Tumor Necrosis Factor-α Induces Functionally Active Hyaluronan-adhesive CD44 by Activating Sialidase through p38 Mitogen-activated Protein Kinase in Lipopolysaccharide-stimulated Human Monocytic Cells
2003; Elsevier BV; Volume: 278; Issue: 39 Linguagem: Inglês
10.1074/jbc.m302309200
ISSN1083-351X
AutoresKatrina Gee, Maya Kozlowski, Ashok Kumar,
Tópico(s)Glycosylation and Glycoproteins Research
ResumoInteraction of CD44, an adhesion molecule, with its ligand, hyaluronan (HA), in monocytic cells plays a critical role in cell migration, inflammation, and immune responses. Most cell types express CD44 but do not bind HA. The biological functions of CD44 have been attributed to the generation of the functionally active, HA-adhesive form of this molecule. Although lipopolysaccharide (LPS) and cytokines induce HA-adhesive CD44, the molecular mechanism underlying this process remains unknown. In this study, we show that LPS-induced CD44-mediated HA (CD44-HA) binding in monocytes is regulated by endogenously produced tumor necrosis factor (TNF)-α and IL-10. Furthermore, p38 mitogen-activated protein kinase (MAPK) activation was required for LPS- and TNF-α-induced, but not IL-10-induced, CD44-HA-binding in normal monocytes. To dissect the signaling pathways regulating CD44-HA binding independently of cross-regulatory IL-10-mediated effects, IL-10-refractory promonocytic THP-1 cells were employed. LPS-induced CD44-HA binding in THP-1 cells was regulated by endogenously produced TNF-α. Our results also suggest that lysosomal sialidase activation may be required for the acquisition of the HA-binding form of CD44 in LPS- and TNF-α-stimulated monocytic cells. Studies conducted to understand the role of MAPKs in the induction of sialidase activity revealed that LPS-induced sialidase activity was dependent on p42/44 MAPK-mediated TNF-α production. Blocking TNF-α production by PD98059, a p42/44 inhibitor, significantly reduced the LPS-induced sialidase activity and CD44-HA binding. Subsequently, TNF-α-mediated p38 MAPK activation induced sialidase activity and CD44-HA binding. Taken together, our results suggest that TNF-α-induced p38 MAPK activation may regulate the induction of functionally active HA-binding form of CD44 by activating sialidase in LPS-stimulated human monocytic cells. Interaction of CD44, an adhesion molecule, with its ligand, hyaluronan (HA), in monocytic cells plays a critical role in cell migration, inflammation, and immune responses. Most cell types express CD44 but do not bind HA. The biological functions of CD44 have been attributed to the generation of the functionally active, HA-adhesive form of this molecule. Although lipopolysaccharide (LPS) and cytokines induce HA-adhesive CD44, the molecular mechanism underlying this process remains unknown. In this study, we show that LPS-induced CD44-mediated HA (CD44-HA) binding in monocytes is regulated by endogenously produced tumor necrosis factor (TNF)-α and IL-10. Furthermore, p38 mitogen-activated protein kinase (MAPK) activation was required for LPS- and TNF-α-induced, but not IL-10-induced, CD44-HA-binding in normal monocytes. To dissect the signaling pathways regulating CD44-HA binding independently of cross-regulatory IL-10-mediated effects, IL-10-refractory promonocytic THP-1 cells were employed. LPS-induced CD44-HA binding in THP-1 cells was regulated by endogenously produced TNF-α. Our results also suggest that lysosomal sialidase activation may be required for the acquisition of the HA-binding form of CD44 in LPS- and TNF-α-stimulated monocytic cells. Studies conducted to understand the role of MAPKs in the induction of sialidase activity revealed that LPS-induced sialidase activity was dependent on p42/44 MAPK-mediated TNF-α production. Blocking TNF-α production by PD98059, a p42/44 inhibitor, significantly reduced the LPS-induced sialidase activity and CD44-HA binding. Subsequently, TNF-α-mediated p38 MAPK activation induced sialidase activity and CD44-HA binding. Taken together, our results suggest that TNF-α-induced p38 MAPK activation may regulate the induction of functionally active HA-binding form of CD44 by activating sialidase in LPS-stimulated human monocytic cells. CD44, an adhesion molecule, comprises a family of 85–200-kDa transmembrane glycoproteins that are widely expressed in a variety of cell types (1Naor D. Sionov R.V. Ish-Shalom D. Adv. Cancer Res. 1997; 71: 241-319Crossref PubMed Google Scholar). CD44 binds to high endothelial venules and to the extracellular matrix via its interaction with its principle ligand, hyaluronan (HA) 1The abbreviations used are: HA, hyaluronan; 2-Neu-Ac, 2,3-dehydro-2-deoxy-N-acetylneuraminic acid; CD44-HA, CD44-mediated hyaluronan binding; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; LPS, lipopolysaccharide; TNF, tumor necrosis factor; mAb, monoclonal antibody; IL, interleukin; PBMC, peripheral blood monocyte.1The abbreviations used are: HA, hyaluronan; 2-Neu-Ac, 2,3-dehydro-2-deoxy-N-acetylneuraminic acid; CD44-HA, CD44-mediated hyaluronan binding; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; LPS, lipopolysaccharide; TNF, tumor necrosis factor; mAb, monoclonal antibody; IL, interleukin; PBMC, peripheral blood monocyte. (1Naor D. Sionov R.V. Ish-Shalom D. Adv. Cancer Res. 1997; 71: 241-319Crossref PubMed Google Scholar, 2Miyake K. Underhill C.B. Lesley J. Kincade P.W. J. Exp. Med. 1990; 172: 69-75Crossref PubMed Scopus (557) Google Scholar, 3Siegelman M.H. DeGrendele H.C. Estess P. J. Leukocyte Biol. 1999; 66: 315-321Crossref PubMed Scopus (179) Google Scholar). CD44-HA interactions have been implicated mainly in cell-cell and cell-matrix adhesion and hence play a key role in a variety of physiological processes, including cell migration, lymphocyte homing, cell activation, and hemopoiesis, as well as in disease processes such as arthritis, inflammation, and tumor metastasis (1Naor D. Sionov R.V. Ish-Shalom D. Adv. Cancer Res. 1997; 71: 241-319Crossref PubMed Google Scholar, 3Siegelman M.H. DeGrendele H.C. Estess P. J. Leukocyte Biol. 1999; 66: 315-321Crossref PubMed Scopus (179) Google Scholar, 4Wittig B. Schwarzler C. Fohr N. Gunthert U. Zoller M. J. Immunol. 1998; 161: 1069-1073PubMed Google Scholar, 5Brocke S. Piercy C. Steinman L. 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The multiple functions of CD44 have been attributed to the existence of numerous CD44 isoforms that are generated by alternative mRNA splicing as well as by extensive post-translational modifications such as N- and O-linked glycosylation and glycosaminoglycan addition (1Naor D. Sionov R.V. Ish-Shalom D. Adv. Cancer Res. 1997; 71: 241-319Crossref PubMed Google Scholar, 3Siegelman M.H. DeGrendele H.C. Estess P. J. Leukocyte Biol. 1999; 66: 315-321Crossref PubMed Scopus (179) Google Scholar, 10Gunthert U. Hofmann M. Rudy W. Reber S. Zoller M. Haussmann I. Matzku S. Wenzel A. Ponta H. Herrlich P. Cell. 1991; 65: 13-24Abstract Full Text PDF PubMed Scopus (1585) Google Scholar, 11Jackson D.G. Buckley J. Bell J.I. J. Biol. Chem. 1992; 267: 4732-4739Abstract Full Text PDF PubMed Google Scholar, 12Dougherty G.J. Landorp P.M. Cooper D.L. Humphries R.K. J. Exp. Med. 1991; 174: 1-5Crossref PubMed Scopus (147) Google Scholar, 13Katoh S. Zheng Z. Oritani K. Shimozato T. Kincade P.W. J. Exp. Med. 1995; 182: 419-429Crossref PubMed Scopus (230) Google Scholar, 14Liu D. Liu T. Li R. Sy M.S. Front. Biosci. 1998; 3: D631-D636Crossref PubMed Google Scholar).The ability of CD44 to bind HA is a tightly regulated process and is dependent on cell type, state of cell activation, and differentiation (1Naor D. Sionov R.V. Ish-Shalom D. Adv. Cancer Res. 1997; 71: 241-319Crossref PubMed Google Scholar, 14Liu D. Liu T. Li R. Sy M.S. Front. Biosci. 1998; 3: D631-D636Crossref PubMed Google Scholar, 15Lesley J. Hascall V.C. Tammi M. Hyman R. J. Biol. Chem. 2000; 275: 26967-26975Abstract Full Text Full Text PDF PubMed Google Scholar, 16Murakami S. Miyake K. June C.H. Kincade P.W. Hodes R.J. J. Immunol. 1990; 145: 3618-3627PubMed Google Scholar, 17Kryworuchko M. Diaz-Mitoma F. Kumar A. Exp. Cell Res. 1999; 250: 241-252Crossref PubMed Scopus (17) Google Scholar). Although most cells express some form of CD44, not all cells constitutively bind HA (17Kryworuchko M. Diaz-Mitoma F. Kumar A. Exp. Cell Res. 1999; 250: 241-252Crossref PubMed Scopus (17) Google Scholar, 18Nandi A. Estess P. Siegelman M.H. J. Biol. Chem. 2000; 275: 14939-14948Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). Acquisition of the HA-binding ability of CD44 thus plays a vital role in determining CD44-mediated biological effects. The HA-binding capacity of CD44 has been suggested to be influenced by multiple factors that include structural variations in the CD44 extracellular domain, oligomerization of CD44 on the cell membrane, and phosphorylation of its cytoplasmic tail (1Naor D. Sionov R.V. Ish-Shalom D. Adv. Cancer Res. 1997; 71: 241-319Crossref PubMed Google Scholar, 10Gunthert U. Hofmann M. Rudy W. Reber S. Zoller M. Haussmann I. Matzku S. Wenzel A. Ponta H. Herrlich P. Cell. 1991; 65: 13-24Abstract Full Text PDF PubMed Scopus (1585) Google Scholar, 11Jackson D.G. Buckley J. Bell J.I. J. Biol. Chem. 1992; 267: 4732-4739Abstract Full Text PDF PubMed Google Scholar, 12Dougherty G.J. Landorp P.M. Cooper D.L. Humphries R.K. J. Exp. Med. 1991; 174: 1-5Crossref PubMed Scopus (147) Google Scholar, 14Liu D. Liu T. Li R. Sy M.S. Front. Biosci. 1998; 3: D631-D636Crossref PubMed Google Scholar, 19Lesley J. English N. Perschl A. Gregoroff J. Hyman R. J. Exp. Med. 1995; 182: 431-437Crossref PubMed Scopus (176) Google Scholar, 20Takahashi K. Stamenkovic I. Cutler M. Dasgupta A. Tanabe K.K. J. Biol. Chem. 1996; 271: 9490-9496Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). It is also known that alterations in the N- and O-linked glycosylation patterns of CD44 play a vital role in the regulation of its binding to HA (13Katoh S. Zheng Z. Oritani K. Shimozato T. Kincade P.W. J. Exp. Med. 1995; 182: 419-429Crossref PubMed Scopus (230) Google Scholar, 19Lesley J. English N. Perschl A. Gregoroff J. Hyman R. J. Exp. Med. 1995; 182: 431-437Crossref PubMed Scopus (176) Google Scholar, 20Takahashi K. Stamenkovic I. Cutler M. Dasgupta A. Tanabe K.K. J. Biol. Chem. 1996; 271: 9490-9496Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 21Bartolazzi A. Nocks A. Aruffo A. Spring F. Stamenkovic I. J. Cell Biol. 1996; 132: 1199-1208Crossref PubMed Scopus (156) Google Scholar). Recently, an inducible sialidase was implicated in CD44-HA binding of lipopolysaccharide (LPS)-stimulated human monocytic cells (22Katoh S. Miyagi T. Taniguchi H. Matsubara Y. Kadota J. Tominaga A. Kincade P.W. Matsukura S. Kohno S. J. Immunol. 1999; 162: 5058-5061PubMed Google Scholar).The lipopolysaccharide component of endotoxin, derived from Gram-negative bacterial cell walls, induces inflammatory responses that contribute to the pathogenesis of sepsis, inflammation, and a number of autoimmune diseases including rheumatoid arthritis. It is well established that mononuclear phagocytes play a major role in the pathogenesis of LPS-induced syndromes. Peripheral blood monocytes express abundant cell surface CD44 yet do not bind HA (23Levesque M.C. Haynes B.F. J. Immunol. 1996; 156: 1557-1565PubMed Google Scholar, 24Levesque M.C. Haynes B.F. J. Immunol. 1997; 159: 6184-6194PubMed Google Scholar). It was recently reported that stimulation of monocytes with LPS up-regulated CD44-mediated HA-binding (23Levesque M.C. Haynes B.F. J. Immunol. 1996; 156: 1557-1565PubMed Google Scholar, 24Levesque M.C. Haynes B.F. J. Immunol. 1997; 159: 6184-6194PubMed Google Scholar). TNF-α, a proinflammatory cytokine, was shown to be an important positive regulator of LPS-induced CD44-HA binding in these cells (24Levesque M.C. Haynes B.F. J. Immunol. 1997; 159: 6184-6194PubMed Google Scholar). Furthermore, ligation of CD44 with HA has been shown to induce a number of proinflammatory cytokines including TNF-α (25Webb D.S. Shimizu Y. Van Seventer G.A. Shaw S. Gerrard T.L. Science. 1990; 249: 1295-1297Crossref PubMed Scopus (276) Google Scholar, 26Noble P.W. McKee C.M. Cowman M. Shin H.S. J. Exp. Med. 1996; 183: 2373-2378Crossref PubMed Scopus (277) Google Scholar). Therefore, the acquisition of HA binding capacity by CD44 expressed on monocytic cells could be critical for their participation in inflammatory responses. Modulation of CD44-HA binding in monocytic cells by endotoxins and inflammatory cytokines may thus have profound effects on the migration of monocytes to sites of inflammation and on the development of immune responses. Hence, understanding the signaling pathways governing the regulation of CD44 expression and the synthesis of the HA-adhesive form of CD44 may lead to the development of strategies for the treatment of autoimmune diseases and cancer.There is very little information available regarding the molecular mechanisms involved in the regulation of HA-adhesive, functionally active CD44 expression (27Fichter M. Hinrichs R. Eissner G. Scheffer B. Classen S. Ueffing M. Oncogene. 1997; 14: 2817-2824Crossref PubMed Scopus (41) Google Scholar). We have recently demonstrated the involvement of c-Jun-N-terminal kinase (JNK) in the regulation of LPS-induced CD44 expression in human monocytic cells (28Gee K. Lim W. Ma W. Diaz-Mitoma F. Kozlowski M. Kumar A. J. Immunol. 2002; 169: 5660-5672Crossref PubMed Scopus (56) Google Scholar). In this study, we examined the mitogen-activated protein kinase (MAPK) transduction pathways involved in the synthesis of the HA-adhesive, functionally active form of CD44 in LPS-stimulated normal human monocytes and in promonocytic THP-1 cells. MAPKs are serine-threonine protein kinases that include p38, p42/44 extracellular signal-regulated kinases (ERKs), and JNK (29Lewis T.S. Shapiro P.S. Ahn N.G. Adv. Cancer Res. 1998; 74: 49-139Crossref PubMed Google Scholar, 30Seger R. Krebs E.G. FASEB J. 1995; 9: 726-735Crossref PubMed Scopus (3188) Google Scholar). MAPKs play a key role in cellular responses such as proliferation, differentiation, and apoptosis (29Lewis T.S. Shapiro P.S. Ahn N.G. Adv. Cancer Res. 1998; 74: 49-139Crossref PubMed Google Scholar, 30Seger R. Krebs E.G. FASEB J. 1995; 9: 726-735Crossref PubMed Scopus (3188) Google Scholar, 31Dong C. Davis R.J. Flavell R.A. Annu. Rev. Immunol. 2002; 20: 55-72Crossref PubMed Scopus (1360) Google Scholar). These three MAPKs are involved in three parallel signaling cascades activated by distinct and sometimes overlapping sets of stimuli. In general, ERKs are activated by growth factors, whereas the p38 and JNK are activated by stress stimuli (29Lewis T.S. Shapiro P.S. Ahn N.G. Adv. Cancer Res. 1998; 74: 49-139Crossref PubMed Google Scholar, 30Seger R. Krebs E.G. FASEB J. 1995; 9: 726-735Crossref PubMed Scopus (3188) Google Scholar). LPS and TNF-α have been shown to induce the expression of several cellular genes via activation of all three classes of MAPKs (29Lewis T.S. Shapiro P.S. Ahn N.G. Adv. Cancer Res. 1998; 74: 49-139Crossref PubMed Google Scholar, 32Hambleton J. Weinstein S.L. Lem L. DeFranco A.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2774-2778Crossref PubMed Scopus (412) Google Scholar, 33Hatzoglou A. Roussel J. Bourgeade M.F. Rogier E. Madry C. Inoue J. Devergne O. Tsapis A. J. Immunol. 2000; 165: 1322-1330Crossref PubMed Scopus (197) Google Scholar, 34Darnay B.G. Aggarwal B.B. J. Leukocyte Biol. 1997; 61: 559-566Crossref PubMed Scopus (168) Google Scholar, 35Ma W. Lim W. Gee K. Aucoin S. Nandan D. Kozlowski M. Diaz-Mitoma F. Kumar A. J. Biol. Chem. 2001; 276: 13664-13674Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar).In this study, we show that LPS-induced CD44-HA binding in normal monocytes is regulated by endogenously produced TNF-α and IL-10. Studies designed to delineate the role of MAPKs revealed that p38 activation was required for LPS- and TNF-α-induced expression of the HA-adhesive CD44. In contrast, IL-10-induced CD44-HA binding was not mediated through either p38 or p42/44 activation. To further dissect the TNF-α- and LPS-induced signaling pathways independent of IL-10-mediated effects, we utilized IL-10 refractory THP-1 cells as a model system. We show that TNF-α production in LPS-stimulated THP-1 cells involves p42/44 MAPK activation, and endogenously produced TNF-α is required for the induction of CD44-mediated HA binding. Our results also show that sialidase activation is required for the acquisition of HA binding capacity by CD44 in LPS- and TNF-α-stimulated monocytic cells. These results prompted us to investigate a role for p38 and p42/44 in LPS- and TNF-α-induced sialidase activity resulting in the synthesis of the HA-binding form of CD44. LPS-induced sialidase activity was dependent on p42/44 MAPK-mediated TNF-α production. Blocking TNF-α production by PD98059, a p42/44 inhibitor, significantly reduced the LPS-induced sialidase activity and CD44-HA binding. Furthermore, TNF-α-induced sialidase activity and CD44-HA binding was found to be regulated by p38 MAPK activation. Taken together, these results suggest a key role for TNF-α in regulating the synthesis of HA-adhesive CD44 by inducing sialidase activity through p38 MAPK activation in LPS-stimulated monocytic cells.EXPERIMENTAL PROCEDURESCell Lines, Cell Culture, and Reagents—THP-1, a promonocytic cell line derived from a human acute lymphocytic leukemia patient, was obtained from the American Type Culture Collection (Manassas, VA). Cells were cultured in Iscove's Dulbecco's medium (Sigma) supplemented with 10% fetal bovine serum (Invitrogen), 100 units/ml penicillin, 100 μg/ml gentamicin, 10 mm HEPES, and 2 mm glutamine. PD98059 (Calbiochem), an inhibitor of MEK1 kinase, selectively blocks the activity of ERK MAPK and has no effect on the activity of other serine-threonine protein kinases, including Raf-1, p38, and JNK MAPKs, or protein kinase C and protein kinase A (36Dudley D.T. Pang L. Decker S.J. Bridges A.J. Saltiel A.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7686-7689Crossref PubMed Scopus (2584) Google Scholar, 37Lee J.C. Young P.R. J. Leukocyte Biol. 1996; 59: 152-157Crossref PubMed Scopus (373) Google Scholar). The pyridinyl imidazole SB202190 (Calbiochem), a potent inhibitor of p38 MAPK, has no significant effect on the activity of the ERK or JNK MAPK subgroups (36Dudley D.T. Pang L. Decker S.J. Bridges A.J. Saltiel A.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7686-7689Crossref PubMed Scopus (2584) Google Scholar, 37Lee J.C. Young P.R. J. Leukocyte Biol. 1996; 59: 152-157Crossref PubMed Scopus (373) Google Scholar). SB202474, an inactive analog of SB202190, was also purchased from Calbiochem. LPS derived from Escherichia coli 0111:B4 (Sigma), recombinant human IL-10 (R&D Systems, Minneapolis, MN), recombinant human TNF-α (BioSource, Montreal, Canada), and human IL-10Rα and human TNF-αR1 antibodies (R&D Systems) capable of neutralizing the biological activities of IL-10 and TNF-α, respectively, were also purchased. Hyaluronan preparation (Calbiochem) was more than 97% pure. Sialidase from Arthrobacter ureafaciens, sialidase inhibitor, 2,3-dehydro-2-deoxy-N-acetylneuraminic acid (2-Neu-Ac), and all other chemicals used for Western blotting were purchased from Sigma. The sialidase inhibitor, 2-Neu-Ac, is a transition state analogue of sialic acid and is a potent inhibitor of viral, bacterial, and mammalian sialidases (38Achyuthan K.E. Achyuthan A.M. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 2001; 129: 29-64Crossref PubMed Scopus (111) Google Scholar).Isolation of Monocytes from PBMCs—PBMCs were isolated from the blood of healthy adult volunteers following approval of the protocol by the Ethics Review Committee of the Children's Hospital of Eastern Ontario (Ottawa, Canada). PBMCs were isolated by density gradient centrifugation over Ficoll-Hypaque (Amersham Biosciences) as previously described (28Gee K. Lim W. Ma W. Diaz-Mitoma F. Kozlowski M. Kumar A. J. Immunol. 2002; 169: 5660-5672Crossref PubMed Scopus (56) Google Scholar, 35Ma W. Lim W. Gee K. Aucoin S. Nandan D. Kozlowski M. Diaz-Mitoma F. Kumar A. J. Biol. Chem. 2001; 276: 13664-13674Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar). Purified, nonactivated monocytes were obtained by a negative selection procedure involving depletion of T cells and B cells using magnetic polystyrene M-450 Dynabeads (Dynal, Oslo, Norway) coated with antibodies specific for CD2 (T cells) or CD19 (B cells), as described earlier (28Gee K. Lim W. Ma W. Diaz-Mitoma F. Kozlowski M. Kumar A. J. Immunol. 2002; 169: 5660-5672Crossref PubMed Scopus (56) Google Scholar, 35Ma W. Lim W. Gee K. Aucoin S. Nandan D. Kozlowski M. Diaz-Mitoma F. Kumar A. J. Biol. Chem. 2001; 276: 13664-13674Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar). Briefly, PBMCs (10 × 106/ml) were resuspended with CD2 and CD19 Dynabeads and were incubated for 30 min on ice with constant mixing. Cells were incubated at 37 °C for 2 h, following which nonadherent cells were removed. The adherent mononuclear cells obtained contained fewer than 1% CD2+ T cells and CD19+ B cells as determined by flow cytometry.Measurement of IL-10 and TNF-α Production in Culture Supernatants by Enzyme-linked Immunosorbent Assay—To determine the effects of p38 and p42/44 MAPK inhibitors on CD44-mediated HA binding and IL-10 and TNF-α production, purified human monocytes (1 × 106/ml) and THP-1 cells (0.5 × 106/ml) were stimulated for 24 h with LPS (1 μg/ml) in the presence or the absence of MAPK inhibitors. Cells were analyzed for CD44-mediated HA binding, and the supernatants were analyzed for IL-10 and TNF-α production by enzyme-linked immunosorbent assay. IL-10 and TNF-α were measured by employing two different monoclonal antibodies (mAbs) recognizing distinct epitopes as described earlier (28Gee K. Lim W. Ma W. Diaz-Mitoma F. Kozlowski M. Kumar A. J. Immunol. 2002; 169: 5660-5672Crossref PubMed Scopus (56) Google Scholar, 35Ma W. Lim W. Gee K. Aucoin S. Nandan D. Kozlowski M. Diaz-Mitoma F. Kumar A. J. Biol. Chem. 2001; 276: 13664-13674Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar). Briefly, the following primary antibodies were used for coating the plates (anti-IL-10 mAb 18551D from BD Pharmingen (Missisauga, Canada); final concentration of 5 μg/ml; anti-TNF-α mAb AHC3712 from BIOSOURCE; final concentration of 5 μg/ml). IL-10 or TNF-α were detected using a second biotinylated mAb (anti-IL-10 antibody 18562D from Pharmingen, final concentration of 4 μg/ml; anti-TNF-α antibody AHC3419 from BIOSOURCE, final concentration of 4 μg/ml). Streptavidin peroxidase was used at a final concentration of 1:1000 (Jackson ImmunoResearch, West Grove, PA). The color reaction was developed by 0-phenylenediamine (Sigma) and hydrogen peroxide, and the absorbance was read at 450 nm. IL-10 (R&D Systems) and TNF-α (BIOSOURCE) were used as standards. The level of sensitivity for both IL-10 and TNF-α production was 16 pg/ml.HA Adhesion Assay—CD44-mediated binding assays were performed as described previously (17Kryworuchko M. Diaz-Mitoma F. Kumar A. Exp. Cell Res. 1999; 250: 241-252Crossref PubMed Scopus (17) Google Scholar, 39Gee K. Kozlowski M. Kryworuchko M. Diaz-Mitoma F. Kumar A. Cell. Immunol. 2001; 211: 131-142Crossref PubMed Scopus (15) Google Scholar). Briefly, stimulated cells (2 × 107 cells/ml) were resuspended in Iscove's Dulbecco's medium 10% fetal bovine serum and were pulsed for 1.5 h with 51Cr (sodium chromate; Amersham Biosciences) at a concentration of 300 μCi/106 cells. After three washes, cells were aliquoted at a concentration of 2 × 106 cells/ml into 96-well microtiter plates (NUNC, Imunomodules, Roskilde, Denmark), which had been coated overnight with a 1 mg/ml concentration of either umbilical cord hyaluronan (Sigma) or, as a negative control, chondroitin sulfate C (Calbiochem). Cells were incubated for 1.5 h at 37 °C and were washed six times with warm Iscove's Dulbecco's medium, 10% fetal bovine serum to remove nonadherent cells. The adherent cells were lysed with 1 n HCl, and radioactivity was measured by scintillation counting using a Microbeta counter (Wallac, Turku, Finland). The percentage of adherent cells was determined as follows. x0026;x0026;&x0025;adherentcells(&x0025;cpmtotalinput)x0026;x0026;=cpmadherent-spontaneousreleasecmpTotalinputcpm-spontaneousreleasecpm×100(Eq. 1) Western Blot Analysis—Phosphorylation of p38 or p42/44 ERK MAPKs was determined by Western blot analysis as previously described (28Gee K. Lim W. Ma W. Diaz-Mitoma F. Kozlowski M. Kumar A. J. Immunol. 2002; 169: 5660-5672Crossref PubMed Scopus (56) Google Scholar, 35Ma W. Lim W. Gee K. Aucoin S. Nandan D. Kozlowski M. Diaz-Mitoma F. Kumar A. J. Biol. Chem. 2001; 276: 13664-13674Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar, 39Gee K. Kozlowski M. Kryworuchko M. Diaz-Mitoma F. Kumar A. Cell. Immunol. 2001; 211: 131-142Crossref PubMed Scopus (15) Google Scholar). Briefly, cells were stimulated at 37 °C for 0–15 min with either LPS, IL-10, or TNF-α. Cell pellets were lysed, and the protein concentration was determined by using the Bio-Rad protein determination assay (Bio-Rad). Total cell proteins were subjected to 8% polyacrylamide SDS gel electrophoresis followed by transfer onto polyvinylidene difluoride membranes (Bio-Rad). The membranes were probed with either rabbit anti-phospho-p38 mAb (New England Biolabs, Mississauga, Canada) or mouse anti-phospho-p42/44 mAb (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), followed by horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse polyclonal antibodies (Bio-Rad). The membranes were incubated in a stripping buffer (62.5 mm Tris-HCl, pH 6.7, 100 mm 2-β-mercaptoethanol, 2% SDS, and 0.7 mm dithiothreitol) for 30 min at 50 °C with gentle agitation. The membranes were washed with TBST buffer (150 mm Tris-HCl, 1 m NaCl, and 1% Tween 20) seven times followed by reprobing with rabbit polyclonal antibodies specific for the unphosphorylated forms of either p38 or p42 MAPKs (Santa Cruz Biotechnology). All immunoblots were visualized by ECL (Amersham Biosciences).Determination of Sialidase Activity—Measurement of lysosomal and plasma membrane sialidase activity was performed as described (22Katoh S. Miyagi T. Taniguchi H. Matsubara Y. Kadota J. Tominaga A. Kincade P.W. Matsukura S. Kohno S. J. Immunol. 1999; 162: 5058-5061PubMed Google Scholar). Cells (15 × 106) were washed with phosphate-buffered saline and resuspended in ice-cold buffer containing 0.25 m sucrose, 1 mm EDTA, and 0.2 mm phenylmethylsulfonyl fluoride. The cell suspension was sonicated on ice for 10 s on a low setting (7% amplitude) (Vibracell™; Sonics and Materials Inc., Newtown, CT) followed by centrifugation at 25,000 × g for 15 min at 4 °C. The resulting supernatant was used to determine the lysosmal sialidase activity. Protein quantification of the supernatant was performed using the Bio-Rad protein determination kit as described above. For the determination of lysosomal sialidase activity, 200 μg of total protein was mixed with 40 nmol of 4-methylumbelliferyl-α-N-acetyl-d-neuraminic acid (Sigma), the lysosmal sialidase-specific substrate, 10 μmol sodium acetate buffer, pH 4.6, and 200 μg of bovine serum albumin in a total volume of 200 μl. The sialidase reaction was allowed to proceed for 1 h at 37 °C and was terminated by the addition of 0.25 m glycine NaOH, pH 10.4. Released 4-methylumbelliferyl-α-N-acetyl-d-neuraminic acid was measured fluorometrically (PerkinElmer Life Sciences) in a sodium carbonate buffer at an excitation wavelength of 365 nm and emission wavelength of 448 nm as described previously (40Miyagi T. Tsuiki S. J. Biol. Chem. 1985; 260: 6710-6716Abstract Full Text PDF PubMed Google Scholar). One unit of sialidase was defined as the amount of enzyme that catalyzed the release of 1 nmol of sialic acid/h. The measurement of lysosmal sialidase activity was optimized using various concentrations of total protein (25, 50, 100, 150, and 200 μg). The maximal sialidase activity was detected when 200 μg of total cellular proteins was used. Furthermore, the optimal incubation time for sialidase reaction was determined to be 1 h.Lysosomal sialidase activity was determined in THP-1 cells stimulated with LPS or TNF-α for varying periods of time. The sialidase activity for these experiments was expressed as a measure of fluorescence intensity. To determine the involvement of MAPKs in the LPS- or TNF-α-induced lysosomal sialidase activity, cells were treated with MAPK inhibitors (SB202190 or PD98059) at concentrations ranging from 5 to 50 μm for 2 h prior to stimulation with either LPS or TNF-α for 16 h. The cells were harvested and analyzed for sialidase activity. In order to clearly illustrate the changes observed upon treatment with the inhibitors, the inhibition of sialidase activity by SB202190 or PD98059 was measured as a percentage change (Δ) of the sialidase activity (units) with respect to that observed with LPS- or TNF-α-stimulated cells. This was calculated as follows. Sialidaseactivity(units)incellstreatedwithMAPKinhibitor±LPSorTNF-αSialidaseactivity(units)incellstreatedwithLPSorTNF-αalone×100(Eq. 2) For the determination of plasma membrane sialidase activity, 200 μg of total cellular protein were mixed with 60 mmol of bovine
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