Infliximab influences the expression of matrix metalloproteinase ( M M P )-2 and-9 in Crohn ' s disease Short title : Infliximab and MMPs in CD

Background and aims: Infliximab, a chimeric IgG1 anti-tumor necrosis factor (TNF)monoclonal antibody, is an effective immunotherapeutic agent for severe Crohn's disease (CD). Matrix metalloproteinases (MMPs) are believed to be actively involved in the pathogenesis of CD. In the present study we assessed the effect of infliximab on the in vitro and in vivo expression of MMP-2 and MMP-9 in CD. Patients and Methods: CD patients with fistulizing (n=10) or active disease (n=7) were administered infliximab and evaluated for serum levels of the MMPs and clinical response for 12 to 14 weeks, in an in-house study. Biopsies from some patients were evaluated immunohistochemically for the MMPs. Serum MMPs levels of fistulizing CD patients (n=42), with a follow-up of 18 weeks, and active CD patients (n=24) followed for 4 weeks, from two international placebo controlled infliximab studies, were also evaluated. In addition, in vitro whole blood cultures stimulated with LPS with/without infliximab were evaluated for MMP mRNA and protein levels by RT-PCR and ELISA, respectively. Results: MMP-2 serum levels in CD patients with either fistulas or active disease increased during follow-up, both in the in-house and in the international study, with a decline at the end of the follow-up in the latter. However, a similar trend was observed in responders and nonresponders to infliximab. Immunohistochemistry showed an immunoreaction to MMP-2 in the extracellular matrix (ECM) of the submucosa and the lamina propria and in endothelial cells without a clear change by the infliximab treatment. In contrast, serum MMP-9 levels showed a consistent pattern of decrease in most CD patients, particularly in the responding patients and less in non-responding patients, although at week 10 the MMP-9 level started to increase again. Immunohistocemically MMP-9 was predominantly present in polymorphonuclear leukocytes (PMNL) and showed a decrease by infliximab in 2 weeks. In vitro short-term 1.5 hours LPS stimulation of whole blood increased the MMP-9 levels in plasma from both CD patients and healthy volunteers, but significantly higher in CD. However, the respective MMP-9 mRNA levels were downregulated to 50% from that of control samples. Infliximab was found not to affect the mRNA and protein levels of this short-term LPS stimulation. LPS stimulation for 24 h did not further increase the MMP-9 plasma levels of the CD patients, whereas in the healthy controls it raised further. The leucocyte MMP-9 mRNA levels simultaneously raised up to16-fold and infliximab did not affect protein but inhibited the MMP-9 mRNA induction by 80%. Conclusion:MMP-2 and MMP-9 in serum of patients with CD exhibit an inverse changing pattern by treatment, i.e. an increase of MMP-2 and a decrease of MMP-9, the latter also in the intestine. However, these changes were not strictly associated with the response to treatment with infliximab, i.e., clinical improvement. In vitro studies indicate that the enhanced leucocyte MMP-9 expression in CD seems to be regulated by TNF-. Infliximab and MMPs in CD 89 Infliximab, a chimeric IgG1 anti-tumor necrosis factor (TNF)monoclonal antibody, is a successful immunotherapeutic agent for Crohn's disease (CD). The treatment with infliximab results in a high clinical efficacy, rapid onset of action and prolonged effect in patients with moderate to severe active CD, which have not responded to conventional therapy, and in fistulizing CD patients. Simultaneously the quality of life of these patients is essentially improved [1-3]. The proposed immunological mechanisms of infliximab include the suppression of TNFbioactivity and the lysis of TNF-producing cells, such as monocytes and lymphocytes, via complement fixation, antibody dependent cellular cytotoxicity (ADCC) and Fc portion binding of the IgG1 antibody. Furthermore, infliximab downregulates mucosal Th1 cytokines, reduces the expression of IFNand other inflammatory molecules, such as intercellular adhesion molecule (ICAM)-1 and vascular adhesion molecule (VCAM)-1 [4-6]. Matrix metalloproteinases (MMPs) compose a family with over 20 members of Zn 2+ containing neutral proteinases [7]. Usually MMPs are synthesized as prepro-enzymes and are secreted in a proenzyme form that requires proteolytic cleavage for activation in most cases [8;9]. The activity of MMPs is precisely regulated within tissues by the balance between zymogen activation and enzyme inhibition. Factors which regulate activity of the MMPs include endogenous inhibitors, -macroglobulins, and tissue inhibitors of metalloproteinases (TIMPs) and the MMPs themselves [10;11]. MMPs are implicated in the inflammatory response, wound healing, tissue remodeling, cell growth, migration, apoptosis, cell-cell communication, tumor invasion and metastasis [12-16]. MMPs exert their activity by the degradation of a class of biological molecules which include not only the components of extracellular matrix (ECM) but also an increasing family of bioactive modulators, such as cytokines, growth factor receptors, other proteinases, coagulation factors, chemotactic molecules, and adhesion molecules [14]. In the pathogenesis of CD, MMPs are believed to be associated with the injury of gut tissue mediated by TNFand Th1 cytokines. One of mechanisms by which TNFcauses intestinal tissue injury is believed to be the enhancement of the MMP production at local sites [17-20]. MMP-2 (72 kDa, gelatinase A) and MMP-9 (92 kDa, gelatinase B) are the two members of the gelatinase subgroup of MMPs. The substrates of MMP-2 and MMP-9 specifically include not only basement membrane (BM) type IV collagen and other components like gelatin, collagen type I, V, VII, X, lastin, laminin and fibronectin, but also numerous bioactive molecules, such as Fibroblast growth factor receptor (FGFR)-1, prointerleukin (IL)-1 and ICAM-1 [21-24]. Previous studies already showed that MMP-2 and -9 are actively involved in the pathophysiological processes in the intestine of IBD patients [17;25-31]. After treatment with infliximab the elevated levels of MMP-1 and -3 in serum of patients with rheumatoid arthritis were reported to be reduced [32]. The role of MMPs in treatment of CD patients with infliximab, however, is still poorly understood. In the present study, we explored the relationship between the clinical efficacy of infliximab and the expression levels of MMP-2 and -9 in patients with CD, and described the in vivo and in vitro regulation of the expression of these two gelatinases by infliximab. Materials and Methods