dexamethasone and interferon-y on steady state levels of FcyRII-specific mRNA in U937 cells and human blood monocytes. We also determined the modulation of FcyRII protein expression following treatment with these agents, in order to correlate the changes in mRNA levels with the changes in cell surface protein. In the current study, we observed a marked, but short-lived, increase in FcyRII mRNA in U937 cells stimulated with interferon-y. This increase could be due to either increased transcription of the FcyRII gene and/or increased stability of FcrRII
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COMBER ET AL. TABLE 1 Equilibrium Binding to FcrRI and FcrRII: Effect of Interferon-y A. FcrRI Number of receptors/cell Experiment
Control
Interferon
Percent change
1 2 3
25,000 18,000 23,600
76,400 87,500 83,800
206 386 256
Mean (+SEM)
22,200 + 2,100
82,600 k 3,300
283 + 54
B. FcrRII Number of receptors/cell Experiment
Control
Interferon
Percent change
1 2 3
67,000 31,400 39,200
150,600 75,600 85,900
125 141 119
45,900 f 10,800
104,000 + 23,500
128k 7
Mean (HEM)
Note. U937 &Is were incubated for 24 hr with either normal medium (control) or 500 U/ml interferon-y. The equilibrium binding of ‘251-labeledmonomeric IgG (FcyRI-dependent) and ‘251-labeledanti-FcyRII was then determined at 37°C. The number of FcrRI (A) and FcyRII (B) sites per cell was determined by Scatchard analysis.
mRNA. A smaller, but also short-lived, change in FcyRII protein expression was also observed on these cells. The time lag and difference in magnitude between the increasein the amount of mRNA and protein following incubation with interferon-y suggestthe involvement of translational or post-translational processesin the regulation of FcyRII protein expression. It is also notable that the 56-72% increase in FcyRII protein is only observed from 16 to 36 hr and was decreased by 48 hr of incubation with interferon-y. The short duration of this interferon-y-induced cell surface expression suggeststhat either (a) the half-life of FcyRII protein is less than 12 hr or (b) the protein is being actively removed from the cell surface. In either case, our data suggest the presence of some regulatory mechanism that affects the translation and/or cell surface expression of the increased FcyRII mRNA present in interferon-y-treated U937 cells. A recent study (54) also examined the expression of FcyRII protein on U937 cells following treatment with interferon-y, but did not observe any change in FcyRII expression. However, protein expression was only examined after 72 hr of incubation with interferon-y. In the current study, the increase in FcyRII protein was seen only at 16 to 36 hr and was significantly decreasedby 48 hr. Thus, the change in receptor expression would not be observable after 72 hr of incubation with interferon-y. Another recent study (5 5) did examine Fey RI1 protein expression after 24 hr of interferon-y treatment, but was performed with polymorphonuclear leukocytes. No changes in FcyRII protein were observed on these neutrophils. Sustained, marked increases
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in FcyRII protein expression have been observed following treatment of U937 with granulocyte or granulocyte-macrophage colony-stimulating factors (CSF) (54). No experiments have as yet been performed with these growth factors to compare their effect with that of interferon-y on FcrRII mRNA levels. When we examined the effects of interferon-y on monocytes, we did not observe any increase in either level of FcyRII mRNA or protein expression. This monocyte nonresponsiveness is not likely due to a generalized resistance of the cells to interferon-y, as receptors for interferon-y have been isolated and quantitated on monocytes (37). Additionally, the expression of FcyRI protein on monocytes was increased by interferon-y (see Fig. 5). The mechanism underlying this difference between monocytes and U937 in their response to interferon-y treatment is not known, but it suggestsa fundamental difference between monocytes and U937 cells in the regulation of FcyRII. In addition to increasing FcrRII protein expression, treatment of U937 cells with interferon-y also increased FcyRI expression, but this modulation differed, in both its kinetics and its extent, from that of FcyRII protein. Maximal stimulation by interferon-y was observed at 16-24 hr for both receptors, but the expression of FcyRI remained increased while the expression of FcyRII decreasedtoward baseline levels. Additionally, the increase in FcyRI protein expression was almost four times that seen for FcrRII. In their recent report of the cloning of FcrRI, Allen and Seed state that FcyRI mRNA levels increase by 6 hr, but decreaseby 24 hr of incubation with interferon-y (12). This is similar to the kinetics we observed for FcyRII mRNA and implies that the differences in the modulation of protein expression following interferon-y treatment are due either to a difference in protein half-life or to different translational or post-translational regulation, and not due to differences in the respective mRNA levels. Further studies to determine the exact mechanisms by which the protein expression of these receptors is regulated are necessary,but these data suggest differences in the post-transcriptional control of these Fey receptor proteins. Since FcyRI and FcyRII bind related ligands, mediate similar functions, and appear to have similar changes in mRNA levels following interferon--y treatment, it is possible the transcription of both genesis coordinately regulated. It will be necessaryto isolate the genomic sequencesfor FcrRI and FcyRII and to analyze their S- and 3’-flanking sequencesanalyzed for the presence of any common potential regulatory sequences. Our studies help to explain the mechanism of the steroid hormone effects on Fey receptors. With U937 cells, the effect of dexamethasone was less marked than that of interferon-y. Dexamethasone alone did not alter FcrRII mRNA levels and only slightly inhibited protein expression. However, dexamethasone partially inhibited the interferon-y-induced increase of FcrRII transcripts and protein. This antagonistic effect of dexamethasone and interferon-y was also observed with FcyRI protein expression on U937 cells. In contrast, dexamethasone and interferon-y increased FcrRI expression on monocytes to a level almost twice as high as that seen with interferon-y alone. The enhancement of the interferon-y effect on monocyte FcrRI has been observed by others (36,56,57) and appears to be due at least in part to a dexamethasone-induced increase in monocyte receptors for interferon-y (58). The mechanism of the inhibitory effect of dexamethasone on FcyRI in U937 cells is not understood. Dexamethasone may not increase interferon-y receptor expression on U937 cells as it does on blood monocytes. Alternatively, the effect of dexamethasone on U937 may be due to an intrinsic difference between monocytes and U937 cells.
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Our Northern blot analyses revealed that in the case of FcyRII on U937 cells, dexamethasone inhibition of the effect of interferon-7 is at least in part due to changes in steady state levels of FcrRII mRNA. In summary, we have demonstrated that treatment with interferon-y increaseslevels of FcyRII-specific mRNA in U937 cells in a time-dependent manner. This modulation of Fc-yRII mRNA is not seen in monocytes treated with interferon-y. The cell surface expression of FcyRII is also increased following interferon-y treatment, but the increase is less than the increase in mRNA levels and occurs after the peak in FcyRII mRNA levels. This suggeststhat translational and/or post-translational regulation is involved in the modulation of FcrRII. Also, dexamethasone partially inhibits the interferon-r-induced increase in both FcrRII mRNA and protein. Additional studies should lead to increased understanding of the mechanisms by which these important cell surface receptors are modulated, and potentially aid in the design of therapeutic interventions for hematologic and autoimmune disorders. ACKNOWLEDGMENTS We thank Mrs. Ruth Rowan for her expert assistancein preparing this manuscript. We also thank Dr. Leonard Braitman and Mr. Delray Schultz for their assistancewith the statistical analysis.
REFERENCES 1. Graziano, R. F., and Fanger, M. W., J. Immunol. 139,3536, 1987. 2. Leslie, R. Cl. Q., Eur. J. Immunol. 10,323, 1980. 3. Graziano, R. F., and Fanger, M. W., J. Immunol. 138,945,1987. 4. Anderson, C. L., and Looney, R. J., Immunol. Today7,264,1986. 5. Unkeless, J. C., Sciglian, E., and Freedman, V. H., Annu. Rev. Zmmunol. 6,25 1, 1988. 6. Hogg, N., Immunol. Today9,185, 1988. 7. Unkeless, J. C., J. Clin. Invest. 83,355, 1989. 8. Anderson, C. L., Guyre, P. M., Whitin, J. C., Ryan, D. H., Looney, R. J., and Fanger, M. W., J. Biol. Chem. 261,12856,1986. 9, Hog& N., and Horton, M. W., In “Leukocyte Typing III. White Cell Differentiation Antigens” (A. J. McMichael, P. C. L. Beverley, S. Cobbold, M. J. Crumpton, W. Gilks, F. M. Gotch, N. Hog& M. Horton, N. Ling, I. C. M. MacLennan, D. Y. Mason, C. Milstein, D. Spiegelhalter, and H. Waldmann, Eds.), pp. 576-602. Oxford Univ. Press,Oxford, 1987. 10. Looney, R. J., Abraham, G. N., and Anderson, C. L., J. Immunol. 136,1641,1986. 11. Lanier, L. L., Le, A. M., Phillips, J. H., Warner, N. L., and Babcock, G. F., J. Immunol. 131, 1789, 1983. 12. Allen, J. M., and Seed, B., Science 243,378, 1989. 13. Hibbq M. L., Bonadonna, L., Scott, B. M., McKenzie, I. F. C., and Hogarth, P. M., Proc. Natl. Acad. Sci. USA 85,2240, 1988. 14. Stuart, S. G., Trounstine, M. L., Vaux, D. J. T., Koch, R., Martens, C. L., Mellman, I., and Moore, K. W.,J. Exp. Med. l&1668,1987. 15. Stengelin, S., Stamenkovic, I., and Seed, B., EMBOJ. 7, 1053, 1988. 16. Simmons, D., and seed, B., Nature(London) 333,568, 1988. 17. Peltz, G. A., Grundy, H. O., Lebo, R. V., Yssel, H., Barsh, G. S., and Moore, K. W., Proc. Natl. Acad. Sci. USA86, 1013, 1989. 18. Atkinson, J. P., Schreiber, A. D., and Frank, M. M., J. Clin. Invest. 52, 1509, 1973. 19. Friedman, D., Nettl, F., and Schreiber, A. D., J. C/in. Invest. 75, 162, 1985. 20. Sanders, M. C,, Levinson, A. I., and Schreiber, A. D., Trans. Assoc. Amer. Physicians 100,268, 1987. 21. Schreiber, A. D., Parson, J., McDermott, P., and Cooper, R. A., J. Clin. Invest. 56, 1189, 1975. 22. Crabtree, G. R., Munck, A., and Smith, K. A., Nature (London) 279,338, 1979. 23. Guyre., P. M., Bodwell, J. E., and Munck, A., J. Steroid B&hem. 15,35, 1981. 24. Nathan, C. F., Murray, H. W., Wiebe.,M. W., and Rubin, B. Y., J. Exp. Med. 158,670, 1983.
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25. Becker, S., J. Immunol. 135,300, 1985. 26. Perussia, B., Dayton, E. T., Lazarus, R., Fanning, V., and Trinchieri, G., J. Exp. Med. 158, 1092, 1983. 27. Guyre, P. M., Morganelli, P. M., and Miller, R., J. Clin. Invest. 72,393, 1983. 28. Rossman, M. D., Chien P., Cassizzi-Cprek, A., Elias, J. A., Holian, A., and Schreiber, A. D., Amer. Rev. Respir. Dis. 133,292, 1986. 29. Jungi, T. W., Lerch, P. G., and Brcic, M., Eur. J. Immunol. 17,735, 1987. 30. Ralph, P., Harris, P. E., Punjabi, C. J., Welte, K., Litcofsky, P. B., Ho, M.-K., Rubin, B. Y., Moore, M. A. S., and Springer, T. A., Blood62,1169,1983. 3 1. Harris, P. E., Ralph, P., Litcofsky, P., and Moore, M. A. S., Cancer Res. 45,9, 1985. 32. Rossman, M. D., Chen, E., Chien, P., and Schreiber, A. D., Cell. Immunol. 119, 174, 1989. 33. Sundstrom, C., and Nilsson, K., Int. J. Cancer 17,565,1976. 34. Schreiber, A. D., Chien, P., Tomaski, A., andcines, D. B., N. Engl. J. Med. 316,503,1987. 35. Chien, P., Pixley, R. A., Stumpo, L. G., Colman, R. W., and Schreiber, A. D., J. Clin. Invest. 82, 1554, 1988. 36. Girard, M. T., Hjaltadottir, S., Fejes-Toth, A. N., and Guyre, P. M., J. Immunol. 138,3235, 1987. 37. Finbloom, D. S., Hoover, D. L., and Wahl, L. M., J. Immunol. 135,300, 1985. 38. Werb, Z., Foley, R., and Munck, A., J. Exp. Med. 147, 1684, 1978. 39. Webel, M. L., Ritts, R. E., Jr., Taswell, A. F., Donadio, Jr., J. V., and Woods, R. E., J. Lab. C/in. Med. 83,383, 1974. 40. Schaffner, A., J. Clin. Invest. 76,1755, 1985.
41. Gunning, P., Ponte, P., Okayama, H., Engel, J., Blau, H., and Kedes, L., Mol. Cell. Biol. 3,787, 1983. 42. Feinberg, A. P., and Bogelstein, B., Anal. B&hem. 132,6, 1983. 43. Maniatis, T., Fritsch, E. F., and Sambrook, J., “Molecular Cloning. A Laboratory Manual.” Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982. 44. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J., and Rutter, W. J., Biochemistry 18,5294, 1979. 45. Lehrach, H., Diamond, D., Wozney, J. M., and Boedtker, H., Biochemistry 16,4743,1977. 46. Wahl, G. M., Stem, M., and Stark, G. R., Proc. Natl. Acad. Sci. USA 76,3683, 1979. 47. Wood, G. S., Warner, N. L., and Wamke, R. A., J. Immunol. 131,212, 1983. 48. Dimitriu-Bona, A., Burmester, G. R., Waters, S. J., and Winchester, R. J., J. Immunol. 130,145,1983. 49. Hunter, W. M., and Greenwood, F. C., Nature (London) 194,495, 1962. 50. Scatchard, G., Ann. N. Y. Acad. Sci. 51,660, 1949. 5 1. Scheffe,H. A., Biometrika 40,87, 1953. 52. Schreiber, A. D., Nettl, F. M., Sanders, M. C., King, M., Szaboics, P., Friedman, D., and Gomez, F., J. Immunol. 141,2959, 1988. 53. Pert&a, B., Kobayashi, M., Rossi, M. E., Anegon, I., and Trinchieri, G., J. Immunol. 138,765, 1987. 54. Liesveld, J. L., Abboud, C. N., Looney, R. J., Ryan, D. H., and Brennan, J. K., J. Immunol. 140, 1527, 1988. 55. Petroni, K. C., Shen, L., and Guyre, P. M., J. Immunol. 140,3467, 1988. 56. Warren, M. K., and Vogel, S. N., J. Immunol. 134,2462,1985. 57. Guyre, P. M., Girard, M. T., Morganelli, P. M., and Manganiello, P. D., J. Steroid Biochem. 30,89, 1988. 58. Strickland, R. W., Wahl, L. M., and Finbloom, D. S., J. Immunol. 137, 1577, 1986.
IMMUNOLOGY
124,292-307( 1989)
Modulation
of Human Mononuclear Phagocyte FcyRll mRNA and Protein’
PAULG.COMBER,*MILTON D. ROSSMAN,*ERIC F. RAPPAPORT,PPAULCHIEN,* P. MARK HOGARTH,* AND ALAN D. SCHRPIBER* *University of Pennsylvania School ofMedicine and TChildren ‘s Hospital, Philadelphia, PennsyIvania 19104, and University ofMeIbourne,$ Melbourne, Australia Received July 10,1989; acceptedAugust 9.1989 Human monocytes and macrophages express three different classesof cell surface receptors for the Fc portion of IgG, Fc+yRI(CD64), FcyRII (CD32), and FcyRIII (CD16). We utilized a cDNA probe for FcyRII to examine the modulation of FcyRII mRNA by dexamethasone, a synthetic glucocorticoid, and interferon-y. We also determined the changesin the expression of both FcyRI and FcyRII protein following treatment with these agents by flow cytometry. In studies performed with the monocyte-like cell line, U937, Northern blot analysis revealed that cells treated with interferon-y showed a 2.5-fold increase in FcyRII mRNA levels that was maximal at 14 hr and declined to 1.4-fold over baseline by 48 hr of incubation. Treatment of U937 cells with dexamethasone did not significantly change the level of FcrRII transcripts, but was able to inhibit by up to 50% the increase seen following interferon-y treatment. The expression of FcyRII protein on U937 cells was increased 56-722 after 16-24 hr of interferon-y treatment, but was only 18%over baseline after 48 hr ofincubation. Treatment with dexamethasone caused a small, but significant, decreasein FcrRII protein, and inhibited by 20-60% the induction of FcyRII by interferon-y. The modulation by dexamethasone and interferon-y of FcyRI protein expression on U937 cells was markedly different from that of FcrRII in both magnitude and kinetics. Interferon-y treatment increased FcyRI expression by 240% at 16 hr, and FcyRI remained elevated through 48 hr. Treatment with dexamethasone decreasedFcyRI expression by 39%, and also inhibited by 40% the increase induced by interferon-y. In contrast to the findings with U937 cells, dexamethasone and/or interferon-y treatment had no significant effect on FcrRII mRNA levels or protein expression in monocytes. However, interferon-y treatment increased FcyRI expression on monocytes, and this increase was further augmented by treatment with dexamethasone. These data indicate that the modulation of FcyRII on U937 cells is at least in part due to changes in steady state levels of FcyRII mRNA. The difference between the magnitude of the changes in FcyRII mRNA and protein suggeststhat some translational or post-translational control is involved in regulating the expression of FcyRII. The differences between FcyRI and FcyRII, as well as between monocytes and U937 cells, in the extent to which they are modulated by dexamethasone and interferon-y indicate that modulation of Fey receptors by these two agents is both cell- and receptor-specific. o 1989 Academic press, IIIC.
INTRODUCTION Surface receptors for the Fc portion of IgG (FcrR) are found on many different human cells. On mononuclear phagocytes (blood monocytes and tissue macro’ This work was partially supported by National Institutes of Health Grant AI/HL-22 193. P.G.C. is the recipient of a National Institutes of Health Medical Scientist Training Program fellowship at the University of Pennsylvania. 292 0008-8749189 $3.00 Copyright 0 1989 by Academic Press,Inc. All rights of reproduction in any form reserved.
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phages), these receptors mediate important effector functions: phagocytosis of opsonized cells (l), clearance of immune complexes (2), and antibody-dependent cell cytotoxicity (3). The stimulation of FcyR on monocytes and macrophages also results in production and extracellular releaseof lysosomal enzymes, neutral proteases,prostaglandins, and superoxide anion. Three different classes of Fcr receptors have been identified on human cells (FcrRI, FcyRII, and FcrRIII) (reviewed in Refs. (4-7)). These receptors have been differentiated on the basis of their size, binding alfmities for various classesof murine IgG, and recognition by monoclonal antibodies. FcyRI (CD64) is a 72-kDa protein, recognized by monoclonal antibody mAb32.2 (8), that is found only on mononuclear phagocytes. It is the only Fey receptor that binds monomeric IgG (Kd = 1O-*- 1O-’ M). FcyRII, which has been designated CD32 (9), is a 40-kDa protein that is recognized by mAbIV.3 ( 10). In addition to being on monocytes and macrophages, FcyRII is also found on platelets, granulocytes, and B-lymphocytes. FcyRIII, a SO-to 70kDa protein formerly called Fc-yR,,, is not present on monocytes but is found on macrophages, granulocytes, natural killer cells, and some T-lymphocytes. FcyRIII is also the leu-1 1 antigen (11) and has been designated CD-16 (9). Recently, cDNA probes encoding for FcyRI ( 12) FcyRII ( 13- 15) and FcyRIII ( 16, 17) have been isolated and cloned. The availability of these clones enables the examination of the expression and regulation of mRNA for these different Fcr receptors. Mononuclear phagocyte Fey receptors have been implicated in the pathogenesis of several hematologic and immunologic diseases,and therefore the modulation of these receptors is of importance. Utilizing an in viva animal model, we observed that glucocorticoids inhibit the clearance of antibody-coated cells from the circulation (an Fey receptor-mediated event) ( 18-20). This finding was confirmed with in vitro studies, which demonstrated that glucocorticoids can inhibit Fey receptor expression and function on mononuclear phagocytes (2 1) and human cell lines (22,23). Interferon-y is a cytokine produced by T-lymphocytes during immune and inflammatory responsesthat has a wide range of effects, including stimulation of macrophage and monocyte oxidative metabolism (24) and differentiation (25). Several in vitro studies have demonstrated that interferon-y treatment of mononuclear phagocytes (26-29) as well as cell lines (30,3 l), markedly increases the expression of the FcrRI protein. We recently reported on the effects of both interferon-y and dexamethasone, a synthetic glucocorticoid, on FcrRI and FcyRII protein expression, and on ligand binding (32). However, the kinetics of these effects on Fey receptor expression have not been studied. Similarly, the modulation of Fcr receptor mRNA has not yet been examined. In this paper, we utilized one of the recently isolated full length FcrRII cDNA probes, HFc3.0 ( 13), to examine the changes in FcyRII mRNA levels following treatment with interferon-y and/or dexamethasone. We also examined the kinetics of the change in protein expression of FcrRI and FcrRII following treatment with these agents. These studies were performed with both fresh blood monocytes and U937 cells (33), a histiocytic lymphoma cell line which has characteristics of immature monocytes and, like monocytes, expressesthe Fcr receptors FcyRI and FcyRII. MATERIALS AND METHODS
Cell Culture U937 cells were obtained from the ATCC (American Type Culture Collection, Rockville, MD). These cells were maintained in continuous culture in RPM1 1640
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(GIBCO Laboratories, Grand Island, NY) culture medium supplemented with 10% heat-inactivated fetal calf serum (FCS), penicillin ( 100 U/ml), streptomycin ( 100 pg/ ml), and glutamine (25 mg/ml). This cell line was originally established from the pleural effision of a patient with a generalized, diffise histiocytic lymphoma (33). Human monocytes were isolated by removing peripheral blood from normal donors, which was anticoagulated with heparin (0.2 U/ml). Mononuclear cells were isolated by layering the blood on Lymphocyte Separation Media (Organon Teknika Corp., Durham, NC) followed by centrifugation at 900g. Cells were collected from the interface and adhered for 45 min at 37°C to T-75 tissue culture flasks that had been precoated with FCS. Nonadherent cells were removed by rinsing five to six times with Hanks’ balanced salt solution (HBSS). The monocytes were then cultured in RPM1 1640 medium containing 10% FCS, penicillin, streptomycin, and glutamine (as for the U937 cells). Monocytes were harvested by striking the flasks against a soft surface to dislodge the cells, followed by rinsing with HBSS (without Ca” or Me) containing 5 mM EDTA, 0.1% gelatin, and 0.1% sodium azide. We have previously shown that this isolation results in a cell population that is 90-95% monocytes as determined by nonspecific esterase staining and phagocytosis of latex particles (34,35). Dexamethasone and Interferon-y Preparations The synthetic glucocorticoid dexamethasone was purchased from Sigma Chemical Co. (St. Louis, MO). A stock solution of 0. I mM concentration was prepared by dissolving lyophilized dexamethasone in water. This stock was prepared fresh prior to each experiment and diluted to a final concentration of 400 n&J. Recombinant human interferon-y was the gift of Genentech, Inc. (South San Francisco, CA). A stock solution of 1.25 X 1O6U/ml was prepared and then diluted to a final concentration of 500 U/ml. All experiments were performed utilizing these dosage levels of 400 ti dexamethasone and 500 U/ml interferon-y for treatment. These doses of dexamethasone and interferon-y utilized in these experiments are the lowest levels at which our laboratory (unpublished observations) and others (36) have seen maximal modulation of FcyRI protein expression in U937 cells and monocytes. Additionally, these dosagesare sufficient to saturate the interferon-y (37) and glucocorticoid (38) receptors on monocytes in vitro. The dose of dexamethasone (400 nM) is well within the range of therapeutic concentrations achievable by pharmacologic administration of glucocorticoids (39,40). cDNA Probes We employed a recently isolated full length cDNA clone for human FcyRII [HFc3.0] (13). HFc3.0 is a full length human FcyRII cDNA clone which encodes a leader sequence,two immunoglobulin-like extracellular domains, a single transmembrane region, and an intracytoplasmic domain. This 1.4-kb clone contains the entire coding sequence for the 28 1 amino acid FcyRII protein, as well as some of the 3’untranslated region. Sequence analysis of overlapping clones has revealed that there is a polyadenylation site just 3’ to the sequence encoded by the HFc3.0 clone, as well as a second polyadenylation site approximately 1 kb further downstream (13). We also utilized a cDNA probe to the constitutively produced protein human y-actin (pHFyA- 1, gift of Larry Kedes) (4 1). The cDNA inserts were isolated by electroelu-
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tion after restriction enzyme digestion. The inserts were radiolabeled to high specific activity (>2 X lo* cpm/pg) utilizing random primer extension by the Klenow fmgment of DNA polymerase I (Random Primed DNA Labeling Kit, Boehringer Mannheim Biochemicals, Indianapolis, IN) in the presence of [32P]CTP (42). Unincorporated radioactivity was removed by spun column chromatography (43) on a prepacked Sephadex G-50 column (Quick Spin, Boehringer Mannheim Biochemicals).
Northern Blot Analysis Total cellular RNA was isolated by lysing the cells in a 4 M guanidine isothiocyanate buffer and pelleting through a 5.7 M cesium chloride gradient, purified by butanol/chloroform extraction, and isolated by ethanol precipitation (44). Equal amounts ( 10 pg), as determined by optical density at 260 nM and ethidium bromide staining, of the RNA samples were electrophoretically separated on a 1% (w/v) agarose/6% formaldehyde gel in a 3% formaldehyde/MOPS buffer solution (42, 45). The RNA was then transferred by capillary blotting in a solution of 1OX standard saline citrate (SSC) to a nylon hybridization membrane (GeneScreen Plus, NEN DuPont, Boston, MA). The blots were rinsed in a solution of 2X SSC and baked at 80°C for 2 hr. The blots were prehybridized for l-4 hr at 42°C in a solution of 50% deionized formamide, 1%sodium dodecyl sulfate (SDS), 1 A4 sodium chloride, and 10%dextran sulfate (46). 32P-labeledFcyRII cDNA was then added to the blots at a concentration of approximately 1 X lo6 cpm/ml and hybridized to the blots overnight at 42°C. The blots were washed twice in 2X SSC for 5 min at 20°C twice in 2X SSC/ 1.O%SDS for 30 min at 65°C and twice in 0.1 X SSC for 30 min at 20°C and then autoradiographed. Probe was stripped from the blots by incubation in a solution of 0.2% SDS/ 0.0 1 mM Tris (pH 7.4) for 60 min at 80°C. The blots were also hybridized with 32Plabeled y-actin cDNA in a similar manner in order to assure that equal amounts of monocyte or U937 RNA were loaded. The autoradiographs were analyzed with a densitometer (UltraScan XL, LKB Products, Bromma, Sweden) to determine the relative amounts FcyRII and y-actin mRNA present in each sample. The measurement of the relative amounts of FcyRII was adjusted for any variation in amount of total RNA loaded as detected by variation in the y-actin signal.
Flow Cytometry Monoclonal antibodies directed against FcyRII [mAbIV.3] (10) and FcyRI [mAb32.2] (8) were utilized in indirect immunofluorescence binding studies to assess protein expression of these receptors. The cells were incubated with the antibodies for 30 min at 4°C and washed twice with phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin and 0.02% sodium azide. Bound antibody was labeled by incubation with an FITC-labeled goat anti-mouse antibody (TAGO, Inc., Burlingame, CA) for 30 min at 4°C. The cells were again washed twice and then fixed with 4% paraformaldehyde until analyzed by flow cytometry. Fluorescence was measured by a FACStar cytometer with Consort-30 software (Becton-Dickinson, Mountain View, CA). For all samples, 10,000 events were recorded on a logarithmic fluorescence scale, and the actual mean fluorescence intensity (MFI) for each sample was determined using the Consort-30 software. In order to correct for autofluorescence, the MFI of a nonreactive murine IgG, antibody [P3 X 631 was subtracted from the
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MFI of the anti-FcyRII- and anti-FcyRI-stained cells. Percentage change in fluorescence intensity was calculated by % change (anti-FcyR MFI-treated cells - P3 X 63 MFI-treated cells) _ 1 x 100 = (anti-FcyR MFI-untreated cells - P3 X 63 MFI-untreated cells) ’
1
Two murine IgGZbantibodies were purchased from Becton-Dickinson and used as isotype controls for anti-FcyRII binding to monocytes [anti-Leud] (47) and U937 cells [anti&u-M31 (48).
Ligand Binding Assays Ligand binding studies were performed in order to determine the number of FcyRI and FcrRII receptor sites. The binding of ‘251-labeledmonomeric IgG and antiFcyRII [mAbIV.3] was utilized to measure FcyRI and FcyRII, respectively. Monomeric IgG was isolated from normal human serum by affinity chromatography utilizing a Sepharosecolumn of murine anti-human IgG antibody, as previously described (35). Both monomeric IgG and anti-FcyRII were radiolabeled with “‘1 by the chloramine-T method (49). Cells were collected, pelleted, resuspended in a serum-free buffer, and incubated for 30 min at 37°C to remove any nascent IgG. The cells were then pelleted and resuspended in HBSS (without Ca2+ or Mg2+) containing 5 mM EDTA, 0.1% gelatin, and 0.1% sodium azide (to prevent receptor endocytosis). The cells were then incubated with increasing amounts of the radiolabeled ligand, with or without an excessof unlabeled ligand, at 37°C for 45 min. We have previously shown that, under these conditions, binding equilibrium is reached within 20-30 min and 96-99% of the ligand is surface bound and not internalized (28, 32,36). Cell-bound ligand was separated from free ligand by centrifugation through silicon oil and quantitated by counting the radioactivity in the cell pellet. Specific binding was determined by subtracting the nonspecific binding (radiolabeled ligand with excessof unlabeled ligand) from total binding (radiolabeled ligand alone). Receptor number and affinity were determined by Scatchard analysis (50).
Statistical Analysis Two-way factorial (3 X 6) analysis of variance (ANOVA) with two groups (time and treatment) was utilized to analyze the results of the flow cytometry of U937 cells. Post hoc analysis was performed with the Scheffe test (51) to assessthe individual time and treatment effects. The Sche& test was chosen becauseit is the most conservative of the post hoc analyses. Since flow cytometry of monocytes was only performed at two time points, the Student t test was used to analyze these results. A paired Student t test was also used to assessany differences between treatments in the ligand binding assay.The ANOVA and ScheG test were performed using the Statistical Analysis System (SAS Institute, Inc., Raleigh, NC) on a Compaq 386/20 computer. The Student t tests were performed using the StatWorks program (Cricket Software, Inc., Philadelphia, PA) on an Apple Macintosh computer.
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A _
DMS
IFN
D+I -
28 s
-
18s
-
28s
FcrRII
-
18 s
7 -Actin
-
18 S
: FcyRII
B _
DMS
IFN
D+I
FIG. 1. Northern blot analysis of U937 cells and monocytes: effect of dexamethasone and interferon~. U937 cells (A) and fresh human blood monocytes (B) were treated with medium alone (-), 400 nMdexamethasone (DMS), 500 U/ml interferon-r (IFN), or both dexamethasone and interferon-y (D + I) for 14 (U937 cells) or 48 (monocytes) hr. Total cellular RNA was isolated and Northern blots were prepared, hybridized with 3zP-labeledcDNA probes to human FcyRII and y-a&n, and autoradiographed. The positions of the 28 S and I8 S ribosomal RNA bands are indicated.
RESULTS
Modulation of FcyRII mRNA Total cellular RNA was isolated from U937 cells and human blood monocytes after incubation with normal medium (control), 400 nM dexamethasone, 500 U/ml interferon-y, and both dexamethasone and interferon-y. Northern blots were prepared and probed with 32P-labeledFcyRII cDNA. Approximately equal amounts of RNA were loaded in each lane of each blot. The blots were also hybridized with a probe for the constitutively produced protein y-actin. Representative autoradiographs of Northern blots from U937 cells and monocytes treated with dexamethasone and/or interferon-y are shown in Fig. 1. These autoradiographs indicate the presence of two transcripts for FcyRII, 2.5 and 1.5 kb in length, which are the same size in both monocytes and U937 cells. This is in agreement with the transcript sizes observed by the laboratories that originally cloned this gene (13- 15). All of the autoradiographs were subjected to densitometric analysis to determine the relative amounts of mRNA present in each sample. As shown in Fig. 1A, treatment with interferon-y
298
COMBER ET AL. Time (hr): Interferon-Y
FcyRII
Y-Actin
-
18 S
FIG. 2. Northern blot analysis of U937 cells: time course of interferon-y effect. U937 cells were treated with either medium alone (-) or 500 U/ml interferon-y (+) for 8 to 48 hr. Total cellular RNA was isolated and Northern blots were prepared, hybridized with ‘*P-labeled cDNA probes to human Fc-yRII and y-actin, and autoradiographed. The positions of the 28 S and 18 S ribosomal RNA bands are indicated.
increased the levels of steady state FcyRII mRNA in U937 cells, and this increase was partially inhibited by cotreatment with dexamethasone. In all of the experiments, both FcrRII transcripts were modulated to the same extent. There was no increase in FcyRII transcripts observed with any of the treatments in monocytes (Fig. 1B). In order to be certain that the autoradiographs were within the linear portion of the response curve of the densitometer, we performed a titration in which a Northern blot was prepared containing samples of from 3 to 75 rg of untreated U937 RNA. This blot was also hybridized with 32P-labeledy-actin and FcyRII cDNA and autoradiographed for an equivalent amount of time as the experimental blots. The densitometric readings for both y-actin and FcyRII increased linearly over this range (data not shown). Thus, the densitometric measurements were linear for changes from 0.3to 7.5-fold as compared to baseline. For the analysis of U937 cells, two complete time course experiments were performed and five separate Northern blots were prepared from each experiment. The results of the densitometric analyses for all of the blots from a particular experiment were compiled and averaged. The data presented represent the means of these compilations. For the monocyte analysis, only one Northern blot was prepared per donor for monocytes incubated for 24 (n = 2) or 48 (n = 4) hr. The analysis of U937 cells indicated that interferon-y treatment increased total FcyRII mRNA an average of 2.5fold (range 2.1-3.0) with a peak effect between 8 and 24 hr of incubation (Fig. 2). By 48 hr, the mRNA levels decreased to l.Cfold (range 1.l- 1.6) greater than untreated cells. The time course of the effect of interferon-y, dexamethasone, and both agents on FcrRII mRNA levels is shown in Fig. 3. Dexamethasone alone did not appear to have an effect on FcyRII mRNA, but did inhibit by up to 50% the increase seen with interferon-y treatment. Interferon-y and dexamethasone altered the levels of the 1.5- and 2.5-kb transcripts to approximately the same degree. In contrast, monocytes demonstrated no significant change in the levels of FcyRII transcript after 24 or 48 hr of incubation with any of the treatments.
MODULATION
0.04 0
. 8
OF FcrRII mRNA AND PROTEIN
, 16
.
.
.
.
24
32
40
48
299
Time (hours) FIG. 3. Modulation of FcrRII mRNA levels in U937 cells. U937 cells were treated with medium alone, 400 n&f dexamethasone (open squares), 500 U/ml interferon-y (solid squares), or both dexamethasone and interferon--y (solid triangles), for 4 to 48 hr. Total cellular RNA was isolated and Northern blots were prepared, hybridized with ‘*P-labeled cDNA probes to human FcrRII and y-actin, and autoradiographed. The relative amount of FcyRII mRNA present at each time point was determined by densitometric analysis. The mean changes (from two experiments, except at 36 hr which is a single experiment) in FcyRII mRNA levels are plotted as the amount of mRNA (fold increase) in treated cells, as compared to untreated cells, at each time point.
Modulation of Fc-yRII and FcyRI Protein Expression U937 cells and monocytes were incubated with medium control, 400 nM dexamethasone, 500 U/ml interferon-y, or both dexamethasone and interferon-y. At various time points, cells were isolated and stained with either anti-FcyRII or anti-FcyRI and analyzed by flow cytometry. In all cases,only single fluorescent peaks were observed, suggesting that treatment with dexamethasone and/or interferon-y was not affecting a specific subpopulation of cells, but rather that the agents were modulating Fey receptors on all of the cells. Additionally, isotype control irrelevant antibodies for both FcrRII and FcrRI bound lessthan 5% of the cells in all treatment groups. The results of staining with anti-FcyRII are summarized in Fig. 4. In U937 cells, there was an overall significant difference between the treatment groups (P < 0.00 1), as well as a significant difference due to incubation time (P < 0.001). Additionally, there was a significant interaction of the effects of time and treatment (P < 0.02), indicating that the magnitude of the differences between the treatment groups varied with time. Therefore, we analyzed the effects of time and treatment separately. This analysis revealed that dexamethasone induced a significant change in FcrRII protein expression over time (P = 0.001) with a significant decreasebetween 4 and 16 hr (P < 0.05) to a level 15 + 3% below baseline. There was also a significant change in FcyRII expression with interferon-y-treated cells over time (P < 0.00 1). The expression of FcrRII protein increased significantly between 4 and 24 hr (P < 0.05), to a
300
COMBER ET AL.
5
50
= i
25
E
-50
8 ; 0
! 4
6
16
24
36
46
Time (hours)
0 H
q
E !: k a
Dexanlethasone Interferon Dexamethasone + Interferon
-50. 24
46
Time (hours)
FIG. 4. Kinetics of the modulation of FcyRII on U937 cells and monocytes. U937 cells (A) and fresh human blood monocytes (B) were incubated with medium alone, 400 nM dexamethasone (open bars), 500 U/ml interferon-y (solid bars), or both dexamethasone and interferon-y (hatched bars) for 4-48 hr (U937 cells) or 24 and 48 hr (monocytes). The amount of FcyRII protein on these cells was then determined by flow cytometry utilizing an anti-FcyRII monoclonal antibody. The changes in FcyRII protein expression are plotted as the mean percentage change in the mean fluorescence intensity in treated cells, as compared to untreated cells, at each time point. The error bars represent the SEM for each point (n = 3-5).
maximum of 72 + 16% as compared to control cells. FcyRII expression then decreasedto only 18 + 4% above baseline after 48 hr of incubation. At each time point from 16 to 48 hr dexamethasone consistently (4/4 experiments) inhibited the interferon-y-induced increase in FcyRII expression, but this difference was not statistically significant. In contrast, when we examined the modulation of FcyRII expression on blood monocytes (Fig. 4B), we did not detect any significant changes in FcyRII expression.
MODULATION
OF FcrRII mRNA AND PROTEIN
301
5 E m c 2w 8 loo
f 5 5 e
0 -100
8
4
00
B
18
24
38
48
Time (hours)
e” e d 800 C t 8 ::0 g = f E ,c
700
600 500 400
3w
2m
g s Jz u
loo
E t
-100
;i a
0 48
24
Tim
(hours)
FIG. 5. Kinetics of the modulation of FcyRI on U937 cells and monocytes. U937 cells (A) and fresh human blood monocytes (B) were incubated with medium alone, 400 n&f dexamethasone (open bars), 500 U/ml interferon-y (solid bars), or both dexamethasone and interferon-y (hatched bars) for 4-48 hr (U937 cells) or 24 and 48 hr (monocytes). The amount of FcrRI protein on these cellswas then determined by flow cytometry utilizing an anti-FcyRI monoclonal antibody. The changesin FcyRI protein expression are plotted as the mean percentage change in the mean fluorescence intensity in treated cells, as compared to untreated cells, at each time point. The error bars represent the SEM for each point (n = 3-5).
The changes in FcyRI protein induced by treatment with dexamethasone and/or interferon-y are shown in Fig. 5. The modulation of FcyRl was markedly different from that of FqRII in both magnitude and kinetics, with much greater changes in protein expression being observed with FcyRI. As with the analysis of FcyRII, there was a significant overall effect of treatment (P < 0.00 1) and time (P c 0.00 1), as well as a significant interaction of these effects(P -C0.00 1). There was a significant change in FcyRI expression over time in U937 cells (Fig. 5A) treated with dexamethasone (P = 0.001) or interferon-y (P = 0.001). The significant change with dexamethasone
302
COMBER ET AL.
treatment occurred between 4 and 16 hr (P < 0.05), when a 39 f 10% decrease in FcyRI expression was observed. Dexamethasone-treated U937 cells maintained this decreasedlevel of receptor expression from 16 to 48 hr of incubation. FcyRI expression on interferon-y-treated U937 cells increased by 240 + 25% from 0 to 16 hr (P < 0.05), and then remained at this elevated level for the remainder of the incubation period. This increase in FcyRI was inhibited by 40% by cotreatment with dexamethasone and required at least 16 hr of incubation. In contrast to the results with U937 cells, dexamethasone alone did not significantly alter FcyRI expression on monocytes (Fig. 5B). However, interferon-y increased the levels of monocyte FcyRI protein by 295 f 82% after 24 hr of incubation (P < 0.05). This increase is of a magnitude similar to that observed with U937 cells. However, in contrast to the effect on U937, cotreatment with dexamethasone resulted in a 155% further increase (P c 0.05) in FcyRI expression as compared to the increase induced by interferon-y alone. This effect was seenafter both 24 and 48 hr of incubation. Induction of FcyRII mRNA and Protein Expression by Interferon-y The above data indicate that there are differences in both the kinetics and the extent of modulation by dexamethasone and/or interferon-y between (a) the two receptors, FcyRI and FcyRII, and (b) the two cell types, U937 cells and monocytes. A comparison of particular interest is that between FcyRII mRNA levels and protein expression in U937 cells incubated with interferon-y. A direct comparison of the modulation of the Fcr receptor transcripts and protein is shown in Fig. 6. At all time points, the increase in mRNA levels was greater than the increase in protein expression. Additionally, the peak increase in FcrRII mRNA levels occurred prior to the increase in protein expression. In order to be certain that the changes observved in anti-Fey receptor immunofluorescence discussed above correlate with changes in actual receptor number, we directly determined the number of FcrRI and FcyRII receptors on U937 cells. U937 were incubated with or without 500 U/ml interferon-y for 24 hr (the time point of maximal increase in fluorescence), and then examined in ligand binding assays.The number of FcyRII receptors was determined by measuring the binding of ‘251-labeled monomeric human IgG to the U937 cells. The number of FcyRII sites on the U937 cells was enumerated by the binding of ‘251-labeledanti-FcyRII monoclonal antibody. There was no significant change following interferon-y treatment in the dissociation constants (&) for either monomeric IgG (3.03 f 0.36 n/M) or anti-FcyRII (0.68 f 0.13 nM). However, treatment with interferon-y increased the average number of FcyRI sites from 22,200 to 82,600, and the FcyRII sites from 45,900 to 104,000 (Table 1). This represents a 283 f 54% increase in FcyRI (P < 0.01) and a 128 & 7% increase in FcyRII (P < 0.05). FACS analysis indicated a 26 1 + 49% increase in antiFcyRI and a 72 f 16% in anti-FcyRII fluorescence after 24 hr of incubation with interferon-y. Thus the interferon-y-induced changes in FcyRI and FcyRII as determined by ligand binding correlate with, and are of a magnitude similar to, those seen by FACS analysis. Even though ligand binding indicated a slightly larger increase in FcyRII expression than did FACS analysis, this difference was not sufficient to explain the discrepancy between the early increase in FcyRII mRNA relative to the rise of protein expression induced by interferon-y.
MODULATION
OF FcrRII mRNA AND PROTEIN
303
Time (hours)
FIG. 6. Effect of interferon-y on FcyRII protein and mRNA levels. U937 cells were incubated with either media alone or 500 U/ml interferon-y for 4 to 48 hr. FcyRII protein expression was determined by flow cytometry with anti-FcyRII, and FcyRII mRNA levels were determined by probing Northern blots with a “P-labeled cDNA probe to human FcyRII. The changes in FcrRII protein expression (open squares) are plotted as the mean percentage change (GEM) in the mean fluorescence intensity in treated cells, as compared to untreated cells, at each time point. The changes in FcrRII mRNA levels (solid squares) are plotted as the mean percentage change (from two experiments) in the relative amount of mRNA in treated cells, as compared to untreated cells, at each time point.
DISCUSSION Fey receptors are important in host defense and autoimmune diseaseand, thus, a thorough understanding of their modulation by both physiologic and pharmacologic signals is important. We studied the effect of two biologic signals on Fey receptors. We chose dexamethasone, a synthetic glucocorticoid, as a model signal because we have observed that Fey receptors are modulated by steroid hormones, such as glucocorticoids (l&21,52). We chose interferon-y because of its known stimulator-y action on Fey receptor functions (26-3 1, 53). We had previously studied the modulation by these agents of FcyRI and FcyRII on U937 cells at a single time point (48 hr) (32). Our current studies extend this work by determining the kinetics of the modulation of human Fcr receptor protein expression. Additionally, we utilized one of the recently isolated cDNA clones for human FcyRII to examine the modulation of this receptor at the mRNA level. Our primary goal was to examine the effects of dexamethasone and interferon-y on steady state levels of FcyRII-specific mRNA in U937 cells and human blood monocytes. We also determined the modulation of FcyRII protein expression following treatment with these agents, in order to correlate the changes in mRNA levels with the changes in cell surface protein. In the current study, we observed a marked, but short-lived, increase in FcyRII mRNA in U937 cells stimulated with interferon-y. This increase could be due to either increased transcription of the FcyRII gene and/or increased stability of FcrRII
304
COMBER ET AL. TABLE 1 Equilibrium Binding to FcrRI and FcrRII: Effect of Interferon-y A. FcrRI Number of receptors/cell Experiment
Control
Interferon
Percent change
1 2 3
25,000 18,000 23,600
76,400 87,500 83,800
206 386 256
Mean (+SEM)
22,200 + 2,100
82,600 k 3,300
283 + 54
B. FcrRII Number of receptors/cell Experiment
Control
Interferon
Percent change
1 2 3
67,000 31,400 39,200
150,600 75,600 85,900
125 141 119
45,900 f 10,800
104,000 + 23,500
128k 7
Mean (HEM)
Note. U937 &Is were incubated for 24 hr with either normal medium (control) or 500 U/ml interferon-y. The equilibrium binding of ‘251-labeledmonomeric IgG (FcyRI-dependent) and ‘251-labeledanti-FcyRII was then determined at 37°C. The number of FcrRI (A) and FcyRII (B) sites per cell was determined by Scatchard analysis.
mRNA. A smaller, but also short-lived, change in FcyRII protein expression was also observed on these cells. The time lag and difference in magnitude between the increasein the amount of mRNA and protein following incubation with interferon-y suggestthe involvement of translational or post-translational processesin the regulation of FcyRII protein expression. It is also notable that the 56-72% increase in FcyRII protein is only observed from 16 to 36 hr and was decreased by 48 hr of incubation with interferon-y. The short duration of this interferon-y-induced cell surface expression suggeststhat either (a) the half-life of FcyRII protein is less than 12 hr or (b) the protein is being actively removed from the cell surface. In either case, our data suggest the presence of some regulatory mechanism that affects the translation and/or cell surface expression of the increased FcyRII mRNA present in interferon-y-treated U937 cells. A recent study (54) also examined the expression of FcyRII protein on U937 cells following treatment with interferon-y, but did not observe any change in FcyRII expression. However, protein expression was only examined after 72 hr of incubation with interferon-y. In the current study, the increase in FcyRII protein was seen only at 16 to 36 hr and was significantly decreasedby 48 hr. Thus, the change in receptor expression would not be observable after 72 hr of incubation with interferon-y. Another recent study (5 5) did examine Fey RI1 protein expression after 24 hr of interferon-y treatment, but was performed with polymorphonuclear leukocytes. No changes in FcyRII protein were observed on these neutrophils. Sustained, marked increases
MODULATION
OF FcyRII mRNA AND PROTEIN
305
in FcyRII protein expression have been observed following treatment of U937 with granulocyte or granulocyte-macrophage colony-stimulating factors (CSF) (54). No experiments have as yet been performed with these growth factors to compare their effect with that of interferon-y on FcrRII mRNA levels. When we examined the effects of interferon-y on monocytes, we did not observe any increase in either level of FcyRII mRNA or protein expression. This monocyte nonresponsiveness is not likely due to a generalized resistance of the cells to interferon-y, as receptors for interferon-y have been isolated and quantitated on monocytes (37). Additionally, the expression of FcyRI protein on monocytes was increased by interferon-y (see Fig. 5). The mechanism underlying this difference between monocytes and U937 in their response to interferon-y treatment is not known, but it suggestsa fundamental difference between monocytes and U937 cells in the regulation of FcyRII. In addition to increasing FcrRII protein expression, treatment of U937 cells with interferon-y also increased FcyRI expression, but this modulation differed, in both its kinetics and its extent, from that of FcyRII protein. Maximal stimulation by interferon-y was observed at 16-24 hr for both receptors, but the expression of FcyRI remained increased while the expression of FcyRII decreasedtoward baseline levels. Additionally, the increase in FcyRI protein expression was almost four times that seen for FcrRII. In their recent report of the cloning of FcrRI, Allen and Seed state that FcyRI mRNA levels increase by 6 hr, but decreaseby 24 hr of incubation with interferon-y (12). This is similar to the kinetics we observed for FcyRII mRNA and implies that the differences in the modulation of protein expression following interferon-y treatment are due either to a difference in protein half-life or to different translational or post-translational regulation, and not due to differences in the respective mRNA levels. Further studies to determine the exact mechanisms by which the protein expression of these receptors is regulated are necessary,but these data suggest differences in the post-transcriptional control of these Fey receptor proteins. Since FcyRI and FcyRII bind related ligands, mediate similar functions, and appear to have similar changes in mRNA levels following interferon--y treatment, it is possible the transcription of both genesis coordinately regulated. It will be necessaryto isolate the genomic sequencesfor FcrRI and FcyRII and to analyze their S- and 3’-flanking sequencesanalyzed for the presence of any common potential regulatory sequences. Our studies help to explain the mechanism of the steroid hormone effects on Fey receptors. With U937 cells, the effect of dexamethasone was less marked than that of interferon-y. Dexamethasone alone did not alter FcrRII mRNA levels and only slightly inhibited protein expression. However, dexamethasone partially inhibited the interferon-y-induced increase of FcrRII transcripts and protein. This antagonistic effect of dexamethasone and interferon-y was also observed with FcyRI protein expression on U937 cells. In contrast, dexamethasone and interferon-y increased FcrRI expression on monocytes to a level almost twice as high as that seen with interferon-y alone. The enhancement of the interferon-y effect on monocyte FcrRI has been observed by others (36,56,57) and appears to be due at least in part to a dexamethasone-induced increase in monocyte receptors for interferon-y (58). The mechanism of the inhibitory effect of dexamethasone on FcyRI in U937 cells is not understood. Dexamethasone may not increase interferon-y receptor expression on U937 cells as it does on blood monocytes. Alternatively, the effect of dexamethasone on U937 may be due to an intrinsic difference between monocytes and U937 cells.
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COMBER ET AL.
Our Northern blot analyses revealed that in the case of FcyRII on U937 cells, dexamethasone inhibition of the effect of interferon-7 is at least in part due to changes in steady state levels of FcrRII mRNA. In summary, we have demonstrated that treatment with interferon-y increaseslevels of FcyRII-specific mRNA in U937 cells in a time-dependent manner. This modulation of Fc-yRII mRNA is not seen in monocytes treated with interferon-y. The cell surface expression of FcyRII is also increased following interferon-y treatment, but the increase is less than the increase in mRNA levels and occurs after the peak in FcyRII mRNA levels. This suggeststhat translational and/or post-translational regulation is involved in the modulation of FcrRII. Also, dexamethasone partially inhibits the interferon-r-induced increase in both FcrRII mRNA and protein. Additional studies should lead to increased understanding of the mechanisms by which these important cell surface receptors are modulated, and potentially aid in the design of therapeutic interventions for hematologic and autoimmune disorders. ACKNOWLEDGMENTS We thank Mrs. Ruth Rowan for her expert assistancein preparing this manuscript. We also thank Dr. Leonard Braitman and Mr. Delray Schultz for their assistancewith the statistical analysis.
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