Karl M. Stuhlmeier

Many alternative treatment approaches, originating from Asia, are becoming increasingly popular in the Western hemisphere. Recently, an article published in a renowned journal reported that venom of apis mellifera (bee venom (BV)) and melittin mediate immune-modulating effects by blocking the activation of the transcription factor NF-κB. Such a modus operandi would corroborate the many claims of beneficial effects of BV treatment and give immediate credit to this form of therapy. Fibroblast-like synoviocytes from rheumatoid arthritis patients and dermal fibroblast cells and white blood cells from healthy volunteers were used to study the effects of BV and melittin on the activation of NF-κB and a series of genes that are markers of inflammation. EMSAs demonstrate that neither BV nor melittin blocked IL-1β-induced NF-κB activation; neither did they affect phosphorylation or degradation of IκB. Contrary to published data, even high concentrations of BV and melittin were without any effect on NF-κB-p50-DNA interactions. More importantly, in fibroblast-like synoviocytes, but also in dermal fibroblasts as well as in mononuclear cells exposed to BV or melittin, mRNA levels of several proinflammatory genes are significantly increased, and Western blot data show elevated cyclooxygenase-2 protein levels. Furthermore, exposure to BV higher than 10 μg/ml resulted in disintegration of all cell types tested. In addition, large quantities of oxygen radicals are produced in a dose-dependent manner in leukocytes exposed to BV. Taken together, data presented in this work do not corroborate an earlier report regarding the effectiveness of BV as an inhibitor of the transcription factor NF-κB.

Hyaluronsäure oder Hyaluronan ist nicht das inaktive "Füllmaterial" für das es lange Zeit gehalten wurde. Im Gegenteil, Hyaluronan, das von zumindest drei Enzymen synthetisiert wird, ist an vielen biologischen Prozessen aktiv beteiligt. Während schon sehr lange bekannt ist, dass zumindest Hyaluronanabbauprodukte eine Reihe negativer Auswirkungen haben können, wurden viele biologische Funktionen der intakten Produkte der drei Hyaluronansynthasen (HAS1, HAS2 und HAS3) erst in den letzten Jahren beschrieben. Die folgende Übersicht liefert eine Zusammenfassung dieser Erkenntnisse. Der Schwerpunkt dieser Arbeit liegt auf der Rolle von Hyaluronan und Hyaluronansynthasen bei Rheumatoider Arthritis, daneben werden noch Aspekte der Hyaluronanbiologie in Atherosklerose, Restenosen, Embryogenese und Tumorentstehung, sowie in der Modulation der Immunantwort beschrieben. HA takes part in a surprisingly large number of biological processes such as embryogenesis, angiogenesis, cell motility, wound healing and cell adhesion. While substantial progress in HA research has indeed been made over the last years, many important questions have not yet been answered. One of the most pertinent questions awaiting an answer is the quest for functional differences of HA synthesized by the three HAS genes. Of similar importance would be investigations into intracellular signaling pathways involved in the activation of this gene family, a field in which to date very little is known. A better understanding of functional differences between the HAS encoding genes not only holds the promise for a better understanding of a series of biological processes but also the opportunity for selective intervention in a number of maladies characterized by abnormalities of HA levels.

Fibroblast-like synoviocytes (FLS) play a key role in the genesis of rheumatoid arthritis (RA). FLS are among the most versatile cells with the potential to activate an array of genes that are able to initiate and propagate inflammation in RA-affected joints. Controlling activation of FLS might hold the key to restraining inflammation in RA-affected joints. In this study, we investigate the effect and mechanisms of short-term hyperthermia on a series of proinflammatory genes in FLS. In vitro experiments demonstrate that exposure of FLS to elevated temperatures for the duration of 30 min prevents activation of a series of genes with proinflammatory properties. Exposure to hyperthermia reduces IL-1β-induced prostaglandin E2 release, suppresses activation of the adhesion molecules VCAM-1, ICAM-1, the cytokines TNFα, IL-1α, IL-1β, IL-8 as well as COX-2 protein synthesis. Real time reverse transcriptase-polymerase chain reaction showed that hyperthermia altered gene expression at the transcriptional level. The amount and the duration of inhibition is gene-specific and lasts for up to 25 h. As to the mechanism of inhibition, electrophoretic mobility shift assay experiments demonstrated that exposure of FLS to hyperthermia prevents IL-1β-induced NF-κB translocation and subsequent DNA binding. Many mechanisms have been shown to be involved in hyperthermia-mediated effects on NF-κB-DNA interactions. We demonstrate by Western blot experiments that in FLS, hyperthermia prevents the phosphorylation and subsequent degradation of IκBα, therefore retaining the NF-κB complex in the cytoplasm. Carefully controlled in vivo tests are certainly needed before one can take full advantage of those phenomena; however, the ease by which the temperature in joints can be modulated might offer an opportunity for manipulating inflammatory processes in joints by simple balneological means.

Macrophages play an important role in rheumatoid arthritis (RA). RA is a disease characterized by the successive accumulation of leukocytes resulting in subsequent destruction of affected joints. Activation of matrix metalloproteinases (MMP) is essential for many physiological as well as many pathological events owing to the essential role of MMP in cell migration. We analyzed the effectiveness of quinacrine as an inhibitor of MMP activation in leukocytes and investigated the mode of action. Leukocytes were isolated and treated with quinacrine with or without phorbol myristic acetate (PMA). ELISA and RT-PCR were used to monitor production of MMP-1, MMP-2, MMP-3, and MMP-8 at the mRNA and protein level. Quinacrine suppressed PMA induced MMP-1 release in mononuclear cells (MNC) in a dose- and time-dependent manner. RT-PCR showed that quinacrine downregulated induced as well as noninduced steady-state mRNA levels of MMP-1, MMP-2, and MMP-8, but had no effect on MMP-3. The observed inhibition was not due to effects of quinacrine on phospholipase A2 (PLA2) activity. Adding exogenous arachidonic acid to reconstitute the blocked PLA2 signaling pathways did not result in restoration of PMA induced mRNA transcription. Inhibition of MMP by quinacrine might, in part, account for its reported immunosuppressive action. Synthesizing more potent derivatives of quinacrine may be a means of suppressing undesired MMP activation.

Despite evidence that points to unfettered hyaluronic acid (HA) production as a culprit in the progression of rheumatic disorders, little is known about differences in regulation and biological functions of the three hyaluronan synthase (HAS) genes. Testing the effects of drugs with proven anti-inflammatory effects could help to clarify biological functions of these genes. In this study, we demonstrate that leflunomide suppresses HA release in fibroblast-like synoviocytes (FLS) in a dose-dependent manner. We further demonstrate that leflunomide suppresses HA synthase activity, as determined by 14C-glucuronic acid incorporation assays. Additional experiments revealed that in FLS, leflunomide specifically blocked the induction of HAS1. HAS2 and HAS3, genes that are, in contrast to HAS1, constitutively expressed in FLS, are not significantly affected. Leflunomide can function as a NF-κB inhibitor. However, EMSA experiments demonstrate that at the concentrations used, leflunomide neither interferes with IL-1β- nor with PMA-induced NF-κB translocation. Furthermore, reconstituting the pyrimidine synthase pathway did not lead to the restoration of IL-1β-induced HAS1 activation. More importantly, two tyrosine kinase inhibitors mimicked the effect of leflunomide in that both blocked IL-1β-induced HAS1 activation without affecting HAS2 or HAS3. These data point at HAS1 activation as the possible cause for unfettered HA production in rheumatoid arthritis and might explain, at least in part, the beneficial effects of leflunomide treatment. These findings also support the concept that IL-1β-induced HAS1 activation depends on the activation of tyrosine kinases, and indicate that leflunomide blocks HA release by suppressing tyrosine kinases rather than through inhibition of NF-κB translocation.

Leflunomide, a potent disease-modifying antirheumatic drug used in the treatment of rheumatoid arthritis (RA), exhibits anti-inflammatory, antiproliferative and immunosuppressive effects. Although most of the beneficial effects of leflunomide have been attributed to its antimetabolite activity, mainly in T cells, other targets accounting for its potency might still exist. Because of mounting evidence for a prominent role of dendritic cells (DCs) in the initiation and maintenance of the immune response in RA, we analyzed the effect of the active metabolite of leflunomide (A77 1726; LEF-M) on phenotype and function of human myleloid DCs at several stages in their life cycle. Importantly, DCs differentiated in the presence of LEF-M exhibited an altered phenotype, with largely reduced surface expression of the critical co-stimulatory molecules CD40 and CD80. Furthermore, treatment of DCs during the differentiation or maturation phase with LEF-M aborted successful DC maturation. Exogenous addition of uridine revealed that DC modulation by LEF-M was independent of its proposed ability as an antimetabolite. In addition, the ability of DCs to initiate T-cell proliferation and to produce the proinflammatory cytokines IL-12 and tumour necrosis factor-α was markedly impaired by LEF-M treatment. As a molecular mechanism, transactivation of nuclear factor-κB, an transcription factor essential for proper DC function, was completely suppressed in DCs treated with LEF-M. These data indicate that interference with several aspects of DC function could significantly contribute to the beneficial effects of leflunomide in inflammatory diseases, including RA.