Marc Feldmann

To investigate the role of the transcription factor nuclear factor-kB (NF-kappaB) in promoting inflammatory and destructive responses in human osteoarthritis (OA) synovial fibroblasts, by assessing the effect of NF-kappaB blockade on the production of cytokines and destructive enzymes. Infection with adenoviruses transferring the beta-galactosidase gene was used to ascertain that the OA fibroblasts could be infected (> 95%). Using an adenovirus transferring the inhibitory subunit IkappaBa, effective inhibition of NF-kappaB was achieved. The expression and production of several pro- and antiinflammatory cytokines and mediators, the major matrix metalloproteinases (MMP 1, 3, and 13), their main inhibitor tissue inhibitor of metalloproteinase-1 (TIMP-1), and the aggrecanases (ADAMTS4 and ADAMTS5) were measured by ELISA and/or reverse transcription-polymerase chain reaction, and their dependence on NF-kappaB evaluated. The production of interleukin 6 (IL-6), monocyte chemoattractant protein-1, and RANTES was potently inhibited by IkBa overexpression, irrespective of stimulus, but IL-8 was unaffected. The p55 soluble tumor necrosis factor (TNF) receptor was unaffected, but the p75 soluble TNF receptor was potently inhibited by IkBa overexpression. MMP-1, MMP-3, and MMP-13 were inhibited by IkappaBa overexpression, at both the mRNA and protein levels, whereas TIMP-1 was unaffected. The mRNA gene expression of ADAMTS4 was also inhibited by IkappaBa overexpression, particularly in IL-1-stimulated cells, but ADAMTS5 was unaffected. In OA synovial fibroblasts, inhibition of NF-kappaB has a beneficial effect on the balance between the expression of proinflammatory cytokines and antiinflammatory mediators. Inhibition of this transcription factor also results in the decreased expression of several destructive metalloproteinases and also the ADAMTS4 aggrecanase.

Antigen presentation is a key rate‐limiting step in the immune response. Dendritic cells (DCs) have been reported to be the most potent antigen‐presenting cells for naïve T cells, but little is known about the biochemical pathways that regulate this function. We here demonstrate that mature murine DC can be infected with adenovirus at high efficiency (>95%) and that an adenovirus transferring the endogenous inhibitor IκBα blocks nuclear factor‐kappa B (NF‐κB) function in murine DC. This result indicates that antigen presentation in the mixed leucocyte reaction is NF‐κB dependent, confirming data with human DC in vitro. However, the importance of this finding depends on verifying that this is true also in vivo. Using delayed type hypersensitivity with allogeneic cells, we show that NF‐κB inhibition had a marked immunosuppressive effect in vivo. These results thus establish NF‐κB as an effective target for blocking DC antigen presentation and hence inhibiting T‐cell‐dependent immune responses. This finding has potential implications for the development of therapeutic agents for use in various pathological conditions of the immune system, including allergy and autoimmunity, and also in transplantation.

Although dendritic cells (DCs) are the most potent antigen-presenting cells involved in numerous physiologic and pathologic processes, little is known about the signaling pathways that regulate DC activation and antigen-presenting function. Recently, we demonstrated that nuclear factor (NF)-κB activation is central to that process, as overexpression of IκBα blocks the allogeneic mixed lymphocyte reaction (MLR), an in vitro model of T-cell activation. In this study, we investigated the role of 2 putative NF-κB–inducing components, NF-κB–inducing kinase (NIK), and IκB kinase 2 (IKK2). Using an adenoviral gene transfer method to efficiently express dominant-negative (dn) forms of these molecules in monocyte-derived DCs, we found that IKK2dn but not NIKdn inhibited the allogeneic MLR. When DCs were fixed, this inhibitory effect of IKK2dn was lost, suggesting that IKK2 is involved in T-cell–derived signals that enhance DC antigen presentation during the allogeneic MLR period and does not have an effect on viability or differentiation state of DCs prior to coculture with T cells. One such signal is likely to be CD40 ligand (CD40L), as IKK2dn blocked CD40L but not lipopolysaccharide (LPS)–induced NF-κB activation, cytokine production, and up-regulation of costimulatory molecules and HLA-DR in DCs. In summary, our results demonstrate that IKK2 is essential for DC activation induced by CD40L or contact with allogeneic T cells, but not by LPS, whereas NIK is not required for any of these signals. In addition, our results support IKK2 as a potential therapeutic target for the down-regulation of unwanted immune responses that may occur during transplantation or autoimmunity.

The transcription factor NF‐κB is a pivotal intracellular regulator of many inflammatory responses and it has been proposed that it represents a potential therapeutic target. As chemokines are important for the progress of an inflammatory response by the recruitment of immuno‐competent cells, the role NF‐κB plays in TNFα‐ or lipopolysaccharides (LPS)‐induced chemokine secretion by human monocyte‐derived macrophages was examined. Secretion of the CXC chemokines IL‐8, GROα and ENA‐78, induced by TNFα, was significantly suppressed by inhibiting NF‐κB, using overexpression of IκBα. However, when induced by LPS the expression of these chemokines was unaffected. In contrast, expression of the CC chemokines MIP‐1α, MCP‐1 and RANTES inducedby TNFα or LPS was significantly inhibited by the overexpression of IκBα. Therefore, there appear to be different mechanisms regulating CC and CXC chemokine secretion by macrophages, depending on the stimulus and that TNFα and LPS can use different signaling mechanisms in macrophages to regulate chemokine synthesis.

The aetiology of systemic, autoimmune, chronic inflammatory diseases — such as rheumatoid arthritis — is not known, and their pathogenesis is complex and multifactorial. However, progress in the characterization of intercellular mediators — proteins that are now known as cytokines — has led to the realization that one cytokine, tumour-necrosis factor (TNF; previously known as TNF-α), has an important role in the pathogenesis of rheumatoid arthritis. This discovery heralded a new era of targeted and highly effective therapeutics for rheumatoid arthritis and, subsequently, other chronic inflammatory diseases.

Dendritic cells (DC) are the most potent antigen-presenting cells for naive T cells, due to their high expression of MHC and costimulatory molecules, but relatively little is known about the biochemical pathways that regulate this function. We used the proteasome inhibitor N-benzyloxycarbonyl-Ile-Glu(O-tert-butyl)-Ala-leucinal (PSI) to demonstrate that DC antigen presentation is NFκB dependent. As PSI is not a specific inhibitor of NFκB, we reproduced this finding using a very specific approach, namely adenoviral gene transfer of IκBα, the naturally occurring inhibitor of NFκB. The mechanism for this inhibition of DC antigen presentation involves at least three aspects of antigen presenting function: down-regulation of HLA class II, down-regulationof CD86, and inhibition of the immunostimulatory cytokines IL-12 and TNF-α. In the light of the marked down-regulation of antigen-presentation cell function, it was of interest to investigate what effects exposure to PSI-treated DC might have on T cell function. It was found that immunological tolerance was induced, as challenge of T cells previously exposed to PSI-treated DC, with normal DC from the same donor did not restore their response, despite the presence of viable T cells. There were also changes in T cell surface markers, with down-regulation of CD3 and CD25 expression, and inhibition of the production of Th1 cytokines like IL-2 and IFN-γ. These results demonstrates that NFκB is an effective target for blocking DC antigen presentation and inhibiting T cell-dependent immune responses, and this has implications for the development of therapeutic agents for use in multiple conditions, including transplantation, allergy and autoimmune diseases.

Rheumatoid arthritis (RA), a systemic disease, is characterized by a chronic inflammatory reaction in the synovium of joints and is associated with degeneration of cartilage and erosion of juxta-articular bone. Many pro-inflammatory cytokines including TNFα, chemokines, and growth factors are expressed in diseased joints. The rationale that TNFα played a central role in regulating these molecules, and their pathophysiological potential, was initially provided by the demonstration that anti-TNFα antibodies added to in vitro cultures of a representative population of cells derived from diseased joints inhibited the spontaneous production of IL-1 and other pro-inflammatory cytokines. Systemic administration of anti-TNFα antibody or sTNFR fusion protein to mouse models of RA was shown to be anti-inflammatory and joint protective. Clinical investigations in which the activcity of TNFα in RA patients was blocked with intravenously administered infliximab, a chimeric anti-TNFα monoclonal antibody (mAB), has provided evidence that TNF regulates IL-6, IL-8, MCP-1, and VEGF production, recruitment of immune and inflammatory cells into joints, angiogenesis, and reduction of blood levels of matrix metalloproteinases-1 and -3. Randomized, placebo-controlled, multi-center clinical trials of human TNFα inhibitors have demonstrated their consistent and remarkable efficacy in controlling signs and symptoms, with a favorable safety profile, in approximately two thirds of patients for up to 2 years, and their ability to retard joint damage. Infliximab (a mAB), and etanercept (a sTNF-R-Fc fusion protein) have been approved by regulatory authorities in the United States and Europe for treating RA, and they represent a significant new addition to available therapeutic options.