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CD45-induced Tumor Necrosis Factor α Production in Monocytes Is Phosphatidylinositol 3-Kinase-dependent and Nuclear Factor-κB-independent*

Open AccessPublished:November 19, 1999DOI:https://doi.org/10.1074/jbc.274.47.33455
      The pro-inflammatory cytokine tumor necrosis factor (TNF)-α plays a pivotal role in the pathogenesis of rheumatoid arthritis. The mechanisms involved in regulating monocyte/macrophage TNFα production are not yet fully understood but are thought to involve both soluble factors and cell/cell contact with other cell types. Ligation of certain cell surface receptors, namely CD45, CD44, and CD58, can induce the production of TNFα in monocytes. In this paper, we investigate further the signaling pathways utilized by cell surface receptors (specifically CD45) to induce monocyte TNFα and compare the common/unique pathways involved with that of lipopolysaccharide. The results indicate that monocyte TNFα induced upon CD45 ligation or lipopolysaccharide stimulation is differentially modulated by phosphatidylinositol 3-kinase and nuclear factor-κB but similarly regulated by p38 mitogen-activated protein kinase. These results demonstrate that both common and unique signaling pathways are utilized by different stimuli for the induction of TNFα. These observations may have a major bearing on approaches to inhibiting TNFα production in disease where the cytokine has a pathogenic role.
      LPS
      lipopolysaccharide
      TNFα
      tumor necrosis factor α
      IL
      interleukin
      PKC
      protein kinase C
      MAPK
      mitogen-activated protein kinase
      JNK
      Jun-N-terminal kinase
      M-CSF
      macrophage colony-stimulating factor
      PI3K
      phosphatidylinositol 3-kinase
      PKB
      protein kinase B
      mAb
      monoclonal antibody
      ELISA
      enzyme-linked immunosorbent assay
      NF-κB
      nuclear factor-κB
      PLA2
      phospholipase A2
      IκB
      inhibitor of κB
      NFAT
      nuclear factor of activated T cells
      CsA
      cyclosporin A
      MOPS
      4-morpholinepropanesulfonic acid
      Lipopolysaccharide (LPS)1 is one of the most potent activators of monocytes/macrophages, resulting in the triggering of a range of cellular responses and the secretion of pro- and anti-inflammatory cytokines, including TNFα, interleukin-1 (IL-1) and IL-6 (
      • Cavaillon J.M.
      • Haeffner-Cavaillon N.
      ,
      • Cavaillon J.M.
      • Fitting C.
      • Haeffner-Cavaillon N.
      • Kirsch S.J.
      • Warren H.S.
      ,
      • Trinchieri G.
      ,
      • Goldfeld A.E.
      • Doyle C.
      • Maniatis T.
      ). LPS, following interaction with serum proteins,e.g. LPS-binding protein and the cell surface receptor, CD14 (
      • Wright S.D.
      • Ramos R.A.
      • Tobias P.S.
      • Ulevitch R.J.
      • Mathison J.C.
      ), activates a number of signaling pathways. These include various tyrosine kinases (
      • Geng Y.
      • Zhang B.
      • Lotz M.
      ,
      • Beaty C.D.
      • Franklin T.L.
      • Uehara Y.
      • Wilson C.B.
      ), protein kinase C (PKC) (
      • Shapira L.
      • Takashiba S.
      • Champagne C.
      • Amar S.
      • Van-Dyke T.E.
      ), the mitogen-activated protein kinases (MAPK) including p38 (
      • Lee J.C.
      • Young P.R.
      ), p44/42 (extracellular signal-regulated kinase) (
      • Liu M.K.
      • Herrera-Velit P.
      • Brownsey R.W.
      • Reiner N.E.
      ), and p54 (stress-activated protein kinase/JNK) (
      • Hambleton J.
      • Weinstein S.L.
      • Lem L.
      • DeFranco A.L.
      ).
      Numerous studies have shown that direct contact between monocytes or monocytic cell lines and prestimulated T cells leads to production of cytokines including, IL-1β, TNFα, IL-12, and IL-10 (
      • Isler P.
      • Vey E.
      • Zhang J.H.
      • Dayer J.M.
      ,
      • Manie S.
      • Kubar J.
      • Limouse M.
      • Ferrua B.
      • Ticchioni M.
      • Breittmayer J.P.
      • Peyron J.F.
      • Schaffar L.
      • Rossi B.
      ,
      • Shu U.
      • Kiniwa M.
      • Wu C.Y.
      • Maliszewski C.
      • Vezzio N.
      • Hakimi J.
      • Gately M.
      • Delespesse G.
      ,
      • Wagner Jr., D.H.
      • Stout R.D.
      • Suttles J.
      ,
      • Sebbag M.
      • Parry S.L.
      • Brennan F.M.
      • Feldmann M.
      ,
      • Parry S.L.
      • Sebbag M.
      • Feldmann M.
      • Brennan F.M.
      ). A variety of T cell-associated cell surface receptors/ligands including CD69, CD40L, CD11b, and CD2 are thought to be important in modulating this monocyte cytokine production (
      • Manie S.
      • Kubar J.
      • Limouse M.
      • Ferrua B.
      • Ticchioni M.
      • Breittmayer J.P.
      • Peyron J.F.
      • Schaffar L.
      • Rossi B.
      ,
      • Shu U.
      • Kiniwa M.
      • Wu C.Y.
      • Maliszewski C.
      • Vezzio N.
      • Hakimi J.
      • Gately M.
      • Delespesse G.
      ,
      • Wagner Jr., D.H.
      • Stout R.D.
      • Suttles J.
      ). Furthermore, direct engagement of certain cell surface receptors, namely CD44, CD58, and CD45, on monocytes induce TNFα production (
      • Webb D.S.A.
      • Shimizu Y.
      • Van Seventer G.A.
      • Shaw S.
      • Gerrard T.L.
      ,
      • Gruber M.F.
      • Williams C.C.
      • Gerrard T.L.
      ), suggesting that receptor engagement may be important in the regulation of cytokines. Potential ligands for CD44 (osteopontin) (
      • Weber G.F.
      • Ashkar S.
      • Glimcher M.J.
      • Cantor H.
      ) and CD58 (CD2) (
      • Dustin M.L.
      • Springer T.A.
      ) are expressed by activated T cells, whereas the ligand for CD45 still remains to be fully clarified, although the B cell adhesion molecule, CD22 (
      • Sgroi D.
      • Koretzky G.A.
      • Stamenkovic I.
      ), and the β-galactosidase-binding protein, galectin-1 (
      • Perillo N.L.
      • Pace K.E.
      • Seilhamer J.J.
      • Baum L.G.
      ), have been proposed to bind to specific isoforms of CD45.
      CD45 is a membrane-anchored protein-tyrosine phosphatases found exclusively on all nucleated hemapoietic cells (
      • Clark E.A.
      • Ledbetter J.A.
      ,
      • Trowbridge I.S.
      • Ostergaard H.
      • Johnson P.
      ). The role of CD45 in T cells has been the subject of much investigation and has been shown to play an important co-stimulatory role in intracellular signal transduction in T lymphocytes (
      • Ledbetter J.A.
      • Tonks N.K.
      • Fischer E.H.
      • Clark E.A.
      ,
      • Justement L.B.
      • Campbell K.S.
      • Chien N.C.
      • Cambier J.C.
      ,
      • Mustelin T.
      • Coggeshall K.M.
      • Altman A.
      ,
      • Pingel J.T.
      • Thomas M.L.
      ,
      • Benatar T.
      • Carsetti R.
      • Furlonger C.
      • Kamalia N.
      • Mak T.
      • Paige C.J.
      ,
      • Byth K.F.
      • Conroy L.A.
      • Howlett S.
      • Smith A.J.
      • May J.
      • Alexander D.R.
      • Holmes N.
      ). While ligation of CD45 on monocytes has been shown to induce synthesis of cytokines, including TNFα, IL-1β, and macrophage-colony stimulating factor (M-CSF) (
      • Webb D.S.A.
      • Shimizu Y.
      • Van Seventer G.A.
      • Shaw S.
      • Gerrard T.L.
      ,
      • Gruber M.F.
      • Williams C.C.
      • Gerrard T.L.
      ), the signaling mechanisms involved and the functional relevance of CD45 on monocyte/macrophages remain unclear.
      We have investigated the signaling pathways utilized upon CD45 ligation on monocytes leading to TNFα production and compared this with the conventional stimulus, LPS. We demonstrate that CD45 ligation (but not LPS) activates the phosphatidylinositol 3-kinase (PI3K) pathway and that inhibitors of PI3K activation block CD45- but not LPS-induced TNFα synthesis. The differences in signaling also extended to nuclear factor-κB (NF-κB), which, unlike LPS, was not required by CD45-induced TNFα synthesis. In contrast, CD45, like LPS, activated p38 MAPK.

      DISCUSSION

      In this paper we investigated the signaling pathway(s) involved in monocyte TNFα production following ligation of the cell surface receptor CD45 or LPS. Our results reveal the unexpected finding that CD45 ligation results in TNFα production that is dependent upon the activation of PI3 kinase but independent of the transcription factor NF-κB. In contrast, LPS-induced TNFα production was dependent upon NF-κB activation as previously reported (
      • Foxwell B.
      • Browne K.
      • Bondeson J.
      • Clarke C.
      • de Martin C.
      • Brennan F.
      • Feldmann M.
      ) while PI3K-independent. These observations indicate that, while NF-κB has previously been shown to be important in TNFα production, it is not always necessary/required.
      The importance of the cell surface receptor, CD45 in the activation of T and B cell antigen receptor-mediated signaling pathways and subsequent cellular responses has been well documented. Engagement of CD45 is known to regulate Src tyrosine kinases (p59 fyn, p56 lck, and p70 zap ) phosphorylation (
      • Biffen M.
      • McMichael-Phillips D.
      • Larson T.
      • Venkitaraman A.
      • Alexander D.
      ,
      • Mustelin T.
      • Pessa-Morikawa T.
      • Autero M.
      • Gassmann M.
      • Andersson L.C.
      • Gahmberg C.G.
      • Burn P.
      ), phospholipase Cγ1 regulation (
      • Kanner S.B.
      • Ledbetter J.A.
      ), inositol phosphate production (
      • Weiss A.
      • Imboden J.B.
      ), diacylglycerol production, PKC activation, and calcium mobilization (
      • Shiroo M.
      • Goff L.
      • Biffen M.
      • Shivnan E.
      • Alexander D.
      ). Ligation of CD45 has previously been shown to induce production of cytokines in monocytes (
      • Webb D.S.A.
      • Shimizu Y.
      • Van Seventer G.A.
      • Shaw S.
      • Gerrard T.L.
      ,
      • Gruber M.F.
      • Williams C.C.
      • Gerrard T.L.
      ); however, the signaling pathways utilized upon CD45 ligation in monocytes have received little attention.
      Ligation of monocyte CD45 results in activation of PI3K and the known downstream effectors PKB and p70 S6K. We have shown the anti-CD45-induced monocyte TNFα production is inhibited by the PI3K inhibitors, wortmannin and LY294002. However the inhibitor of p70 S6K activation, rapamycin, did not inhibit anti-CD45-induced TNFα production. These findings suggest that TNFα production is p70 S6K-independent and other, as yet unidentified, downstream components of PI3K pathway are involved.
      In contrast, wortmannin but not LY294002 enhanced LPS-induced monocyte TNFα production, suggesting that the effects observed with wortmannin are not specific to PI3K activation. Wortmannin has other targets including PLA2 (
      • Cross M.J.
      • Stewart A.
      • Hodgkin M.N.
      • Kerr D.J.
      • Wakelam M.J.
      ), and we have shown that the PLA2 inhibitor, AKTA, also enhances LPS-induced TNFα production in monocytes,3 suggesting that the effect of wortmannin on LPS-induced TNFα production may be due to PLA2 inhibition. How PLA2 negatively regulates TNFα production is unclear, but this enzyme is required for synthesis of PGE2, an inhibitor of TNFα production (
      • Rola-Pleszczynski M.
      • Thivierge M.
      • Gagnon N.
      • Lacasse C.
      • Stankova J.
      ). Wortmannin is known to stimulate the stress-activated protein kinase pathway (
      • Kharbanda S.
      • Saleem A.
      • Shafman T.
      • Emoto Y.
      • Taneja N.
      • Rubin E.
      • Weichselbaum R.
      • Woodgett J.
      • Avruch T.
      • Kyriakis J.
      • Kufe D.
      ), and this may also have a positive effect on TNFα production. Furthermore, we observed only a weak increase in PI3K and p70 S6K activity following LPS stimulation, suggesting that neither of these pathways play a major role in LPS-mediated events in monocytes. These findings contradict with those performed by Herrera et al.(
      • Herrera-Velit P.
      • Reiner N.E.
      ), in which LPS was demonstrated to induce PI3K activity in monocytes, using similar methods to those described here. The reason for these apparently contradicting findings remain unclear. These studies have focused upon class 1A PI3Ks, specifically those involving the p85α subunit and the involvement of other PI3K subclasses including those regulated by G-proteins and those which are wortmannin-insensitive have not been investigated.
      p70 S6K and PKB are known downstream effectors of PI3K (
      • Burgering B.M.
      • Coffer P.J.
      ,
      • Monfar M.
      • Lemon K.P.
      • Grammer T.C.
      • Cheatham L.
      • Chung J.
      • Vlahos C.J.
      • Blenis J.
      ,
      • Franke T.F.
      • Kaplan D.R.
      • Cantley L.C.
      • Toker A.
      ,
      • Pai S.-Y.
      • Calvo V.
      • Wood M.
      • Bierer B.E.
      ); however, our studies indicate that CD45-induced TNFα production in monocytes is p70 S6K-independent. Furthermore, while ligation of CD45 induces phosphorylation of PKB, the involvement of PKB in monocyte TNFα production at this stage cannot be verified due to the lack of specific PKB inhibitors. These findings indicate that there must be a bifurcation of the signaling pathways downstream of PI3K that regulate TNFα production. Several signaling molecules have been shown to directly and/or indirectly regulate PI3K, leading to the activation of transcription factors, e.g. atypical PKCζ and PKCλ. (
      • Akimoto K.
      • Takahashi R.
      • Moriya S.
      • Nishioka N.
      • Takayanagi J.
      • Kimura K.
      • Fukui Y.
      • Osada S.
      • Mizuno K.
      • Hirai S.
      • Kazlauskas A.
      • Ohno S.
      ). Unfortunately, inhibitors of PKC were found to be toxic to monocytes and as such the involvement of PKC in anti-CD45-induced TNFα production has not been assessed. Other potential downstream effectors include Rac, Rab5 (
      • Hawkins P.T.
      • Eguinoa A.
      • Qiu R.G.
      • Stokoe D.
      • Cooke F.T.
      • Walters R.
      • Wennstrom S.
      • Claesson-Welsh L.
      • Evans T.
      • Symons M.
      • et al.
      ,
      • Goldfeld A.E.
      • Strominger J.L.
      • Doyle C.
      ), Bruton's tyrosine kinase (
      • Tamagnone L.
      • Lahtinen I.
      • Mustonen T.
      • Virtaneva K.
      • Francis F.
      • Muscatelli F.
      • Alitalo R.
      • Smith C.I.
      • Larsson C.
      • Alitalo K.
      ,
      • Qui Y.
      • Robinson D.
      • Pretlow T.G.
      • Kung H.-J.
      ), and JNK/stress-activated protein kinase (
      • Klippel A.
      • Reinhard C.
      • Kavanaugh W.M.
      • Apell G.
      • Escobedo M.A.
      • Williams L.T.
      ,
      • Logan S.K.
      • Falasca M.
      • Hu P.
      • Schlessinger J.
      ). The involvement of these molecules in PI3K-dependent TNFα production still remains to be determined.
      Several studies have suggested that LPS-induced TNFα production in monocytes/macrophages is NF-κB-dependent. Protease inhibitors, gliotoxin, and free radical scavengers have all been used to block NF-κB activity; however, the lack of specificity of these reagents remains a constant problem. More recently, the over expression of IκBα following adenoviral infection (AdvIκBα) has been demonstrated to inhibit LPS-induced TNFα production in monocytes (
      • Foxwell B.
      • Browne K.
      • Bondeson J.
      • Clarke C.
      • de Martin C.
      • Brennan F.
      • Feldmann M.
      ). Curiously, we demonstrated that ligation of CD45 induced IκBα degradation (results not shown) but only a weak NF-κB binding activity; the reasons for this remain unclear, although it suggests further complexity of the NF-κB system. Overexpression of AdvIκBα did not inhibit anti-CD45-induced TNFα production. These findings indicate that other, as yet unidentified, transcription factors are involved in anti-CD45-induced TNFα production monocytes.
      In T cells, induction of TNFα gene expression is regulated by the nuclear factor of activated T cells (NFAT), not NF-κB (
      • Goldfeld A.E.
      • Strominger J.L.
      • Doyle C.
      ,
      • Goldfeld A.E.
      • Tsai E.
      • Kincaid R.
      • Belshaw P.J.
      • Schrieber S.L.
      • Strominger J.L.
      • Rao A.
      ,
      • Tsai E.Y.
      • Yie J.
      • Thanos D.
      • Goldfeld A.E.
      ). NFAT binds to the κ3 element of the TNFα gene (located −97 and −88 nucleotides relative to the TNFα start site), in association with ATF-2 and c-Jun proteins, which bind to the cyclic AMP response element site (
      • Goldfeld A.E.
      • McCaffrey P.G.
      • Strominger J.L.
      • Rao A.
      ). NFAT DNA binding activity in activated T cells is prevented by the immunosuppressive drugs cyclosporin A (CsA), and FK506 (
      • Mattila P.S.
      • Ullman K.S.
      • Fiering S.
      • Emmel E.A.
      • McCutcheon M.
      • Crabtree G.R.
      • Herzenberg L.A.
      ,
      • Brabletz T.
      • Pietrowski I.
      • Serfling E.
      ,
      • Tsai E.Y.
      • Yie J.
      • Thanos D.
      • Goldfeld A.E.
      ). CsA and FK506 form complexes with their intracellular receptors (immunophilins), and inhibit the activity of calcineurin (protein phosphatase 2B), a ubiquitous calcium- and calmodulin-dependent phosphatase (reviewed in Ref.
      • Liu J.
      ). Induction of TNFα mRNA gene transcription in T cells can be blocked by CsA and FK506 (
      • Goldfeld A.E.
      • McCaffrey P.G.
      • Strominger J.L.
      • Rao A.
      ), and expression of calcineurin is sufficient to activate a reporter gene whose transcription is driven by the TNFα promoter (
      • Goldfeld A.E.
      • Tsai E.
      • Kincaid R.
      • Belshaw P.J.
      • Schrieber S.L.
      • Strominger J.L.
      • Rao A.
      ). The involvement of NFAT in monocyte TNFα production remains to be confirmed. However, CsA and FK506 failed to inhibit anti-CD45-induced TNFα production in monocytes (results not shown), but this does not discount the involvement of CsA-insensitive NFAT in the regulation of monocyte TNFα production. These findings suggest that NFAT, like NF-κB, is not required for anti-CD45-induced TNFα production.
      LPS has previously been shown to activate the three major mammalian MAPK pathways, p42/44 (extracellular signal-regulated kinases 1/2), p38, and p54 MAPK (stress-activated protein kinase), in monocyte/macrophages (
      • Lee J.C.
      • Young P.R.
      ,
      • Liu M.K.
      • Herrera-Velit P.
      • Brownsey R.W.
      • Reiner N.E.
      ,
      • Hambleton J.
      • Weinstein S.L.
      • Lem L.
      • DeFranco A.L.
      ). However, the relationship between the activation of these signaling molecules cytokine expression remains to be clarified. p38 MAPK is the only kinase that has been shown to play a pivotal role in the production of TNFα (
      • Lee J.C.
      • Laydon J.T.
      • McDonnell P.C.
      • Gallagher T.F.
      • Kumar S.
      • Green D.
      • McNulty D.
      • Blumenthal M.J.
      • Heys J.R.
      • Landvatter S.W.
      • et al.
      ). Previous studies have suggested that the post-transcriptional regulation of TNFα is mediated through adenosine-uridine (AU)-rich elements present within the 3′-untranslated region of the TNFα mRNA (
      • Kruys V.
      • Marinx O.
      • Shaw G.
      • Deschamps J.
      • Huez G.
      ). Deletion of this region leads to the constitutive synthesis of TNFα in cell lines (
      • Kruys V.
      • Kemmer K.
      • Shakhov A.
      • Jongeneel V.
      • Beutler B.
      ) and transgenic animals (
      • Keffer J.
      • Probert L.
      • Cazlaris H.
      • Georgopoulos S.
      • Kaslaris E.
      • Kioussis D.
      • Kollias G.
      ). TNFα reporter gene constructs that do not contain the 3′-AU-rich element regions lose their sensitivity to inhibition by the p38 inhibitor, SB203580, and it has been suggested that the p38 MAPK cascade is mediating the release of translational repression of TNFα (
      • Lee J.C.
      • Laydon J.T.
      • McDonnell P.C.
      • Gallagher T.F.
      • Kumar S.
      • Green D.
      • McNulty D.
      • Blumenthal M.J.
      • Heys J.R.
      • Landvatter S.W.
      • et al.
      ). The pyridinyl imidazole compound, SB203580, has been used to determine the involvement of p38 MAPK in the regulation of numerous pro-inflammatory cytokines including IL-1, IL-6, and TNFα (
      • Lee J.C.
      • Young P.R.
      ). Recently, SB203580 has been shown to inhibit TNFα protein and mRNA induced by LPS, suggesting that TNFα is being inhibited at the pre-translational level (
      • Dean J.L.
      • Brook M.
      • Clark A.R.
      • Saklatvala J.
      ,
      • Saklatvala J.
      • Dean J.
      • Finch A.
      ). We have demonstrated that monocyte TNFα production is regulated by distinct transcriptional mechanisms. Furthermore, we have demonstrated that both LPS- and anti-CD45-induced TNFα production is regulated by p38 MAPK suggesting that both stimuli utilize similar translational mechanisms to regulate TNFα production. We observed that ligation of CD45 resulted in activation of the MAPKs p38 and p42/p44 (results not shown) with similar kinetics to that observed with LPS. Furthermore, inhibitors of p38 MAPK (SB203580) and p42/44 MAPK (PD98059) (results not shown) were shown to block both anti-CD45 and LPS-induced TNFα production. At higher concentrations SB203580 is known to inhibit the activity of JNK2 and JNK3 (
      • Whitmarsh A.J.
      • Yang S.H.
      • Su M.S.
      • Sharrocks A.D.
      • Davis R.J.
      ); however, the IC50 values observed for SB203580 inhibition of anti-CD45- and LPS-induced monocyte TNFα synthesis are consistent with its effects on p38 MAPK and not JNK, although the nonspecific actions of this drugs cannot be disregarded. These findings indicate that TNFα production is regulated by distinct transcriptional signaling mechanisms, while the translational mechanisms appear to be identical.
      In summary, this study demonstrates that TNFα production in monocytes is regulated by multiple signaling pathways. The initiating signals for TNFα production in inflammatory disorders such as rheumatoid arthritis are unknown. However, these findings suggest that engagement of specific cell surface receptors may be important in regulating TNFα production via distinct signaling pathways and investigation of these mechanisms in both physiological and pathological systems is currently being investigated.
      F. M. Brennan, A. L. Mayes, C. J. Ciesielski, P. Green, B. M. J. Foxwell, and M. Feldman, manuscript in preparation.

      Acknowledgments

      We thank Dr. C. Ciesielski for assistance with adenoviral infection of monocytes.

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