- Email: [email protected]
TNFα and IL-4 Regulation of Hyaluronan Binding to Monocyte CD44 Involves Posttranslational Modification of CD44
Cellular Immunology 193, 209 –218 (1999) Article ID cimm.1999.1456, available online at http://www.idealibrary.com on
TNFa and IL-4 Regulation of Hyaluronan Binding to Monocyte CD44 Involves Posttranslational Modification of CD44 1 Marc C. Levesque 2 and Barton F. Haynes Department of Medicine, Division of Rheumatology, Allergy and Clinical Immunology; Department of Immunology; and Duke University Arthritis Center, Duke University Medical Center, Durham, North Carolina 27710 Received October 26, 1998; accepted January 4, 1999
INTRODUCTION Our previous studies have identified TNFa as a positive regulator and IL-4 as a negative regulator of human monocyte CD44 –HA binding. In order to determine the mechanisms of IL-4- and TNFa-mediated regulation of monocyte HA binding, we measured HA binding and CD44 expression on peripheral blood monocytes following monocyte treatment with TNFa or IL-4, as well as following monocyte treatment with inhibitors of protein synthesis, N- and O-linked glycosylation, and chondroitin sulfation. IL-4 decreased CD44 –HA binding on monocytes initially treated with TNFa. Similarly, pretreatment of monocytes with IL-4 prevented subsequent TNFa-mediated HA binding. Cycloheximide (protein synthesis inhibitor), tunicamycin (N-linked glycosylation inhibitor), and b-D-xyloside (chondroitin sulfation inhibitor) all inhibited IL4-mediated downregulation of TNFa-induced monocyte HA binding. Western blot analysis of CD44 from TNFa-treated monocytes revealed a 5–10 M r decrease in the standard isoform of CD44. In contrast, IL-4 treatment of monocytes inhibited CD44 –HA binding and reversed the 5- to 10-kDa decrease in monocyte CD44 M r. Finally, studies with F10.44.2, a CD44 mab that enhances CD44 –HA binding, indicated that IL-4 treatment of monocytes not only diminished constitutive HA binding, but also diminished CD44 mab-induced HA binding. Taken together, these data suggested that IL-4-mediated inhibition of TNFa-induced monocyte HA binding was dependent not only on protein synthesis, but also on N-linked glycosylation and chondroitin-sulfate modification of either CD44 or, alternatively, another monocyte protein(s) that may regulate the ability of CD44 to bind HA. © 1999 Academic Press
1 This work was supported by the Specialized Center of Research in Rheumatoid Arthritis Grant AR39162 and by NIH Grant AR01918-02. 2 To whom reprint requests should be addressed at Box 3258, Duke University Medical Center, Durham, NC 27710.
Several phases of inflammatory immune responses are dependent on interactions of the cell surface proteoglycan CD44 with the extracellular matrix glycosaminoglycan hyaluronan (HA) 3 (1– 8). CD44 –HA interactions on lymphocytes and monocyte-lineage cells influence Langerhan’s and dendritic cell migration to lymph nodes following antigen uptake (1) and mediate T lymphocyte extravasation into inflammatory sites (9). Because human peripheral blood (PB) monocytes do not constitutively bind HA (10, 11), we have suggested that HA binding to monocyte CD44 is a cytokine-regulated event (11, 12). Our previous studies have identified TNFa as a positive regulator and IL-4 and IL-13 as negative regulators of HA binding to monocyte CD44 (11, 12). However, the molecular mechanisms involved in cytokine-regulated HA binding to monocyte CD44 have remained unknown. CD44 has been shown to undergo several posttranslational modifications including glycosylation (13–16), palmitoylation (17), chondroitin and keratan sulfation (14, 18, 19), phosphorylation (20), and cytoskeletal binding (21–24). Of these modifications, decreased Nlinked glycosylation (16), increased N-linked glycosylation (13), decreased sialyation (16), decreased O-glycosylation (25), and decreased chondroitin and keratan sulfation (19, 26) have all been associated with increased HA binding to CD44 in various nonmonocyte systems. Of these mechanisms involved in HA binding to CD44, chondroitin sulfate modification may be different than other modifications, because HA binding to CD44 in the presence of chondroitin may require another cell surface protein(s) besides CD44 (26). Cytoskeletal binding (27, 28) and phosphorylation (28) have also been associated with increased HA binding to 3 Abbreviations used: CD44S, standard form of CD44; CD44V, variant forms of CD44; HA, hyaluronan; HA–FITC, hyaluronan conjugated to fluorescein isothiocyanate; MFI, mean fluorescence intensity; PBMC, peripheral blood mononuclear cells; RA, rheumatoid arthritis; TNFa, tumor necrosis factor a.
209
0008-8749/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
210
LEVESQUE AND HAYNES
CD44 on mouse T lymphoma (BW5147) and thymoma (AKR1.G10) cells, but recent data have questioned these findings (29, 30). The utilization of spliced CD44 isoforms for HA binding has been studied as well. Many investigators have found the unspliced 85-kDa standard CD44 form, CD44S, can bind HA (31–34). Galluzzo et al. demonstrated that cell surface expression of the CD44V isoforms V6 and V9 are required for HA binding to lymphocytes (35). Stamenkovic et al. found a role for CD44S but not for V9-containing CD44 variant isoforms in binding HA (32). However, others have demonstrated that cells transfected with a V9-containing CD44V could bind HA (33, 34), and Liao et al. showed V9-containing CD44 variant isoform-transfected Jurkat T cells bound HA following ligation with CD44 mab A1G3 (31). Thus, under certain circumstances, spliced CD44 variant isoforms can also bind HA. Finally, others have postulated that CD44 undergoes a conformational change that induces HA binding, perhaps related to clustering of cell surface CD44 (31, 36 – 38). These latter studies have been based on the observation that a subset of CD44 mabs can induce HA binding to cell surface CD44 and to CD44 fusion proteins (31, 36, 37). Several studies have also presented data that suggest that CD44 dimerization is necessary for HA binding. These latter studies have been based on the observation that CD44 molecules without cytoplasmic tails do not bind HA (31, 37), yet tailess CD44 dimers do bind CD44 (38) and cysteine 286 in the transmembrane domain is necessary for HA binding to CD44 (39, 40). Together, these studies have led Lesley and co-workers to propose that CD44 can exist in one of three possible activation states with regard to HA binding: constitutive active, activatable, and nonactivatable (26). Finally, a cellular cofactor, i.e., another cell surface protein, has been proposed to be required for HA binding to CD44, although attempts to identify such a cofactor have not been successful (26, 41, 42). Therefore, we undertook these studies to study the mechanisms of TNFa- and IL-4-mediated regulation of HA binding to monocyte CD44. Our studies suggested that cytokine-regulated HA binding to CD44 was not dependent on the level of total CD44 expression, but rather was dependent on posttranslational modifications of monocyte CD44. MATERIALS AND METHODS Antibodies and cytokines. The CD44 mabs A3D8 and F10.44.2 and the control mab (P3 3 63/Ag8) were used as described (43– 45). Anti-CD14 –PE, IgG–FITC, and IgG2a–PE were obtained from Dako (Glostrup, Denmark). The FITC-conjugated anti-CD44 mab, MEM-85, was purchased from Caltag (Burlingame, CA). IL-4 and TNFa were obtained from R&D Systems (Minneapolis, MN).
Flow cytometry and HA binding assay. Surface expression of CD44 on PBMC was analyzed by flow cytometry and direct immunofluorescence assays as described (46). HA binding to PB monocytes was assayed using saturating amounts of soluble HA conjugated with fluorescein (HA–FITC) in double-labeling protocols gating on CD14 1 cells (12). For experiments involving CD44 mab F10.44.2 mediated induction of HA binding, either a saturating amount of mouse ascites containing F10.44.2 or an equal quantity of mouse ascites containing the control mab (P3 3 63/Ag8) was added to cells 15 min prior to the addition of the HA– FITC and anti-CD14 –PE. The HA–FITC (gift of Anika Therapeutics, Woburn, MA) was derived from rooster comb and had an average molecular weight of 6.9 3 10 5Da. Unless otherwise stated, there were no subpopulations of monocytes with differential HA binding, i.e., analysis of HA binding to monocytes revealed uniform shifts in the entire monocyte population with cytokine and antibody treatments. Samples were analyzed on a FACStar Plus (Becton–Dickinson, Mountain View, CA). The binding level of HA–FITC to freshly isolated or cultured monocytes that could not be blocked by unlabeled HA or anti-CD44 mabs was considered nonspecific. Binding of HA to monocytes was confirmed to be specifically binding to CD44 since 100% of HA binding could be inhibited by the CD44 mab 5F12 (12). Monocyte separation. Monocytes were purified from PBMC using a combination of magnetic beads for removal of lymphocytes and adherence to plastic. Briefly, PBMC were treated with a mixture of magnetic beads coated with antibodies to CD2 and CD19 (Dynal, Lake Success, NY) according to the manufacturer’s instructions. Further purification of monocytes was obtained by plating the remaining cells and removing nonadherent cells after culture for 2 h at 37°C. This procedure resulted in preparations of monocytes that were 92 6 2.5% (mean 6 SEM) monocytes as determined by nonspecific esterase staining. Monocyte cultures. Monocytes (5 3 10 5) were cultured for 72 h in 48-well plates in RPMI supplemented with 10% v/v autologous serum at 37°C, 5% CO 2 in air for HA binding assays and CD44 mab phenotyping experiments (31). Autologous human serum was obtained simultaneously with peripheral blood and used in monocyte culture experiments. Cytokines and metabolic inhibitors were added to monocyte cultures following removal of nonadherent cells. Metabolic inhibitor studies. Monocytes were initially cultured for 72 h with TNFa (10 ng/ml). The medium was then removed, adherent cells were washed with RPMI, and fresh medium containing IL-4 (10 ng/ml) was added to the wells along with the protein synthesis inhibitor cycloheximide (10 mg/ml)
HYALURONAN BINDING TO MONOCYTE CD44
(Sigma, St. Louis, MO), the N-glycosylation inhibitor tunicamycin (1 mg/ml) (Sigma), the glucosidase I and II inhibitor castanospermine (100 mg/ml) (CalBiochem, La Jolla, CA), the mannosidase I inhibitor deoxymannojirimycin (100 mg/ml) (CalBiochem), the mannosidase II inhibitor swainsonine (10 mg/ml) (CalBiochem), the O-glycosylation inhibitor benzyl 2-acetamido-2-deoxy-a-D-galactopyranoside (2 mM) (Sigma), or the chondroitin sulfation inhibitor 4-methylumbelliferyl b-D-xyloside (0.5 mM) (Sigma) (47). Cells were harvested after an additional 24 h of culture for all of the inhibitors tested and also after 72 h for castanospermine, deoxymannojirimycin, swainsonine, benzyl 2-acetamido-2-deoxy-a-D-galactopyranoside, and 4-methylumbelliferyl b-D-xyloside (b-D-xyloside). Longer incubation times were not performed for cycloheximide and tunicamycin because these metabolic inhibitors induced monocyte toxicity after 24 h of treatment. HA binding and CD44 expression were measured as described earlier. Western blot analysis. Western blot analysis of PBMC lysates was performed as described (48). Briefly, PBMC were lysed in a buffer containing 10 mM Tris, pH 7.4, 150 mM NaCl, 1% NP-40, 20 mM iodoacetamide, and 1 mM PMSF. A soluble protein extract was prepared by centrifugation at 10,000g (20 min, 4°C). For each sample, protein from 2 3 10 5 cell equivalents was separated by electrophoresis on 6% polyacrylamide gels and electrophoretically transferred to nitrocellulose filters. CD44 on nitrocellulose filters was detected using CD44 mab A3D8 and indirect immunoperoxidase staining using horseradish peroxidase-conjugated goat anti-mouse IgG antibodies (Promega, Madison, WI). Protein bands were visualized by incubation with Rennaisance chemiluminescence reagent (DuPont NEN, Boston, MA) followed by exposure to film. RESULTS Effect of IL-4 on TNFa-induced monocyte HA binding and CD44 cell surface expression. Our previous studies indicated that the simultaneous addition of IL-4 to PB monocyte cultures treated with TNFa resulted in no HA binding to monocytes (12). To study mechanisms by which TNFa induced HA binding, and mechanisms whereby IL-4 blocked or prevented TNFainduced HA binding, we measured HA binding and CD44 expression on monocytes treated sequentially either with TNFa, no cytokine, or IL-4 for the first 72 h followed by treatment with either TNFa, no cytokine, or IL-4 for another 72 h (Fig. 1). Treatment of monocytes with TNFa alone for the first 72 h resulted in upregulation of HA binding compared to treatment with no cytokine or IL-4 (Figs. 1A, 1C, and 1E). Total cell surface CD44 increased over time on monocytes
211
treated with either TNFa, no cytokine, or IL-4 (Figs. 1B, 1D, and 1F). Subsequent treatment of monocytes initially treated with TNFa for 72 h with IL-4 or no cytokine resulted in loss of HA binding compared to continued monocyte treatment with TNFa (Fig. 1A). Interestingly, CD44 cell surface levels on monocytes intially treated with TNFa declined a similar amount when treated with either no cytokine, TNFa, or IL-4 during the second 72 h (Fig. 1B). Like the increase in HA binding induced by TNFa (12), the decrease in HA binding induced by IL-4 by monocytes initially treated with TNFa for 72 h was gradual; at 96 and 120 h, only intermediate levels of HA binding were present (data not shown). As previously reported (11), the in vitro culture of monocytes with no cytokine for 72 h resulted in lowlevel HA binding (Fig. 1C). In contrast, initial treatment of monocytes with IL-4 for 72 h resulted in no HA binding and prevented upregulation of HA binding when these IL-4-treated monocytes were treated for a subsequent 72 h with TNFa (Fig. 1E). However, following initial treatment of monocytes with either no cytokine or IL-4, TNFa treatment resulted in further upregulation of CD44 expression (Figs. 1D and 1F) to levels seen with the initial TNFa treatment of monocytes (72 h time point in Fig. 1A). The results shown in Fig. 1 established three important points about the mechanism of HA binding to moncyte CD44. First, these data confirmed that HA binding did not always correlate with CD44 cell surface expression levels (e.g., Figs. 1C vs 1D and 1E vs 1F). Second, IL-4 treatment of PB monocytes prevented subsequent TNFa-mediated HA binding (Fig. 1E) and IL-4-induced decreased monocyte CD44 HA binding on monocytes initially treated with TNFa (Fig. 1A). Third, the mechanisms of TNFa-induction and IL-4 inhibition of HA binding were processes that occurred over hours to days. In other systems, regulation of ligand binding to receptors by phosphorylation, palmitoylation, and cytoskeletal binding occured rapidly, in minutes to hours, and occured in the absence of new protein synthesis (49). In contrast, changes in glycosylation, glycosoaminoglycan modification, and isoform expression require new protein synthesis and “replacement” of current cell surface CD44 with a time course requiring hours to days (49), a time course more compatible with the lasting effects of TNFa induction and IL-4 inhibition of HA binding to monocyte CD44. Effect of the protein synthesis inhibitor cycloheximide on IL-4-mediated downregulation of TNFa-induced HA binding to monocytes. Mechanisms that regulate cytokine-mediated HA binding to CD44 that involve posttranslational modifications such as glycosylation and glycosoaminoglycan modification require new protein synthesis. Therefore, monocytes were treated for 72 h with TNFa and then treated with IL-4 and cyclohexi-
212
LEVESQUE AND HAYNES
FIG. 1. Effect of sequential TNFa and IL-4 treatment on monocyte HA binding and CD44 expression. Purified monocytes were cultured for 72 h with TNFa (10 ng/ml) (A, B), no cytokine (C, D), or IL-4 (10 ng/ml) (E, F) and the medium was then removed, the wells were washed with RPMI, and fresh medium was added containing no cytokine, TNFa, or IL-4. Monocyte DMFI values for HA binding to CD14 1 cells were determined as follows: DMFI 5 [(MFI for HA–FITC) 2 (MFI for IgG–FITC)]. Monocyte DMFI values for CD44 expression on CD14 1 cells were determined as follows: DMFI 5 [(MFI for anti-CD44 –FITC binding) 2 (MFI for IgG1–FITC binding)] Data represent the mean 6 SEM of three experiments.
mide (10 mg/ml) for a further 24 h. (Cycloheximide treatment for longer than 24 h was toxic to cultured monocytes.) HA binding and CD44 levels were then assessed and compared to TNFa-treated monocytes harvested at the time IL-4 and cycloheximide were added or to TNFa-treated monocytes treated with IL-4 alone. As shown in Fig. 2, cycloheximide prevented IL-4-mediated downregulation of HA binding to CD44 (cycloheximide vs no inhibitor, P 5 0.02) despite cycloheximide-induced decreases in CD44 cell surface ex-
pression. Decreases in monocyte cell surface expression of CD44 that occurred in this setting with cycloheximide treatment likely resulted from turnover of cell surface CD44. Studies from other laboratories have shown that the half-life of cell surface CD44 ranges from 24 to 48 h (50). Effect of N- and O-glycosylation inhibitors on IL-4mediated downregulation of TNFa-induced HA binding to monocytes. Studies performed by Katoh et al.
HYALURONAN BINDING TO MONOCYTE CD44
213
FIG. 2. Effect of cycloheximide treatment on IL-4-mediated downregulation of TNFa-induced monocyte HA binding. Purified monocytes were cultured for 72 h with TNFa (10 ng/ml) and then treated for 24 h with fresh medium containing IL-4 (10 ng/ml) and either no inhibitor or cycloheximide (10 mg/ml). Cycloheximide treatment for longer than 24 h decreased monocyte cell viability. Monocyte DMFI values for CD44 expression and HA binding to CD14 1 cells were determined as described in Fig. 1. Data represent the mean 6 SEM of three experiments. For HA binding, P 5 0.02 for cycloheximide treatment vs no inhibitor.
suggested that changes in N-linked glycosylation modulated HA binding to CHO cells (16). Likewise, Camp et al. have shown that in mice, exudative compared to resident peritoneal macrophages can be characterized by expression of CD44 molecules with less N-linked glycosylation (14). While this latter study did not correlate changes in CD44 N-linked glycosylation with changes in HA binding, given the data presented in Figs. 1 and 2, combined with the results of Katoh et al. we postulated that alterations in N-linked glycosylation might be involved in the TNFa- and IL-4-induced changes in HA binding to monocyte CD44. In order to test this possibilty, studies were performed with the N-linked glycosylation inhibitor, tunicamycin, the glucosidase I and II inhibitor, castanospermine, the mannosidase I inhibitor, deomannojirimycin, and the mannosidase II inhibitor, swainsonine (47). Monocytes were treated with TNFa for 72 h as in Fig. 2, then treated with IL-4 plus the inhibitors shown in Fig. 3 for an additional 24 or 72 h. (Tunicamycin treatment for longer than 24 h was toxic to the monocytes.) HA binding and CD44 levels were assessed and compared to TNFa-treated monocytes harvested at the time IL-4 plus the inhibitors were added, or compared with TNFa-treated monocytes treated with IL-4 alone. We found that tunicamycin prevented IL-4-mediated downregulation of HA binding to CD44 (tunicamycin vs no inhibitor, P 5 0.013) despite significant decreases in CD44 cell surface expression (tunicamycin vs no inhibitor, P 5 0.002) (Fig. 3). Unlike tunicamycin, castanospermine, deoxymannojirimycin, or swainsonine treatment of TNFa-treated monocytes for a further
24 h did not prevent IL-4-induced downregulation of HA binding to monocytes (Fig. 3). TNFa-treated monocytes treated with IL-4 and castanospermine, deoxymannojirimycin, or swainsonine for 72 h yielded results similar to those for 24-h treatment (data not shown). One study has suggested that decreased O-glycosylation was associated with increased HA binding to CD44 (25). Therefore, similar experiments were also performed with the O-glycosylation inhibitor, benzyl 2-acetamido-2-deoxy-a-D-galactopyranoside. As shown in Fig. 3, a-D-galactopyranoside did not prevent IL-4induced downregulation of HA binding to monocyte CD44. Effect of the chondroitin sulfation inhibitor, b-D-xyloside, on IL-4- mediated downregulation of TNFa-induced HA binding to monocytes. Several studies have shown that the 85-kDa standard isoform of CD44 (CD44S) does not contain chondroitin sulfate, although this isoform of CD44 can undergo modification to produce an 180- to 200-kDa molecule (14, 26). Interestingly, a study by Lesley et al. revealed that b-D-xyloside treatment or chondroitinase digestion of intact CD44 expressing pre-B cells (Raw 253) and fibroblasts (L cells) augmented HA binding, without an accompanying change in CD44 M r (26). Furthermore, CD44 has been shown to bind to at least two chondroitin sulfatemodified proteins, the chondroitin sulfate form of invariant chain (51) and a soluble chondroitin-modified proteoglycan, serglycin (52). In order to test the possibility that chondroitin sul-
214
LEVESQUE AND HAYNES
FIG. 3. Effect of N- and O-glycosylation inhibitors on IL-4-mediated downregulation of TNFa-induced monocyte HA binding. Purified monocytes were cultured for 72 h with TNFa (10 ng/ml) and then treated for 24 h with fresh medium containing IL-4 and no inhibitor, tunicamycin (1 mg/ml), castanospermine (100 mg/ml), deoxymannojirimycin (100 mg/ml), swainsonine (10 mg/ml), or benzyl 2-acetamido-2deoxy-a-D-galactopyranoside (2 mM). Tunicamycin treatment for longer than 24 h decreased monocyte cell viability. Monocyte DMFI values for CD44 expression and HA binding to CD14 1 cells were determined as described in Fig. 1. Treatment of monocytes for 72 h with TNFa and then with cytokines and inhibitors for 72 h gave similar results. Data represent the mean 6 SEM of three experiments. For HA binding, P 5 0.013 for tunicamycin vs no inhibitor, and for CD44 cell surface expression, P 5 0.002 for tunicamycin vs no inhibitor.
fation may regulate HA binding to monocyte CD44, the chondroitin sulfation inhibitor, b-D-xyloside, was added to monocytes treated with TNFa for 72 h, followed by treatment for either 24 or 72 h with no cytokine, with TNFa, or with IL-4. We found that for TNFa-treated monocytes in all settings (no cytokine, TNFa addition, and IL-4 addition), b-D-xyloside treatment for 24 h induced upregulation of HA binding to monocyte CD44 (Fig. 4). TNFa-treated monocytes treated with either no cytokine, IL-4, or TNFa and b-D-xyloside for 72 h yielded results similar to those for 24-h treatment (data not shown). There was a trend toward increased HA binding under all cytokine treatment conditions with b-D-xyloside treatment vs no inhibitor and significant increases in HA binding following treatment with IL-4 and b-D-xyloside for 24 h. (P 5 0.003 for b-D-xyloside treatment vs no inhibitor). Thus, inhibition of chondroitin sulfation by b-D-xyloside inhibited the ability of IL-4 to downregulate TNFa-induced ability of monocyte CD44 to bind HA. Western blot analysis of cytokine-treated monocytes. Our previous studies (11) and work presesented by Katoh et al. (16) and Camp et al. (14) revealed an approximately 5- to 10-kDa decrease in the M r of the standard isoform of CD44 in association with monocyte HA binding, or when comparing exudative to resident macrophages. This change in M r was consistent with
the finding that there were differences in N-linked glycosylation between the two populations of cells compared in each of these analyses. Therefore, Western blot analysis was performed on monocytes treated with TNFa for 72 h followed by treatment with either TNFa, IL-4, or no cytokine for an additional 72 h. As shown in Fig. 5, there was a consistent decrease in relative molecular mass of CD44 by approximately 5 to 10 kDa in monocyte preparations that were treated with TNFa and bound HA (lanes 3 and 5) vs those that were treated with no cytokine or IL-4 and did not bind high levels of HA (lanes 2 and 6). Also evident in lanes 3, 4, and 5 is another isoform of CD44 with a M r of approximately 117 kDa that was present in increased amounts in monocyte preparations that bound HA. Whether the effects of the chondroitin sulfation inhibitor b-D-xyloside on HA binding to IL-4-treated monocytes were mediated by changes in chondroitin sulfation of CD44 remains unresolved, since no 180- to 200-kDa bands (chondroitinated forms) were evident on these Western blot analyses. Effect of the CD44 mab F10.44.2 on HA binding to monocytes treated with TNFa or IL-4. To determine whether a conformation-based mechanism may in part explain IL-4 modulation of HA binding, we cultured monocytes with either no cytokine, IL-4, or TNFa for 72 h and then measured HA binding to these mono-
215
HYALURONAN BINDING TO MONOCYTE CD44
FIG. 4. Effect of the chondroitin-sulfate inhibitor, 4-methylumbelliferyl b-D-xyloside, on IL-4-mediated downregulation of TNFa-induced monocyte HA binding. Monocytes were cultured for 72 h with TNFa (10 ng/ml) and then treated for 24 or 72 h with fresh medium containing no cytokine, TNFa (10 ng/ml), or IL-4 (10 ng/ml) and either no inhibitor or 4-methylumbelliferyl b-D-xyloside (0.5 mM). Monocyte DMFI values for CD44 expression and HA binding to CD14 1 cells were determined as described in Fig. 1. Data represent the mean 6 SEM of three experiments. For HA binding, P 5 0.003 for IL-4 and b-D-xyloside treatment vs IL-4 and no inhibitor treatment.
cytes in the presence of either a control mab (constitutive HA binding) or saturating amounts of mab F10.44.2, a CD44 mab that has previously been shown to induce CD44-HA binding (inducible HA binding) (44, 45). We found that monocytes treated with TNFa for 72 h constitutively bound high levels of HA, and this population of monocytes could not be further induced to bind more HA by mab F10.44.2 treatment (Fig. 6A). Monocytes treated for 72 h with no cytokine constitutively bound lower levels of HA, and monocytes treated with IL-4 for 72 h did not bind HA. Mab F10.44.2 induced small increases in HA binding in no cytokine and IL-4-treated monocytes (Fig. 6A). The data in Fig. 6A demonstrated that IL-4 pretreatment of monocytes not only diminished constitutive HA binding but IL-4 also diminished inducible HA binding. Therefore, TNFa effectively “desensitized” inducible monocyte–HA binding.
with IL-4 did not constitutively bind HA, and monocytes initially treated for 72 h with TNFa and then treated with no cytokine or TNFa for an additional 72 h bound low levels of HA (Fig. 6B). The anti-CD44 mab F10.44.2 induced high-level HA binding in these latter two treatment groups (Fig. 6B). Mab F10.44.2 induced low level HA binding in monocytes initially treated with TNFa for 72 h followed by treatment with IL-4 for 72 h. The data in Fig. 6B demonstrated that IL-4-mediated inhibition of TNFa-induced HA binding to monocyte CD44 could not be reversed by CD44 mabs such as F10.44.2 that induce HA binding. Together, the data in Figs. 6A and 6B suggest that a conformation-based mechanism cannot explain IL-4-mediated inhibition of TNFa-induced CD44 –HA binding.
Effect of the CD44 mab F10.44.2 on HA binding to monocytes treated initially with TNFa, followed by treatment with TNFa, IL-4, or no cytokine. To further understand IL-4-mediated downregulation of TNFainduced monocyte HA binding, we measured HA binding in the presence of either a control mab or mab F10.44.2 on monocytes treated initially for 72 h with TNFa and then treated with no cytokine, IL-4, or TNFa for 72 h. We found that monocytes intially treated for 72 h with TNFa and then treated for 72 h
We performed these studies to study the mechanisms governing TNFa- and IL-4-mediated regulation of HA binding to monocyte CD44. As expected from our previous studies (12), initial treatment of monocytes for 72 h with TNFa resulted in high-level HA binding and increased CD44 expression. In this current study, we have shown that subsequent treatment of monocytes for an additional 72 h with TNFa maintained HA binding while subsequent treatment for 72 h with no cytokine or with IL-4 resulted in downregulation of monocyte HA binding.
DISCUSSION
216
LEVESQUE AND HAYNES
FIG. 5. Western blot analysis of CD44 on monocytes treated with TNFa and IL-4. Cell lysates were prepared as described under Materials and Methods from 2 3 10 5 cell equivalents/lane of freshly isolated monocytes (lane 1) and of monocytes cultured with either no cytokine or TNFa (10 ng/ml) for 72 h and either harvested immediately (lanes 2 and 3, respectively) or treated for another 72 h with fresh medium containing no cytokine (lane 4), TNFa (10 ng/ml) (lane 5), or IL-4 (10 ng/ml) (lane 6). Following electrophoresis and transfer to nitrocellulose, Western blot analysis was performed with the CD44 mab A3D8 and isotype-matched control antibody, P3. At the exposure time shown, no bands were visible on the control antibody blot and only results for the CD44 mab blot are shown. The CD44 band in lane 1 was not visible at the exposure time shown for lanes 2 to 6. The CD44 band in lane 1 is from an exposure time that was 153 longer than that shown in lanes 2 to 6. The Western blot shown is representative of results from three experiments. Monocyte DMFI values for CD44 expression and HA binding to CD14 1 cells were determined as described in Fig. 1.
Further, we found that the protein synthesis inhibitor cycloheximide, the N-glycosylation inhibitor, tunicamycin, and the chondroitin sulfation inhibitor, b-D-xyloside, inhibited IL-4-mediated downregulation of TNFa-induced monocyte HA binding. As has been reported in other systems, our studies suggest that HA binding to monocyte CD44 is regulated by a variety of mechanisms (13, 16, 19, 25–28, 31, 35– 40). Cytokine-regulated HA binding to monocyte CD44 may be regulated by N-glycosylation of CD44 and possibly by chondroitin-sulfate modification of CD44 or another cell surface molecule. Our data support the idea that the functional HA binding status of any given cell may be determined by the sum total of several different events including the amount of CD44 present, the CD44 configuration on the cell surface, posttranslational modifications of CD44, and perhaps CD44 association with other cellular proteins. It is clear from Fig. 5 that in monocytes the standard CD44S 85-kDa form is approximately 5–10 kDa smaller when HA is able to bind to monocyte CD44. Taken together with our data that tunicamycin increased HA binding to IL-4-treated monocytes, these data suggest that N-glycosylation of CD44 is one mechanism of maintaining the “inactive” or “nonbinding” form of CD44. Monocyte activation by TNFa would be
predicted to decrease N-glycosylation of CD44, and IL-4 would be predicted to increase this process, postulates that are compatible with data shown in Figs. 3 and 5. Work presented by others has shown that the 85kDa standard isoform of CD44 does not contain chondroitin sulfate, although this isoform of CD44 can undergo chondroitination to produce a 180- to 200kDa molecule (14, 26). Interestingly, a study by Lesley et al. revealed that b-D-xyloside treatment or chondroitinase digestion of intact CD44 on murine pre-B cells and fibroblasts augmented HA binding, without an accompanying change in CD44 M r (26). These and our studies suggest that either a chondroitin-sulfate-modified CD44 or another chondroitinsulfated cellular protein may regulate or participate in HA binding to CD44. Extracellular matrix HA exists in a protein complex with aggrecan and link protein; aggrecan is heavily modified with chondroitin sulfate (53, 54). A similar structure, i.e., the hyaluronan coat, exists on the surface of cells in which cell surface CD44 is complexed with HA and aggrecan or an aggrecanlike protein (55, 56). In this model, IL-4 would be expected to either promote production of the chondroitin sulfate modified protein and/or stimulate production of HA, thereby inducing formation of a hy-
217
HYALURONAN BINDING TO MONOCYTE CD44
FIG. 6. Effect of CD44 mab F10.44.2 on HA binding to monocytes treated with IL-4 or TNFa. (A) Monocytes were cultured for 72 h with no cytokine, IL-4 (10 ng/ml), or TNFa (10 ng/ml) and then harvested. (B) Monocytes were cultured for 72 h with TNFa (10 ng/ml) and the medium was then removed, the wells were washed with RPMI, and fresh medium was added containing no cytokine, IL-4 (10 ng/ml), or TNFa (10 ng/ml) for a further 72 h and then monocytes were harvested. Following harvesting, monocyte DMFI values were determined as described in Fig. 1 except monocytes were initially treated with a saturating amount of mouse ascites containing either CD44 mab F10.44.2 or control mab (P3 3 63/Ag8) prior to staining with HA–FITC and anti-CD14 –PE. Data represent the mean 6 SEM of three experiments. For F10.44.2 induced HA binding, P 5 0.068 for monocytes treated with no cytokine vs TNFa-treated monocytes treated for a further 72 h with no cytokine and P 5 0.048 for monocytes treated with IL-4 vs TNFa-treated monocytes treated for a further 72 h with IL-4.
aluronan coat. Indeed, studies have shown that TNFa diminishes proteoglycan production by chondrocytes (57). Furthermore, IL-4 promotes proteoglycan synthesis by fibroblasts (58) and reverses TNFamediated inhibition of cartilage proteoglycan synthesis (59). The presence of cell surface HA would be expected to block binding of exogenous FITC-labeled HA giving the impression that IL-4 treated monocytes do not bind HA. An alternative hypothesis is that IL-4 induces production of a chondroitin-sulfate modified protein that binds to CD44 and blocks HA binding to CD44. Lesley and co-workers have suggested that CD44 can exist in one of three possible activation states with regard to HA binding: constitutive active, activatible, and nonactivatible (26). TNFa has effects on both constitutive and inducible HA binding, and these effects could be separated in our studies. Thus our studies suggest a diversity of mechanisms governing HA binding to monocyte CD44. Given the proinflammatory consequences of CD44 –HA binding, these studies suggest several new sites for potential inhibition of CD44 proinflammatory activity. Studies of IL-4 in vivo in the treatment of RA may provide an opportunity to determine the effect of IL-4 in vivo on human monocyte ability to bind HA and to determine the ability of IL-4 to modify clinical inflammation in human synovitis.
ACKNOWLEDGMENTS The authors thank Dr. Larry Liao for insightful discussions, Mary Misukonis for performing monocyte nonspecific esterase stains, and Erlina Siragusa of the CFAR Cell Sorter Facility.
REFERENCES 1.
2. 3. 4. 5.
6.
7. 8.
9.
Weiss, J. M., Sleeman, J., Renkl, A. C., Dittmar, H., Termeer, C. C., Taxis, S., Howells, N., Hofmann, M., Kohler, G., Schopf, E., Ponta, H., Herrlich, P., and Simon, J. C., J. Cell Biol. 137, 1137, 1997. Clark, R. A., Alon, R., and Springer, T. A., J. Cell Biol. 134, 1075, 1996. Galandrini, R., Galluzzo, E., Albi, N., Grossi, C. E., and Velardi, A., J. Immunol. 153, 21, 1994. DeGrendele, H. C., Estess, P., Picker, L. J., and Siegelman, M. H., J. Exp. Med. 183, 1119, 1996. Trochon, V., Mabilat, C., Bertrand, P., Legrand, Y., SmadjaJoffe, F., Soria, C., Delpech, B., and Lu, H., Int. J. Cancer 66, 664, 1996. Hodge-Dufour, J., Noble, P. W., Horton, M. R., Bao, C., Wysoka, M., Burdick, M. D., Strieter, R. M., Trinchieri, G., and Pure, E., J. Immunol. 159, 2492, 1997. Noble, P. W., Lake, F. R., Henson, P. M., and Riches, D. W., J. Clin. Invest. 91, 2368, 1993. McKee, C. M., Penno, M. B., Cowman, M., Burdick, M. D., Strieter, R. M., Bao, C., and Noble, P. W., J. Clin. Invest. 98, 2403, 1996. Degrendele, H. C., Estess, P., and Siegelman, M. H., Science 278, 672, 1997.
218 10. 11. 12. 13. 14. 15.
16. 17. 18. 19. 20. 21. 22. 23. 24. 25.
26. 27. 28. 29. 30.
31.
32. 33.
LEVESQUE AND HAYNES Hyman, R., Lesley, J., and Schulte, R., Immunogenetics 33, 392, 1991. Levesque, M. C., and Haynes, B. F., J. Immunol. 156, 1557, 1996. Levesque, M. C., and Haynes, B. F., J. Immunol. 159, 6184, 1997. Bartolazzi, A., Nocks, A., Aruffo, A., Spring, F., and Stamenkovic, I., J. Cell Biol. 132, 1199, 1996. Camp, R. L., Kraus, T. A., and Pure, E., J. Cell Biol. 115, 1283, 1991. Campanero, M. R., Pulido, R., Alonso, J. L., Pivel, J. P., Pimentel-Muinos, F. X., Fresno, M., and Sanchez-Madrid, F., Eur. J. Immunol. 21, 3045, 1991. Katoh, S., Zheng, Z., Oritani, K., Shimozato, T., and Kincade, P. W., J. Exp. Med. 182, 419, 1995. Guo, Y. J., Lin, S. C., Wang, J. H., Bigby, M., and Sy, M. S., Int. Immunol. 6, 213, 1994. Jackson, D. G., Bell, J. I., Dickinson, R., Timans, J., Shields, J., and Whittle, N., J. Cell Biol. 128, 673, 1995. Takahashi, K., Stamenkovic, I., Cutler, M., Dasgupta, A., and Tanabe, K. K., J. Biol. Chem. 271, 9490, 1996. Neame, S. J., and Isacke, C. M., EMBO J. 11, 4733, 1992. Isacke, C. M., J. Cell Sci. 107, 2353, 1994. Kalomiris, E. L., and Bourguignon, L. Y., J. Cell Biol. 106, 319, 1988. Kalomiris, E. L., and Bourguignon, L. Y., J. Biol. Chem. 264, 8113, 1989. Tsukita, S., Oishi, K., Sato, N., Sagara, J., Kawai, A., and Tsukita, S., J. Cell Biol. 126, 391, 1994. Bennett, K. L., Modrell, B., Greenfield, B., Bartolazzi, A., Stamenkovic, I., Peach, R., Jackson, D. G., Spring, F., and Aruffo, A., J. Cell Biol. 131, 1623, 1995. Lesley, J., English, N., Perschl, A., Gregoroff, J., and Hyman, R., J. Exp. Med. 182, 431, 1995. Bourguignon, L. Y., Lokeshwar, V. B., Chen, X., and Kerrick, W. G., J. Immunol. 151, 6634, 1993. Pure, E., Camp, R. L., Peritt, D., Panettieri, R. A., Jr., Lazaar, A. L., and Nayak, S., J. Exp. Med. 181, 55, 1995. Uff, C. R., Neame, S. J., and Isacke, C. M., Eur. J. Immunol. 25, 1883, 1995. Murakami, S., Shimabukuro, Y., Miki, Y., Saho, T., Hino, E., Kasai, D., Nozaki, T., Kusumoto, Y., and Okada, H., J. Immunol. 152, 467, 1994. Liao, H. X., Levesque, M. C., Patton, K., Bergamo, B., Jones, D., Moody, M. A., Telen, M. J., and Haynes, B. F., J. Immunol. 151, 6490, 1993. Stamenkovic, I., Aruffo, A., Amiot, M., and Seed, B., EMBO J. 10, 343, 1991. He, Q., Lesley, J., Hyman, R., Ishihara, K., and Kincade, P. W., J. Cell Biol. 119, 1711, 1992.
34. 35.
36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59.
Dougherty, G. J., Cooper, D. L., Memory, J. F., and Chiu, R. K., J. Biol. Chem. 269, 9074, 1994. Galluzzo, E., Albi, N., Fiorucci, S., Merigiola, C., Ruggeri, L., Tosti, A., Grossi, C. E., and Velardi, A., Eur. J. Immunol. 25, 2932, 1995. Lesley, J., Kincade, P. W., and Hyman, R., Eur. J. Immunol. 23, 1902, 1993. Lesley, J., He, Q., Miyake, K., Hamann, A., Hyman, R., and Kincade, P. W., J. Exp. Med. 175, 257, 1992. Perschl, A., Lesley, J., English, N., Trowbridge, I., and Hyman, R., Eur. J. Immunol. 25, 495, 1995. Liu, D., and Sy, M. S., J. Exp. Med. 183, 1987, 1996. Liu, D. C., and Sy, M. S., J. Immunol. 159, 2702, 1997. Guo, Y., Wu, Y., Shinde, S., Sy, M. S., Aruffo, A., and Liu, Y., J. Exp. Med. 184, 955, 1996. Conrad, P., Rothman, B. L., Kelley, K. A., and Blue, M. L., J. Immunol. 149, 1833, 1992. Telen, M. J., Eisenbarth, G. S., and Haynes, B. F., J. Clin. Invest. 71, 1878, 1983. Liao, H. X., Telen, M. J., and Haynes, B. F., in “Leukocyte Typing V,” Oxford Univ. Press, Oxford, 1995. Dalchau, R., Kirkley, J., and Fabre, J. W., Eur. J. Immunol. 10, 745, 1980. Haynes, B. F., Metzgar, R. S., Minna, J. D., and Bunn, P. A., N. Engl. J. Med. 304, 1319, 1981. Elbein, A. D., FASEB J. 5, 3055, 1991. Burnette, W. N., Anal. Biochem. 112, 195, 1981. Krishna, R. G., and Wold, F., “Advances in Enzymology & Related Areas of Molecular Biology,” 67, 265, 1993. Goebeler, M., Kaufmann, D., Brocker, E. B., and Klein, C. E., J. Cell Sci. 109, 1957, 1996. Naujokas, M. F., Morin, M., Anderson, M. S., Peterson, M., and Miller, J., Cell 74, 257, 1993. Toyama-Sorimachi, N., and Miyasaka, M., J. Dermatol. 21, 795, 1994. Hardingham, T. E., and Fosang, A. J., FASEB J. 6, 861, 1992. Morgelin, M., Paulsson, M., and Engel, J., Biochem. Soc. Trans. 18, 204, 1990. Knudson, C. B., J. Cell Biol. 120, 825, 1993. Knudson, W., Bartnik, E., and Knudson, C. B., Proc. Natl. Acad. Sci. USA 90, 4003, 1993. Hardingham, T. E., Rayan, V., and Lewthwaite, J. C., Rev. Rheum. 61, 93S, 1994. Wegrowski, Y., Paltot, V., Gillery, P., Kalis, B., Randoux, A., and Maquart, F. X., Biochem. J. 307, 673, 1995. van Roon, J. A., van Roy, J. L., Gmelig-Meyling, F. H., Lafeber, F. P., and Bijlsma, J. W., Arthritis Rheum. 39, 829, 1996.
TNFa and IL-4 Regulation of Hyaluronan Binding to Monocyte CD44 Involves Posttranslational Modification of CD44 1 Marc C. Levesque 2 and Barton F. Haynes Department of Medicine, Division of Rheumatology, Allergy and Clinical Immunology; Department of Immunology; and Duke University Arthritis Center, Duke University Medical Center, Durham, North Carolina 27710 Received October 26, 1998; accepted January 4, 1999
INTRODUCTION Our previous studies have identified TNFa as a positive regulator and IL-4 as a negative regulator of human monocyte CD44 –HA binding. In order to determine the mechanisms of IL-4- and TNFa-mediated regulation of monocyte HA binding, we measured HA binding and CD44 expression on peripheral blood monocytes following monocyte treatment with TNFa or IL-4, as well as following monocyte treatment with inhibitors of protein synthesis, N- and O-linked glycosylation, and chondroitin sulfation. IL-4 decreased CD44 –HA binding on monocytes initially treated with TNFa. Similarly, pretreatment of monocytes with IL-4 prevented subsequent TNFa-mediated HA binding. Cycloheximide (protein synthesis inhibitor), tunicamycin (N-linked glycosylation inhibitor), and b-D-xyloside (chondroitin sulfation inhibitor) all inhibited IL4-mediated downregulation of TNFa-induced monocyte HA binding. Western blot analysis of CD44 from TNFa-treated monocytes revealed a 5–10 M r decrease in the standard isoform of CD44. In contrast, IL-4 treatment of monocytes inhibited CD44 –HA binding and reversed the 5- to 10-kDa decrease in monocyte CD44 M r. Finally, studies with F10.44.2, a CD44 mab that enhances CD44 –HA binding, indicated that IL-4 treatment of monocytes not only diminished constitutive HA binding, but also diminished CD44 mab-induced HA binding. Taken together, these data suggested that IL-4-mediated inhibition of TNFa-induced monocyte HA binding was dependent not only on protein synthesis, but also on N-linked glycosylation and chondroitin-sulfate modification of either CD44 or, alternatively, another monocyte protein(s) that may regulate the ability of CD44 to bind HA. © 1999 Academic Press
1 This work was supported by the Specialized Center of Research in Rheumatoid Arthritis Grant AR39162 and by NIH Grant AR01918-02. 2 To whom reprint requests should be addressed at Box 3258, Duke University Medical Center, Durham, NC 27710.
Several phases of inflammatory immune responses are dependent on interactions of the cell surface proteoglycan CD44 with the extracellular matrix glycosaminoglycan hyaluronan (HA) 3 (1– 8). CD44 –HA interactions on lymphocytes and monocyte-lineage cells influence Langerhan’s and dendritic cell migration to lymph nodes following antigen uptake (1) and mediate T lymphocyte extravasation into inflammatory sites (9). Because human peripheral blood (PB) monocytes do not constitutively bind HA (10, 11), we have suggested that HA binding to monocyte CD44 is a cytokine-regulated event (11, 12). Our previous studies have identified TNFa as a positive regulator and IL-4 and IL-13 as negative regulators of HA binding to monocyte CD44 (11, 12). However, the molecular mechanisms involved in cytokine-regulated HA binding to monocyte CD44 have remained unknown. CD44 has been shown to undergo several posttranslational modifications including glycosylation (13–16), palmitoylation (17), chondroitin and keratan sulfation (14, 18, 19), phosphorylation (20), and cytoskeletal binding (21–24). Of these modifications, decreased Nlinked glycosylation (16), increased N-linked glycosylation (13), decreased sialyation (16), decreased O-glycosylation (25), and decreased chondroitin and keratan sulfation (19, 26) have all been associated with increased HA binding to CD44 in various nonmonocyte systems. Of these mechanisms involved in HA binding to CD44, chondroitin sulfate modification may be different than other modifications, because HA binding to CD44 in the presence of chondroitin may require another cell surface protein(s) besides CD44 (26). Cytoskeletal binding (27, 28) and phosphorylation (28) have also been associated with increased HA binding to 3 Abbreviations used: CD44S, standard form of CD44; CD44V, variant forms of CD44; HA, hyaluronan; HA–FITC, hyaluronan conjugated to fluorescein isothiocyanate; MFI, mean fluorescence intensity; PBMC, peripheral blood mononuclear cells; RA, rheumatoid arthritis; TNFa, tumor necrosis factor a.
209
0008-8749/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
210
LEVESQUE AND HAYNES
CD44 on mouse T lymphoma (BW5147) and thymoma (AKR1.G10) cells, but recent data have questioned these findings (29, 30). The utilization of spliced CD44 isoforms for HA binding has been studied as well. Many investigators have found the unspliced 85-kDa standard CD44 form, CD44S, can bind HA (31–34). Galluzzo et al. demonstrated that cell surface expression of the CD44V isoforms V6 and V9 are required for HA binding to lymphocytes (35). Stamenkovic et al. found a role for CD44S but not for V9-containing CD44 variant isoforms in binding HA (32). However, others have demonstrated that cells transfected with a V9-containing CD44V could bind HA (33, 34), and Liao et al. showed V9-containing CD44 variant isoform-transfected Jurkat T cells bound HA following ligation with CD44 mab A1G3 (31). Thus, under certain circumstances, spliced CD44 variant isoforms can also bind HA. Finally, others have postulated that CD44 undergoes a conformational change that induces HA binding, perhaps related to clustering of cell surface CD44 (31, 36 – 38). These latter studies have been based on the observation that a subset of CD44 mabs can induce HA binding to cell surface CD44 and to CD44 fusion proteins (31, 36, 37). Several studies have also presented data that suggest that CD44 dimerization is necessary for HA binding. These latter studies have been based on the observation that CD44 molecules without cytoplasmic tails do not bind HA (31, 37), yet tailess CD44 dimers do bind CD44 (38) and cysteine 286 in the transmembrane domain is necessary for HA binding to CD44 (39, 40). Together, these studies have led Lesley and co-workers to propose that CD44 can exist in one of three possible activation states with regard to HA binding: constitutive active, activatable, and nonactivatable (26). Finally, a cellular cofactor, i.e., another cell surface protein, has been proposed to be required for HA binding to CD44, although attempts to identify such a cofactor have not been successful (26, 41, 42). Therefore, we undertook these studies to study the mechanisms of TNFa- and IL-4-mediated regulation of HA binding to monocyte CD44. Our studies suggested that cytokine-regulated HA binding to CD44 was not dependent on the level of total CD44 expression, but rather was dependent on posttranslational modifications of monocyte CD44. MATERIALS AND METHODS Antibodies and cytokines. The CD44 mabs A3D8 and F10.44.2 and the control mab (P3 3 63/Ag8) were used as described (43– 45). Anti-CD14 –PE, IgG–FITC, and IgG2a–PE were obtained from Dako (Glostrup, Denmark). The FITC-conjugated anti-CD44 mab, MEM-85, was purchased from Caltag (Burlingame, CA). IL-4 and TNFa were obtained from R&D Systems (Minneapolis, MN).
Flow cytometry and HA binding assay. Surface expression of CD44 on PBMC was analyzed by flow cytometry and direct immunofluorescence assays as described (46). HA binding to PB monocytes was assayed using saturating amounts of soluble HA conjugated with fluorescein (HA–FITC) in double-labeling protocols gating on CD14 1 cells (12). For experiments involving CD44 mab F10.44.2 mediated induction of HA binding, either a saturating amount of mouse ascites containing F10.44.2 or an equal quantity of mouse ascites containing the control mab (P3 3 63/Ag8) was added to cells 15 min prior to the addition of the HA– FITC and anti-CD14 –PE. The HA–FITC (gift of Anika Therapeutics, Woburn, MA) was derived from rooster comb and had an average molecular weight of 6.9 3 10 5Da. Unless otherwise stated, there were no subpopulations of monocytes with differential HA binding, i.e., analysis of HA binding to monocytes revealed uniform shifts in the entire monocyte population with cytokine and antibody treatments. Samples were analyzed on a FACStar Plus (Becton–Dickinson, Mountain View, CA). The binding level of HA–FITC to freshly isolated or cultured monocytes that could not be blocked by unlabeled HA or anti-CD44 mabs was considered nonspecific. Binding of HA to monocytes was confirmed to be specifically binding to CD44 since 100% of HA binding could be inhibited by the CD44 mab 5F12 (12). Monocyte separation. Monocytes were purified from PBMC using a combination of magnetic beads for removal of lymphocytes and adherence to plastic. Briefly, PBMC were treated with a mixture of magnetic beads coated with antibodies to CD2 and CD19 (Dynal, Lake Success, NY) according to the manufacturer’s instructions. Further purification of monocytes was obtained by plating the remaining cells and removing nonadherent cells after culture for 2 h at 37°C. This procedure resulted in preparations of monocytes that were 92 6 2.5% (mean 6 SEM) monocytes as determined by nonspecific esterase staining. Monocyte cultures. Monocytes (5 3 10 5) were cultured for 72 h in 48-well plates in RPMI supplemented with 10% v/v autologous serum at 37°C, 5% CO 2 in air for HA binding assays and CD44 mab phenotyping experiments (31). Autologous human serum was obtained simultaneously with peripheral blood and used in monocyte culture experiments. Cytokines and metabolic inhibitors were added to monocyte cultures following removal of nonadherent cells. Metabolic inhibitor studies. Monocytes were initially cultured for 72 h with TNFa (10 ng/ml). The medium was then removed, adherent cells were washed with RPMI, and fresh medium containing IL-4 (10 ng/ml) was added to the wells along with the protein synthesis inhibitor cycloheximide (10 mg/ml)
HYALURONAN BINDING TO MONOCYTE CD44
(Sigma, St. Louis, MO), the N-glycosylation inhibitor tunicamycin (1 mg/ml) (Sigma), the glucosidase I and II inhibitor castanospermine (100 mg/ml) (CalBiochem, La Jolla, CA), the mannosidase I inhibitor deoxymannojirimycin (100 mg/ml) (CalBiochem), the mannosidase II inhibitor swainsonine (10 mg/ml) (CalBiochem), the O-glycosylation inhibitor benzyl 2-acetamido-2-deoxy-a-D-galactopyranoside (2 mM) (Sigma), or the chondroitin sulfation inhibitor 4-methylumbelliferyl b-D-xyloside (0.5 mM) (Sigma) (47). Cells were harvested after an additional 24 h of culture for all of the inhibitors tested and also after 72 h for castanospermine, deoxymannojirimycin, swainsonine, benzyl 2-acetamido-2-deoxy-a-D-galactopyranoside, and 4-methylumbelliferyl b-D-xyloside (b-D-xyloside). Longer incubation times were not performed for cycloheximide and tunicamycin because these metabolic inhibitors induced monocyte toxicity after 24 h of treatment. HA binding and CD44 expression were measured as described earlier. Western blot analysis. Western blot analysis of PBMC lysates was performed as described (48). Briefly, PBMC were lysed in a buffer containing 10 mM Tris, pH 7.4, 150 mM NaCl, 1% NP-40, 20 mM iodoacetamide, and 1 mM PMSF. A soluble protein extract was prepared by centrifugation at 10,000g (20 min, 4°C). For each sample, protein from 2 3 10 5 cell equivalents was separated by electrophoresis on 6% polyacrylamide gels and electrophoretically transferred to nitrocellulose filters. CD44 on nitrocellulose filters was detected using CD44 mab A3D8 and indirect immunoperoxidase staining using horseradish peroxidase-conjugated goat anti-mouse IgG antibodies (Promega, Madison, WI). Protein bands were visualized by incubation with Rennaisance chemiluminescence reagent (DuPont NEN, Boston, MA) followed by exposure to film. RESULTS Effect of IL-4 on TNFa-induced monocyte HA binding and CD44 cell surface expression. Our previous studies indicated that the simultaneous addition of IL-4 to PB monocyte cultures treated with TNFa resulted in no HA binding to monocytes (12). To study mechanisms by which TNFa induced HA binding, and mechanisms whereby IL-4 blocked or prevented TNFainduced HA binding, we measured HA binding and CD44 expression on monocytes treated sequentially either with TNFa, no cytokine, or IL-4 for the first 72 h followed by treatment with either TNFa, no cytokine, or IL-4 for another 72 h (Fig. 1). Treatment of monocytes with TNFa alone for the first 72 h resulted in upregulation of HA binding compared to treatment with no cytokine or IL-4 (Figs. 1A, 1C, and 1E). Total cell surface CD44 increased over time on monocytes
211
treated with either TNFa, no cytokine, or IL-4 (Figs. 1B, 1D, and 1F). Subsequent treatment of monocytes initially treated with TNFa for 72 h with IL-4 or no cytokine resulted in loss of HA binding compared to continued monocyte treatment with TNFa (Fig. 1A). Interestingly, CD44 cell surface levels on monocytes intially treated with TNFa declined a similar amount when treated with either no cytokine, TNFa, or IL-4 during the second 72 h (Fig. 1B). Like the increase in HA binding induced by TNFa (12), the decrease in HA binding induced by IL-4 by monocytes initially treated with TNFa for 72 h was gradual; at 96 and 120 h, only intermediate levels of HA binding were present (data not shown). As previously reported (11), the in vitro culture of monocytes with no cytokine for 72 h resulted in lowlevel HA binding (Fig. 1C). In contrast, initial treatment of monocytes with IL-4 for 72 h resulted in no HA binding and prevented upregulation of HA binding when these IL-4-treated monocytes were treated for a subsequent 72 h with TNFa (Fig. 1E). However, following initial treatment of monocytes with either no cytokine or IL-4, TNFa treatment resulted in further upregulation of CD44 expression (Figs. 1D and 1F) to levels seen with the initial TNFa treatment of monocytes (72 h time point in Fig. 1A). The results shown in Fig. 1 established three important points about the mechanism of HA binding to moncyte CD44. First, these data confirmed that HA binding did not always correlate with CD44 cell surface expression levels (e.g., Figs. 1C vs 1D and 1E vs 1F). Second, IL-4 treatment of PB monocytes prevented subsequent TNFa-mediated HA binding (Fig. 1E) and IL-4-induced decreased monocyte CD44 HA binding on monocytes initially treated with TNFa (Fig. 1A). Third, the mechanisms of TNFa-induction and IL-4 inhibition of HA binding were processes that occurred over hours to days. In other systems, regulation of ligand binding to receptors by phosphorylation, palmitoylation, and cytoskeletal binding occured rapidly, in minutes to hours, and occured in the absence of new protein synthesis (49). In contrast, changes in glycosylation, glycosoaminoglycan modification, and isoform expression require new protein synthesis and “replacement” of current cell surface CD44 with a time course requiring hours to days (49), a time course more compatible with the lasting effects of TNFa induction and IL-4 inhibition of HA binding to monocyte CD44. Effect of the protein synthesis inhibitor cycloheximide on IL-4-mediated downregulation of TNFa-induced HA binding to monocytes. Mechanisms that regulate cytokine-mediated HA binding to CD44 that involve posttranslational modifications such as glycosylation and glycosoaminoglycan modification require new protein synthesis. Therefore, monocytes were treated for 72 h with TNFa and then treated with IL-4 and cyclohexi-
212
LEVESQUE AND HAYNES
FIG. 1. Effect of sequential TNFa and IL-4 treatment on monocyte HA binding and CD44 expression. Purified monocytes were cultured for 72 h with TNFa (10 ng/ml) (A, B), no cytokine (C, D), or IL-4 (10 ng/ml) (E, F) and the medium was then removed, the wells were washed with RPMI, and fresh medium was added containing no cytokine, TNFa, or IL-4. Monocyte DMFI values for HA binding to CD14 1 cells were determined as follows: DMFI 5 [(MFI for HA–FITC) 2 (MFI for IgG–FITC)]. Monocyte DMFI values for CD44 expression on CD14 1 cells were determined as follows: DMFI 5 [(MFI for anti-CD44 –FITC binding) 2 (MFI for IgG1–FITC binding)] Data represent the mean 6 SEM of three experiments.
mide (10 mg/ml) for a further 24 h. (Cycloheximide treatment for longer than 24 h was toxic to cultured monocytes.) HA binding and CD44 levels were then assessed and compared to TNFa-treated monocytes harvested at the time IL-4 and cycloheximide were added or to TNFa-treated monocytes treated with IL-4 alone. As shown in Fig. 2, cycloheximide prevented IL-4-mediated downregulation of HA binding to CD44 (cycloheximide vs no inhibitor, P 5 0.02) despite cycloheximide-induced decreases in CD44 cell surface ex-
pression. Decreases in monocyte cell surface expression of CD44 that occurred in this setting with cycloheximide treatment likely resulted from turnover of cell surface CD44. Studies from other laboratories have shown that the half-life of cell surface CD44 ranges from 24 to 48 h (50). Effect of N- and O-glycosylation inhibitors on IL-4mediated downregulation of TNFa-induced HA binding to monocytes. Studies performed by Katoh et al.
HYALURONAN BINDING TO MONOCYTE CD44
213
FIG. 2. Effect of cycloheximide treatment on IL-4-mediated downregulation of TNFa-induced monocyte HA binding. Purified monocytes were cultured for 72 h with TNFa (10 ng/ml) and then treated for 24 h with fresh medium containing IL-4 (10 ng/ml) and either no inhibitor or cycloheximide (10 mg/ml). Cycloheximide treatment for longer than 24 h decreased monocyte cell viability. Monocyte DMFI values for CD44 expression and HA binding to CD14 1 cells were determined as described in Fig. 1. Data represent the mean 6 SEM of three experiments. For HA binding, P 5 0.02 for cycloheximide treatment vs no inhibitor.
suggested that changes in N-linked glycosylation modulated HA binding to CHO cells (16). Likewise, Camp et al. have shown that in mice, exudative compared to resident peritoneal macrophages can be characterized by expression of CD44 molecules with less N-linked glycosylation (14). While this latter study did not correlate changes in CD44 N-linked glycosylation with changes in HA binding, given the data presented in Figs. 1 and 2, combined with the results of Katoh et al. we postulated that alterations in N-linked glycosylation might be involved in the TNFa- and IL-4-induced changes in HA binding to monocyte CD44. In order to test this possibilty, studies were performed with the N-linked glycosylation inhibitor, tunicamycin, the glucosidase I and II inhibitor, castanospermine, the mannosidase I inhibitor, deomannojirimycin, and the mannosidase II inhibitor, swainsonine (47). Monocytes were treated with TNFa for 72 h as in Fig. 2, then treated with IL-4 plus the inhibitors shown in Fig. 3 for an additional 24 or 72 h. (Tunicamycin treatment for longer than 24 h was toxic to the monocytes.) HA binding and CD44 levels were assessed and compared to TNFa-treated monocytes harvested at the time IL-4 plus the inhibitors were added, or compared with TNFa-treated monocytes treated with IL-4 alone. We found that tunicamycin prevented IL-4-mediated downregulation of HA binding to CD44 (tunicamycin vs no inhibitor, P 5 0.013) despite significant decreases in CD44 cell surface expression (tunicamycin vs no inhibitor, P 5 0.002) (Fig. 3). Unlike tunicamycin, castanospermine, deoxymannojirimycin, or swainsonine treatment of TNFa-treated monocytes for a further
24 h did not prevent IL-4-induced downregulation of HA binding to monocytes (Fig. 3). TNFa-treated monocytes treated with IL-4 and castanospermine, deoxymannojirimycin, or swainsonine for 72 h yielded results similar to those for 24-h treatment (data not shown). One study has suggested that decreased O-glycosylation was associated with increased HA binding to CD44 (25). Therefore, similar experiments were also performed with the O-glycosylation inhibitor, benzyl 2-acetamido-2-deoxy-a-D-galactopyranoside. As shown in Fig. 3, a-D-galactopyranoside did not prevent IL-4induced downregulation of HA binding to monocyte CD44. Effect of the chondroitin sulfation inhibitor, b-D-xyloside, on IL-4- mediated downregulation of TNFa-induced HA binding to monocytes. Several studies have shown that the 85-kDa standard isoform of CD44 (CD44S) does not contain chondroitin sulfate, although this isoform of CD44 can undergo modification to produce an 180- to 200-kDa molecule (14, 26). Interestingly, a study by Lesley et al. revealed that b-D-xyloside treatment or chondroitinase digestion of intact CD44 expressing pre-B cells (Raw 253) and fibroblasts (L cells) augmented HA binding, without an accompanying change in CD44 M r (26). Furthermore, CD44 has been shown to bind to at least two chondroitin sulfatemodified proteins, the chondroitin sulfate form of invariant chain (51) and a soluble chondroitin-modified proteoglycan, serglycin (52). In order to test the possibility that chondroitin sul-
214
LEVESQUE AND HAYNES
FIG. 3. Effect of N- and O-glycosylation inhibitors on IL-4-mediated downregulation of TNFa-induced monocyte HA binding. Purified monocytes were cultured for 72 h with TNFa (10 ng/ml) and then treated for 24 h with fresh medium containing IL-4 and no inhibitor, tunicamycin (1 mg/ml), castanospermine (100 mg/ml), deoxymannojirimycin (100 mg/ml), swainsonine (10 mg/ml), or benzyl 2-acetamido-2deoxy-a-D-galactopyranoside (2 mM). Tunicamycin treatment for longer than 24 h decreased monocyte cell viability. Monocyte DMFI values for CD44 expression and HA binding to CD14 1 cells were determined as described in Fig. 1. Treatment of monocytes for 72 h with TNFa and then with cytokines and inhibitors for 72 h gave similar results. Data represent the mean 6 SEM of three experiments. For HA binding, P 5 0.013 for tunicamycin vs no inhibitor, and for CD44 cell surface expression, P 5 0.002 for tunicamycin vs no inhibitor.
fation may regulate HA binding to monocyte CD44, the chondroitin sulfation inhibitor, b-D-xyloside, was added to monocytes treated with TNFa for 72 h, followed by treatment for either 24 or 72 h with no cytokine, with TNFa, or with IL-4. We found that for TNFa-treated monocytes in all settings (no cytokine, TNFa addition, and IL-4 addition), b-D-xyloside treatment for 24 h induced upregulation of HA binding to monocyte CD44 (Fig. 4). TNFa-treated monocytes treated with either no cytokine, IL-4, or TNFa and b-D-xyloside for 72 h yielded results similar to those for 24-h treatment (data not shown). There was a trend toward increased HA binding under all cytokine treatment conditions with b-D-xyloside treatment vs no inhibitor and significant increases in HA binding following treatment with IL-4 and b-D-xyloside for 24 h. (P 5 0.003 for b-D-xyloside treatment vs no inhibitor). Thus, inhibition of chondroitin sulfation by b-D-xyloside inhibited the ability of IL-4 to downregulate TNFa-induced ability of monocyte CD44 to bind HA. Western blot analysis of cytokine-treated monocytes. Our previous studies (11) and work presesented by Katoh et al. (16) and Camp et al. (14) revealed an approximately 5- to 10-kDa decrease in the M r of the standard isoform of CD44 in association with monocyte HA binding, or when comparing exudative to resident macrophages. This change in M r was consistent with
the finding that there were differences in N-linked glycosylation between the two populations of cells compared in each of these analyses. Therefore, Western blot analysis was performed on monocytes treated with TNFa for 72 h followed by treatment with either TNFa, IL-4, or no cytokine for an additional 72 h. As shown in Fig. 5, there was a consistent decrease in relative molecular mass of CD44 by approximately 5 to 10 kDa in monocyte preparations that were treated with TNFa and bound HA (lanes 3 and 5) vs those that were treated with no cytokine or IL-4 and did not bind high levels of HA (lanes 2 and 6). Also evident in lanes 3, 4, and 5 is another isoform of CD44 with a M r of approximately 117 kDa that was present in increased amounts in monocyte preparations that bound HA. Whether the effects of the chondroitin sulfation inhibitor b-D-xyloside on HA binding to IL-4-treated monocytes were mediated by changes in chondroitin sulfation of CD44 remains unresolved, since no 180- to 200-kDa bands (chondroitinated forms) were evident on these Western blot analyses. Effect of the CD44 mab F10.44.2 on HA binding to monocytes treated with TNFa or IL-4. To determine whether a conformation-based mechanism may in part explain IL-4 modulation of HA binding, we cultured monocytes with either no cytokine, IL-4, or TNFa for 72 h and then measured HA binding to these mono-
215
HYALURONAN BINDING TO MONOCYTE CD44
FIG. 4. Effect of the chondroitin-sulfate inhibitor, 4-methylumbelliferyl b-D-xyloside, on IL-4-mediated downregulation of TNFa-induced monocyte HA binding. Monocytes were cultured for 72 h with TNFa (10 ng/ml) and then treated for 24 or 72 h with fresh medium containing no cytokine, TNFa (10 ng/ml), or IL-4 (10 ng/ml) and either no inhibitor or 4-methylumbelliferyl b-D-xyloside (0.5 mM). Monocyte DMFI values for CD44 expression and HA binding to CD14 1 cells were determined as described in Fig. 1. Data represent the mean 6 SEM of three experiments. For HA binding, P 5 0.003 for IL-4 and b-D-xyloside treatment vs IL-4 and no inhibitor treatment.
cytes in the presence of either a control mab (constitutive HA binding) or saturating amounts of mab F10.44.2, a CD44 mab that has previously been shown to induce CD44-HA binding (inducible HA binding) (44, 45). We found that monocytes treated with TNFa for 72 h constitutively bound high levels of HA, and this population of monocytes could not be further induced to bind more HA by mab F10.44.2 treatment (Fig. 6A). Monocytes treated for 72 h with no cytokine constitutively bound lower levels of HA, and monocytes treated with IL-4 for 72 h did not bind HA. Mab F10.44.2 induced small increases in HA binding in no cytokine and IL-4-treated monocytes (Fig. 6A). The data in Fig. 6A demonstrated that IL-4 pretreatment of monocytes not only diminished constitutive HA binding but IL-4 also diminished inducible HA binding. Therefore, TNFa effectively “desensitized” inducible monocyte–HA binding.
with IL-4 did not constitutively bind HA, and monocytes initially treated for 72 h with TNFa and then treated with no cytokine or TNFa for an additional 72 h bound low levels of HA (Fig. 6B). The anti-CD44 mab F10.44.2 induced high-level HA binding in these latter two treatment groups (Fig. 6B). Mab F10.44.2 induced low level HA binding in monocytes initially treated with TNFa for 72 h followed by treatment with IL-4 for 72 h. The data in Fig. 6B demonstrated that IL-4-mediated inhibition of TNFa-induced HA binding to monocyte CD44 could not be reversed by CD44 mabs such as F10.44.2 that induce HA binding. Together, the data in Figs. 6A and 6B suggest that a conformation-based mechanism cannot explain IL-4-mediated inhibition of TNFa-induced CD44 –HA binding.
Effect of the CD44 mab F10.44.2 on HA binding to monocytes treated initially with TNFa, followed by treatment with TNFa, IL-4, or no cytokine. To further understand IL-4-mediated downregulation of TNFainduced monocyte HA binding, we measured HA binding in the presence of either a control mab or mab F10.44.2 on monocytes treated initially for 72 h with TNFa and then treated with no cytokine, IL-4, or TNFa for 72 h. We found that monocytes intially treated for 72 h with TNFa and then treated for 72 h
We performed these studies to study the mechanisms governing TNFa- and IL-4-mediated regulation of HA binding to monocyte CD44. As expected from our previous studies (12), initial treatment of monocytes for 72 h with TNFa resulted in high-level HA binding and increased CD44 expression. In this current study, we have shown that subsequent treatment of monocytes for an additional 72 h with TNFa maintained HA binding while subsequent treatment for 72 h with no cytokine or with IL-4 resulted in downregulation of monocyte HA binding.
DISCUSSION
216
LEVESQUE AND HAYNES
FIG. 5. Western blot analysis of CD44 on monocytes treated with TNFa and IL-4. Cell lysates were prepared as described under Materials and Methods from 2 3 10 5 cell equivalents/lane of freshly isolated monocytes (lane 1) and of monocytes cultured with either no cytokine or TNFa (10 ng/ml) for 72 h and either harvested immediately (lanes 2 and 3, respectively) or treated for another 72 h with fresh medium containing no cytokine (lane 4), TNFa (10 ng/ml) (lane 5), or IL-4 (10 ng/ml) (lane 6). Following electrophoresis and transfer to nitrocellulose, Western blot analysis was performed with the CD44 mab A3D8 and isotype-matched control antibody, P3. At the exposure time shown, no bands were visible on the control antibody blot and only results for the CD44 mab blot are shown. The CD44 band in lane 1 was not visible at the exposure time shown for lanes 2 to 6. The CD44 band in lane 1 is from an exposure time that was 153 longer than that shown in lanes 2 to 6. The Western blot shown is representative of results from three experiments. Monocyte DMFI values for CD44 expression and HA binding to CD14 1 cells were determined as described in Fig. 1.
Further, we found that the protein synthesis inhibitor cycloheximide, the N-glycosylation inhibitor, tunicamycin, and the chondroitin sulfation inhibitor, b-D-xyloside, inhibited IL-4-mediated downregulation of TNFa-induced monocyte HA binding. As has been reported in other systems, our studies suggest that HA binding to monocyte CD44 is regulated by a variety of mechanisms (13, 16, 19, 25–28, 31, 35– 40). Cytokine-regulated HA binding to monocyte CD44 may be regulated by N-glycosylation of CD44 and possibly by chondroitin-sulfate modification of CD44 or another cell surface molecule. Our data support the idea that the functional HA binding status of any given cell may be determined by the sum total of several different events including the amount of CD44 present, the CD44 configuration on the cell surface, posttranslational modifications of CD44, and perhaps CD44 association with other cellular proteins. It is clear from Fig. 5 that in monocytes the standard CD44S 85-kDa form is approximately 5–10 kDa smaller when HA is able to bind to monocyte CD44. Taken together with our data that tunicamycin increased HA binding to IL-4-treated monocytes, these data suggest that N-glycosylation of CD44 is one mechanism of maintaining the “inactive” or “nonbinding” form of CD44. Monocyte activation by TNFa would be
predicted to decrease N-glycosylation of CD44, and IL-4 would be predicted to increase this process, postulates that are compatible with data shown in Figs. 3 and 5. Work presented by others has shown that the 85kDa standard isoform of CD44 does not contain chondroitin sulfate, although this isoform of CD44 can undergo chondroitination to produce a 180- to 200kDa molecule (14, 26). Interestingly, a study by Lesley et al. revealed that b-D-xyloside treatment or chondroitinase digestion of intact CD44 on murine pre-B cells and fibroblasts augmented HA binding, without an accompanying change in CD44 M r (26). These and our studies suggest that either a chondroitin-sulfate-modified CD44 or another chondroitinsulfated cellular protein may regulate or participate in HA binding to CD44. Extracellular matrix HA exists in a protein complex with aggrecan and link protein; aggrecan is heavily modified with chondroitin sulfate (53, 54). A similar structure, i.e., the hyaluronan coat, exists on the surface of cells in which cell surface CD44 is complexed with HA and aggrecan or an aggrecanlike protein (55, 56). In this model, IL-4 would be expected to either promote production of the chondroitin sulfate modified protein and/or stimulate production of HA, thereby inducing formation of a hy-
217
HYALURONAN BINDING TO MONOCYTE CD44
FIG. 6. Effect of CD44 mab F10.44.2 on HA binding to monocytes treated with IL-4 or TNFa. (A) Monocytes were cultured for 72 h with no cytokine, IL-4 (10 ng/ml), or TNFa (10 ng/ml) and then harvested. (B) Monocytes were cultured for 72 h with TNFa (10 ng/ml) and the medium was then removed, the wells were washed with RPMI, and fresh medium was added containing no cytokine, IL-4 (10 ng/ml), or TNFa (10 ng/ml) for a further 72 h and then monocytes were harvested. Following harvesting, monocyte DMFI values were determined as described in Fig. 1 except monocytes were initially treated with a saturating amount of mouse ascites containing either CD44 mab F10.44.2 or control mab (P3 3 63/Ag8) prior to staining with HA–FITC and anti-CD14 –PE. Data represent the mean 6 SEM of three experiments. For F10.44.2 induced HA binding, P 5 0.068 for monocytes treated with no cytokine vs TNFa-treated monocytes treated for a further 72 h with no cytokine and P 5 0.048 for monocytes treated with IL-4 vs TNFa-treated monocytes treated for a further 72 h with IL-4.
aluronan coat. Indeed, studies have shown that TNFa diminishes proteoglycan production by chondrocytes (57). Furthermore, IL-4 promotes proteoglycan synthesis by fibroblasts (58) and reverses TNFamediated inhibition of cartilage proteoglycan synthesis (59). The presence of cell surface HA would be expected to block binding of exogenous FITC-labeled HA giving the impression that IL-4 treated monocytes do not bind HA. An alternative hypothesis is that IL-4 induces production of a chondroitin-sulfate modified protein that binds to CD44 and blocks HA binding to CD44. Lesley and co-workers have suggested that CD44 can exist in one of three possible activation states with regard to HA binding: constitutive active, activatible, and nonactivatible (26). TNFa has effects on both constitutive and inducible HA binding, and these effects could be separated in our studies. Thus our studies suggest a diversity of mechanisms governing HA binding to monocyte CD44. Given the proinflammatory consequences of CD44 –HA binding, these studies suggest several new sites for potential inhibition of CD44 proinflammatory activity. Studies of IL-4 in vivo in the treatment of RA may provide an opportunity to determine the effect of IL-4 in vivo on human monocyte ability to bind HA and to determine the ability of IL-4 to modify clinical inflammation in human synovitis.
ACKNOWLEDGMENTS The authors thank Dr. Larry Liao for insightful discussions, Mary Misukonis for performing monocyte nonspecific esterase stains, and Erlina Siragusa of the CFAR Cell Sorter Facility.
REFERENCES 1.
2. 3. 4. 5.
6.
7. 8.
9.
Weiss, J. M., Sleeman, J., Renkl, A. C., Dittmar, H., Termeer, C. C., Taxis, S., Howells, N., Hofmann, M., Kohler, G., Schopf, E., Ponta, H., Herrlich, P., and Simon, J. C., J. Cell Biol. 137, 1137, 1997. Clark, R. A., Alon, R., and Springer, T. A., J. Cell Biol. 134, 1075, 1996. Galandrini, R., Galluzzo, E., Albi, N., Grossi, C. E., and Velardi, A., J. Immunol. 153, 21, 1994. DeGrendele, H. C., Estess, P., Picker, L. J., and Siegelman, M. H., J. Exp. Med. 183, 1119, 1996. Trochon, V., Mabilat, C., Bertrand, P., Legrand, Y., SmadjaJoffe, F., Soria, C., Delpech, B., and Lu, H., Int. J. Cancer 66, 664, 1996. Hodge-Dufour, J., Noble, P. W., Horton, M. R., Bao, C., Wysoka, M., Burdick, M. D., Strieter, R. M., Trinchieri, G., and Pure, E., J. Immunol. 159, 2492, 1997. Noble, P. W., Lake, F. R., Henson, P. M., and Riches, D. W., J. Clin. Invest. 91, 2368, 1993. McKee, C. M., Penno, M. B., Cowman, M., Burdick, M. D., Strieter, R. M., Bao, C., and Noble, P. W., J. Clin. Invest. 98, 2403, 1996. Degrendele, H. C., Estess, P., and Siegelman, M. H., Science 278, 672, 1997.
218 10. 11. 12. 13. 14. 15.
16. 17. 18. 19. 20. 21. 22. 23. 24. 25.
26. 27. 28. 29. 30.
31.
32. 33.
LEVESQUE AND HAYNES Hyman, R., Lesley, J., and Schulte, R., Immunogenetics 33, 392, 1991. Levesque, M. C., and Haynes, B. F., J. Immunol. 156, 1557, 1996. Levesque, M. C., and Haynes, B. F., J. Immunol. 159, 6184, 1997. Bartolazzi, A., Nocks, A., Aruffo, A., Spring, F., and Stamenkovic, I., J. Cell Biol. 132, 1199, 1996. Camp, R. L., Kraus, T. A., and Pure, E., J. Cell Biol. 115, 1283, 1991. Campanero, M. R., Pulido, R., Alonso, J. L., Pivel, J. P., Pimentel-Muinos, F. X., Fresno, M., and Sanchez-Madrid, F., Eur. J. Immunol. 21, 3045, 1991. Katoh, S., Zheng, Z., Oritani, K., Shimozato, T., and Kincade, P. W., J. Exp. Med. 182, 419, 1995. Guo, Y. J., Lin, S. C., Wang, J. H., Bigby, M., and Sy, M. S., Int. Immunol. 6, 213, 1994. Jackson, D. G., Bell, J. I., Dickinson, R., Timans, J., Shields, J., and Whittle, N., J. Cell Biol. 128, 673, 1995. Takahashi, K., Stamenkovic, I., Cutler, M., Dasgupta, A., and Tanabe, K. K., J. Biol. Chem. 271, 9490, 1996. Neame, S. J., and Isacke, C. M., EMBO J. 11, 4733, 1992. Isacke, C. M., J. Cell Sci. 107, 2353, 1994. Kalomiris, E. L., and Bourguignon, L. Y., J. Cell Biol. 106, 319, 1988. Kalomiris, E. L., and Bourguignon, L. Y., J. Biol. Chem. 264, 8113, 1989. Tsukita, S., Oishi, K., Sato, N., Sagara, J., Kawai, A., and Tsukita, S., J. Cell Biol. 126, 391, 1994. Bennett, K. L., Modrell, B., Greenfield, B., Bartolazzi, A., Stamenkovic, I., Peach, R., Jackson, D. G., Spring, F., and Aruffo, A., J. Cell Biol. 131, 1623, 1995. Lesley, J., English, N., Perschl, A., Gregoroff, J., and Hyman, R., J. Exp. Med. 182, 431, 1995. Bourguignon, L. Y., Lokeshwar, V. B., Chen, X., and Kerrick, W. G., J. Immunol. 151, 6634, 1993. Pure, E., Camp, R. L., Peritt, D., Panettieri, R. A., Jr., Lazaar, A. L., and Nayak, S., J. Exp. Med. 181, 55, 1995. Uff, C. R., Neame, S. J., and Isacke, C. M., Eur. J. Immunol. 25, 1883, 1995. Murakami, S., Shimabukuro, Y., Miki, Y., Saho, T., Hino, E., Kasai, D., Nozaki, T., Kusumoto, Y., and Okada, H., J. Immunol. 152, 467, 1994. Liao, H. X., Levesque, M. C., Patton, K., Bergamo, B., Jones, D., Moody, M. A., Telen, M. J., and Haynes, B. F., J. Immunol. 151, 6490, 1993. Stamenkovic, I., Aruffo, A., Amiot, M., and Seed, B., EMBO J. 10, 343, 1991. He, Q., Lesley, J., Hyman, R., Ishihara, K., and Kincade, P. W., J. Cell Biol. 119, 1711, 1992.
34. 35.
36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59.
Dougherty, G. J., Cooper, D. L., Memory, J. F., and Chiu, R. K., J. Biol. Chem. 269, 9074, 1994. Galluzzo, E., Albi, N., Fiorucci, S., Merigiola, C., Ruggeri, L., Tosti, A., Grossi, C. E., and Velardi, A., Eur. J. Immunol. 25, 2932, 1995. Lesley, J., Kincade, P. W., and Hyman, R., Eur. J. Immunol. 23, 1902, 1993. Lesley, J., He, Q., Miyake, K., Hamann, A., Hyman, R., and Kincade, P. W., J. Exp. Med. 175, 257, 1992. Perschl, A., Lesley, J., English, N., Trowbridge, I., and Hyman, R., Eur. J. Immunol. 25, 495, 1995. Liu, D., and Sy, M. S., J. Exp. Med. 183, 1987, 1996. Liu, D. C., and Sy, M. S., J. Immunol. 159, 2702, 1997. Guo, Y., Wu, Y., Shinde, S., Sy, M. S., Aruffo, A., and Liu, Y., J. Exp. Med. 184, 955, 1996. Conrad, P., Rothman, B. L., Kelley, K. A., and Blue, M. L., J. Immunol. 149, 1833, 1992. Telen, M. J., Eisenbarth, G. S., and Haynes, B. F., J. Clin. Invest. 71, 1878, 1983. Liao, H. X., Telen, M. J., and Haynes, B. F., in “Leukocyte Typing V,” Oxford Univ. Press, Oxford, 1995. Dalchau, R., Kirkley, J., and Fabre, J. W., Eur. J. Immunol. 10, 745, 1980. Haynes, B. F., Metzgar, R. S., Minna, J. D., and Bunn, P. A., N. Engl. J. Med. 304, 1319, 1981. Elbein, A. D., FASEB J. 5, 3055, 1991. Burnette, W. N., Anal. Biochem. 112, 195, 1981. Krishna, R. G., and Wold, F., “Advances in Enzymology & Related Areas of Molecular Biology,” 67, 265, 1993. Goebeler, M., Kaufmann, D., Brocker, E. B., and Klein, C. E., J. Cell Sci. 109, 1957, 1996. Naujokas, M. F., Morin, M., Anderson, M. S., Peterson, M., and Miller, J., Cell 74, 257, 1993. Toyama-Sorimachi, N., and Miyasaka, M., J. Dermatol. 21, 795, 1994. Hardingham, T. E., and Fosang, A. J., FASEB J. 6, 861, 1992. Morgelin, M., Paulsson, M., and Engel, J., Biochem. Soc. Trans. 18, 204, 1990. Knudson, C. B., J. Cell Biol. 120, 825, 1993. Knudson, W., Bartnik, E., and Knudson, C. B., Proc. Natl. Acad. Sci. USA 90, 4003, 1993. Hardingham, T. E., Rayan, V., and Lewthwaite, J. C., Rev. Rheum. 61, 93S, 1994. Wegrowski, Y., Paltot, V., Gillery, P., Kalis, B., Randoux, A., and Maquart, F. X., Biochem. J. 307, 673, 1995. van Roon, J. A., van Roy, J. L., Gmelig-Meyling, F. H., Lafeber, F. P., and Bijlsma, J. W., Arthritis Rheum. 39, 829, 1996.