Cyclosporine A Enhances Leukocyte Binding by Human Intestinal Microvascular Endothelial Cells through Inhibition of p38 MAPK and iNOS

The calcineurin inhibitor cyclosporine A (CsA) modulates leukocyte cytokine production but may also effect nonimmune cells, including microvascular endothelial cells, which regulate the inflammatory process through leukocyte recruitment. We hypothesized that CsA would promote a proinflammatory phenotype in human intestinal microvascular endothelial cells (HIMEC), by inhibiting inducible nitric-oxide synthase (iNOS, NOS2)-derived NO, normally an important mechanism in limiting endothelial activation and leukocyte adhesion. Primary cultures of HIMEC were used to assess CsA effects on endothelial activation, leukocyte interaction, and the expression of iNOS as well as cell adhesion molecules. CsA significantly increased leukocyte binding to activated HIMEC, but paradoxically decreased endothelial expression of cell adhesion molecules (E-selectin, intercellular adhesion molecule 1, and vascular cell adhesion molecule-1). In contrast, CsA completely inhibited the expression of iNOS in tumor necrosis factor-α/lipopolysaccharide-activated HIMEC. CsA blocked p38 MAPK phosphorylation in activated HIMEC, a key pathway in iNOS expression, but failed to inhibit NFκB activation. These studies demonstrate that CsA exerts a proinflammatory effect on HIMEC by blocking iNOS expression. CsA exerts a proinflammatory effect on the microvascular endothelium, and this drug-induced endothelial dysfunction may help explain its lack of efficacy in the long-term treatment of chronically active inflammatory bowel disease. The calcineurin inhibitor cyclosporine A (CsA) modulates leukocyte cytokine production but may also effect nonimmune cells, including microvascular endothelial cells, which regulate the inflammatory process through leukocyte recruitment. We hypothesized that CsA would promote a proinflammatory phenotype in human intestinal microvascular endothelial cells (HIMEC), by inhibiting inducible nitric-oxide synthase (iNOS, NOS2)-derived NO, normally an important mechanism in limiting endothelial activation and leukocyte adhesion. Primary cultures of HIMEC were used to assess CsA effects on endothelial activation, leukocyte interaction, and the expression of iNOS as well as cell adhesion molecules. CsA significantly increased leukocyte binding to activated HIMEC, but paradoxically decreased endothelial expression of cell adhesion molecules (E-selectin, intercellular adhesion molecule 1, and vascular cell adhesion molecule-1). In contrast, CsA completely inhibited the expression of iNOS in tumor necrosis factor-α/lipopolysaccharide-activated HIMEC. CsA blocked p38 MAPK phosphorylation in activated HIMEC, a key pathway in iNOS expression, but failed to inhibit NFκB activation. These studies demonstrate that CsA exerts a proinflammatory effect on HIMEC by blocking iNOS expression. CsA exerts a proinflammatory effect on the microvascular endothelium, and this drug-induced endothelial dysfunction may help explain its lack of efficacy in the long-term treatment of chronically active inflammatory bowel disease. The calcineurin inhibitor cyclosporine A (CsA) 1The abbreviations used are: CsA, cyclosporine A; HIMEC, human intestinal microvascular endothelial cells; NO, nitric oxide; NOS, nitric-oxide synthase; VCAM-1, vascular cell adhesion molecule-1; ICAM-1, intercellular adhesion molecule-1; MAPK, mitogen-activated protein kinases; SAPK, stress-activated protein kinase; JNK, c-Jun NH2-terminal kinase; ERK, extracellular signal-regulated kinase; TNF-α, tumor necrosis factor α; LPS, lipopolysaccharide; NFκB, nuclear factor κB; IBD, inflammatory bowel disease; DCF-DA, dichlorodihydrofluorescein diacetate; l-NMMA, NG-monomethyl-l-arginine; l-NIL, N-iminoethyl-l-lysine; PEG-SOD, polyethylene glycol-conjugated superoxide dismutase; ROS, reactive oxygen species; PBS, phosphate-buffered saline; CAM, cell adhesion molecule; iNOS, inducible nitric-oxide synthase. is a potent immunosuppressive agent that has formed the pharmacologic cornerstone of solid organ transplantation. CsA prevents the activation of lymphokine genes essential for T cell proliferation by disrupting calcium-dependent signal transduction pathways in leukocytes. Although pharmacologic studies of CsA have focused primarily on T cell responses, there is emerging evidence that this agent may exert potent effects on blood vessels, where it disturbs production of nitric oxide (NO) ultimately promoting arterial hypertension, inducing long-term vascular dysfunction, and contributing to obliterative vasculopathy in chronic transplant rejection (1Gao S.Z. Schroeder J.S. Alderman E.L. Hunt S.A. Valantine H.A. Wiederhold V. Stinson E.B. Circulation. 1989; 80: 100-105Google Scholar, 2Textor S.C. Canzanello V.J. Taler S.J. Wilson D.J. Schwartz L.L. Augustine J.E. Raymer J.M. Romero J.C. Wiesner R.H. Krom R.A. Mayo Clin. Proc. 1994; 69: 1182-1193Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 3Ventura H.O. Malik F.S. Mehra M.R. Stapleton D.D. Smart F.W. Curr. Opin. 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However, the effect of CsA on microvascular endothelial activation has We the effect of CsA on the activation of human intestinal microvascular endothelial cells (HIMEC), the microvascular that leukocyte during chronic intestinal inflammation in We hypothesized that CsA would exert a effect on HIMEC, through effects on during inflammatory activation, to a of NO production and leukocyte adhesion. this cultures of HIMEC to assess the effect of CsA on We focused on the effect of CsA on mechanisms that regulate the inflammatory activation of HIMEC, the expression of iNOS and cell adhesion as well as the signal transduction pathways that underlie inflammatory activation of The of this demonstrate that CsA the production of NO in activated HIMEC, normally a key mechanism for limiting endothelial activation by tumor necrosis and CsA inhibited signal transduction pathways during HIMEC activation, blocking activation of p38 MAPK, a mechanism for iNOS J. J. Biol. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar), ultimately in a proinflammatory effect of this normally powerful immunosuppressive for HIMEC were from bowel The of human tissues by the of the of HIMEC from microvascular M. R. In Biol. PubMed Scopus Google Scholar, K. S.N. Gastroenterology. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). In the and of intestinal were were in and were to to of microvascular endothelial cells, which were and in HIMEC and endothelial cell microvascular endothelial cell were and a Endothelial cultures were by and expression of factor R.A. S. J. Invest. 1992; Full Text PDF PubMed Google Scholar). HIMEC were cells and HIMEC from intestinal from were for this cells, a human cell from a were from and in and The cells underwent and were used and in CsA to the endothelial cultures for a of were including and The of signal transduction pathways to HIMEC activation and expression were defined p38 MAPK inhibitor MAPK inhibitor which the and the and the inhibitor of NFκB activation were to endothelial the of as of nitric-oxide In a of which iNOS S. R.A. S.C. Wilson Am. J. Physiol. Google Scholar). glycol-conjugated superoxide to HIMEC to inhibit production of superoxide during the activation and to the HIMEC for to and during the activation of adhesion to HIMEC were as K. S.N. Gastroenterology. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). In endothelial cells were HIMEC and to in the endothelial cells were a of and of activation, HIMEC were and cells were on endothelial and to in a a cells were and cells were phosphate-buffered by of the were and a and were in an as of In to the adhesion an adhesion used to assess HIMEC activation and The endothelial on the of and S.M. 1992; Full Text PDF PubMed Scopus Google Scholar), which a of the endothelial Endothelial cells were HIMEC and to Endothelial cells were a of and were and in a S.M. 1992; Full Text PDF PubMed Scopus Google Scholar, M.B. PubMed Google Scholar). cells of HIMEC a of were the endothelial and to and adhesion a to an for were by the of a and adhesion as of iNOS expression in and activated cultures of HIMEC, CsA and pharmacologic Endothelial cells were a of and for and by of in a of of and of and In the iNOS were in the as an for the of the and the of used in the of a step an step and an step a of by an step The for human iNOS were on A. J. Biol. 1992; Full Text PDF PubMed Google Scholar, Wood 1993; PubMed Scopus Google Scholar). The and were as and were on and and HIMEC were in the of and were NO by to NO and of a NO and activated were and were as of NO were as the of NO production of endothelial cell protein of reactive oxygen a V.J. K. 1995; Scopus Google Scholar). of HIMEC were on which in and Endothelial cells were in the of CsA to cells were a that into is by cellular to the which in the of reactive oxygen and is to the of HIMEC were and on a of HIMEC were an a of to for HIMEC were cells well and for in of a Endothelial were activation. HIMEC were CsA as of human ICAM-1, and were used to HIMEC expression of cell adhesion molecules. a adhesion molecule were and for to cell extensive to to the R. de J. Clin. Invest. 1994; PubMed Scopus Google Scholar). HIMEC were in and in a from were as the a were in and were a of treatment to the cell and were of protein were by and to J. Proc. Natl. Acad. Sci. U. S. A. 76: PubMed Scopus Google Scholar). The were blocked and MAPK New and iNOS as by to and in used to the cell of protein in of in of PBS, and for were to protein and the the were by the were and the were of were to the as of cellular protein from HIMEC were of MAPK the in of the and of protein for The by of treatment and were by and were by of cellular protein from HIMEC were of the the and of protein as a for phosphorylation by as were as A. V. K. Malik J. Biol. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). HIMEC were in A and of and were and the in A of and of and were to the The in of and of and were on for of to the were as were an NFκB for in The were on were in for in were to to NFκB nuclear HIMEC were on to HIMEC were activated as for were for in PBS, and blocked in for by a and were and a were and for were were as multiple were of used as by the Primary cultures of HIMEC cells were used to assess the effect of CsA on endothelial activation, and to the that CsA would a proinflammatory effect leukocyte binding in the endothelial of HIMEC used to assess endothelial in including their ability to activation and binding of through of cell adhesion molecules. cells were used as a leukocyte for as this cell is to the cell adhesion molecules and which binding to their endothelial and on activated HIMEC. 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HIMEC were CsA, and activated there a but in the of expression of These were the in leukocyte by CsA in activated HIMEC. These that mechanisms of expression the leukocyte binding and proinflammatory effect of CsA on activated HIMEC. A mechanism endothelial activation and homeostasis nitric oxide production from increased expression of the inducible nitric-oxide synthase (iNOS, We have demonstrated that NO activation cytokine and activation of HIMEC S. R.A. S.C. Wilson Am. J. Physiol. Google Scholar). the effect of CsA on HIMEC NO production and its were adhesion and CsA in the and of the as an for the multiple and prevents NO production from in to HIMEC, there a in HIMEC HIMEC CsA activated demonstrated leukocyte binding to the effect of and CsA were to HIMEC to activation, there effect S. R.A. S.C. Wilson Am. J. Physiol. Google of the inhibitor in CsA effects to l-NMMA, in leukocyte binding that NO the of NO production in activated HIMEC. Because the effect of CsA on activated HIMEC to that of the inhibitor l-NMMA, which is to NO to the effect of CsA on NO production by activated HIMEC We have that HIMEC of NO, which activation. of of NO S. R.A. S.C. Wilson Am. J. Physiol. Google Scholar), where the NO and are the of NO of HIMEC CsA to activation for demonstrated a of increased NO production These that CsA would NO production in activated HIMEC. Because iNOS a central role in the of NO in activated HIMEC, focused on the effect of CsA on HIMEC activation and iNOS expression. demonstrated that CsA a effect on HIMEC during activation, but on the endothelial the effect of CsA on HIMEC activation, focused on the MAPK which have in of multiple cells including endothelial We defined the that activation in HIMEC, of cell of the MAPK for MAPK p38 MAPK, and of signal transduction molecules phosphorylation in HIMEC activation. demonstrated that p38 MAPK underwent a and activation, which MAPK activation and iNOS expression inflammatory an role in the of HIMEC activation. of CsA on the expression of iNOS activation of HIMEC in a signal for the iNOS and CsA inhibited expression of iNOS were to the role of MAPK in the expression of iNOS The p38 MAPK inhibitor completely inhibited the expression of iNOS in activated HIMEC, to the effect of The MAPK inhibitor inhibited HIMEC iNOS expression These on signal transduction pathways the expression of iNOS in HIMEC that p38 MAPK the role in expression, and the effect of CsA to the p38 MAPK inhibitor of cell demonstrated that the effect of CsA on expression of iNOS to the p38 MAPK inhibitor of failed to inhibit iNOS expression in HIMEC that CsA inhibiting activation of p38 MAPK in HIMEC, MAPK used to p38 MAPK in and HIMEC. in as a and to p38 MAPK in HIMEC, in activation of and this activation by CsA and the p38 MAPK inhibitor MAPK activation inhibited by CsA of HIMEC which inhibited by and However, CsA effect on and this pathway NFκB a key role in cytokine and activation of endothelial cells R.D. J. Leukocyte Biol. Google Scholar). the effect of CsA on NFκB activation in HIMEC. activated NFκB in HIMEC, as demonstrated by as well as nuclear of the of NFκB by CsA of HIMEC failed to inhibit activation of NFκB, and which blocked NFκB activation. Because CsA significantly increased activated binding of cell adhesion molecule the mechanisms of leukocyte adhesion. have demonstrated that endothelial activation in a in the of superoxide and X.F. Smith C.W. Kubes P. Circ. Res. 1994; 74: 1133-1140Crossref PubMed Scopus (315) Google Scholar, M. Grisham M.B. Granger D.N. J. Leukocyte Biol. 1991; 50: PubMed Scopus (78) Google Scholar). We hypothesized that the of NO in the HIMEC would to activation of cells through an assess this used to assess the of reactive oxygen in HIMEC activation and is an that reactive oxygen and by in HIMEC HIMEC were but were However, HIMEC were CsA the of activation, there a in reactive oxygen Because CsA and blocked production of NO and increased were to this mechanism to the binding CsA leukocyte binding the result of increased superoxide in the HIMEC, a pharmacologic of superoxide the In the adhesion treatment of HIMEC completely the leukocyte binding by CsA These that superoxide in the activated CsA a role in the leukocyte calcineurin and pharmacologic have in transplant the potent ability to rejection, and have in of chronic inflammation and is to inhibit protein and proliferation of is a potent and immunosuppressive a the protein a to the of and of ultimately inhibiting cell and of populations, to its immunosuppressive effect in transplantation. and have in endothelial cell dysfunction, to the effect of CsA M. P. M. G. S. M. S. M. Med. PubMed Scopus Google Scholar). We to the proinflammatory effect of CsA on the microvascular would in the used potent immunosuppressive were on HIMEC CsA, and and a of which to CsA in HIMEC activated that endothelial dysfunction and inflammatory activation is a by of immunosuppressive The mechanisms the leukocyte binding in the and endothelial cells were We have demonstrated that the immunosuppressive agent CsA expression in human intestinal microvascular endothelial cells inflammatory activation. The CsA effect through of ultimately in a proinflammatory phenotype by leukocyte of HIMEC and in activation of the MAPK and NFκB, and expression of the proinflammatory cell adhesion molecules ICAM-1, VCAM-1, and HIMEC demonstrated an which an important early step in the inflammatory response. In to the increased expression of proinflammatory cell adhesion activation also the expression of iNOS and increased NO a key mechanism that ultimately the of HIMEC activation. Although treatment of HIMEC CsA a in the expression of proinflammatory molecules it a and of the iNOS in microvascular endothelial The effect of CsA on HIMEC activation a of NO production and a proinflammatory in leukocyte an effect for this which is normally used to inhibit inflammatory Although CsA has demonstrated to exert an effect on the expression of iNOS in cell including vascular cells and cells, this is the of its effect on the microvascular endothelium, a nonimmune cell that a regulatory role in inflammatory The effect of CsA on iNOS expression in nonimmune cells has completely which of the mechanisms in HIMEC activation and the effect of CsA on activation the important of this on the of of MAPK pathways by CsA inhibited p38 MAPK during the activation of intestinal microvascular endothelial cells, which ultimately a proinflammatory phenotype in important of this the of proinflammatory and expression by cytokine and activation of microvascular endothelial and activated MAPK as well as NFκB in HIMEC. MAPK and NFκB activation in proinflammatory expression of CAM, and a expression of iNOS, a key regulatory mechanism for limiting activation in HIMEC. Although CsA multiple expression it its powerful effect on the expression of the regulatory mechanism iNOS through a potent of p38 MAPK activation. CsA is as an but has demonstrated in the treatment of long term chronic inflammatory bowel disease. of the the efficacy of CsA has focused on the effect of this on T cells and the of on S. N. L. J. I. D. J. Clin. 1996; 80: PubMed Scopus Google Scholar). of the pharmacologic of CsA on nonimmune cell that this also exerts potent effects on microvascular endothelial populations, which play key regulatory in the inflammatory that CsA exerts multiple effects on the blood vessel the result of which to an of inflammation through increased leukocyte binding of signal transduction pathways is a emerging in molecular of which effect cellular activation pathways disease limiting adverse is the of of and p38 MAPK has demonstrated to clinical efficacy in short term of Crohn's disease D. G. M. S. Gastroenterology. Full Text Full Text PDF PubMed Scopus Google Scholar). the short term of intravenous CsA has efficacy in the treatment of ulcerative colitis and Crohn's disease S.B. Smith M.B. Am. J. Gastroenterol. 1993; 88: 646-649PubMed Google Scholar, 8Lichtiger S. Present D.H. Kornbluth A. Gelernt I. Bauer J. Galler G. Michelassi F. Hanauer S. New Engl. J. Med. 1994; 330: 1841-1845Crossref PubMed Scopus (1560) Google Scholar), long term has failed to demonstrate clinical Although the role of the in of intestinal inflammation are there are important in the nitric oxide biology of which may for a of of human microvascular during intestinal inflammation Gastroenterology. Full Text Full Text PDF PubMed Scopus Google Scholar). in human intestinal inflammation on in and the of microvascular endothelial cell cultures an essential for into molecular and cellular mechanisms by pharmacologic of HIMEC activation and the proinflammatory effect of CsA may a mechanism for the lack of efficacy of long term CsA in We have demonstrated the lack of NO in endothelial cells from chronically of bowel in to of of NO the ability of microvascular endothelial cells to down-regulate inflammatory activation, and may to chronic intestinal NO is in of in that an ability to NO in the microvasculature prevents of the disease process P. S. Wilson Biol. Med. PubMed Scopus Google Scholar). that CsA NO production from the intestinal vascular to leukocyte and chronic intestinal The effect of CsA on HIMEC activation may also into the of in solid organ where transplant long term calcineurin inhibitor CsA and of clinical in the T.R. J. Am. J. Gastroenterol. 1997; Google Scholar). the and effects of pharmacologic an important in may a a proinflammatory effect of CsA on the microvascular endothelium, and the of this in the long term treatment of chronic inflammatory bowel disease. CsA the of NO in the activated intestinal NO pharmacologic NO may a long term for calcineurin In have that NO as an to superoxide during HIMEC activation. Endothelial of in to CsA to a pharmacologic NO may the of NO production inhibited by of NO pharmacologic has as has and has demonstrated efficacy and in the treatment of of N. S. K. P. A. S. Gastroenterology. Full Text Full Text PDF PubMed Scopus Google Scholar). We of for of the and for the