T-cell contact-dependent regulation of CC and CXC chemokine production in monocytes through differential involvement of NFκB: implications for rheumatoid arthritis

We and others have reported that rheumatoid arthritis (RA) synovial T cells can activate human monocytes/macrophages in a contact-dependent manner to induce the expression of inflammatory cytokines, including tumour necrosis factor alpha (TNFα). In the present study we demonstrate that RA synovial T cells without further activation can also induce monocyte CC and CXC chemokine production in a contact-dependent manner. The transcription factor NFκB is differentially involved in this process as CXC chemokines but not CC chemokines are inhibited after overexpression of IκBα, the natural inhibitor of NFκB. This effector function of RA synovial T cells is also shared by T cells activated with a cytokine cocktail containing IL-2, IL-6 and TNFα, but not T cells activated by anti-CD3 cross-linking that mimics TCR engagement. This study demonstrates for the first time that RA synovial T cells as well as cytokine-activated T cells are able to induce monocyte chemokine production in a contact-dependent manner and through NFκB-dependent and NFκB-independent mechanisms, in a process influenced by the phosphatidyl-inositol-3-kinase pathway. Moreover, this study provides further evidence that cytokine-activated T cells share aspects of their effector function with RA synovial T cells and that their targeting in the clinic has therapeutic potential

High-efficiency gene transfer into nontransformed cells: utility for studying gene regulation and analysis of potential therapeutic targets

The elucidation of the signalling pathways involved in inflammatory diseases, such as rheumatoid arthritis, could provide long sought after targets for therapeutic intervention. Gene regulation is complex and varies depending on the cell type, as well as the signal eliciting gene activation. However, cells from certain lineages, such as macrophages, are specialised to degrade exogenous material and consequently do not easily transfect. Methods for high-efficiency gene transfer into primary cells of various lineages and disease states are desirable, as they remove the uncertainties associated with using transformed cell lines. Significant research has been undertaken into the development of nonviral and viral vectors for basic research, and as vehicles for gene therapy. We briefly review the current methods of gene delivery and the difficulties associated with each system. Adenoviruses have been used extensively to examine the role of various cytokines and signal transduction molecules in the pathogenesis of rheumatoid arthritis. This review will focus on the involvement of different signalling molecules in the production of tumour necrosis factor alpha by macrophages and in rheumatoid synovium. While the NF-kappaB pathway has proven to be a major mediator of tumour necrosis factor alpha production, it is not exclusive and work evaluating the involvement of other pathways is ongoing

Bruton's Tyrosine Kinase Is Required For Lipopolysaccharide-induced Tumor Necrosis Factor α Production

Lipopolysaccharide (LPS), a product of Gram-negative bacteria, is potent mediator of tumor necrosis factor (TNF)α production by myeloid/macrophage cells. Inhibitors capable of blocking the signaling events that result in TNFα production could provide useful therapeutics for treating septic shock and other inflammatory diseases. Broad spectrum tyrosine inhibitors are known to inhibit TNFα production, however, no particular family of tyrosine kinases has been shown to be essential for this process. Here we show that the Bruton's tyrosine kinase (Btk)-deficient mononuclear cells from X-linked agammaglobulinemia patients have impaired LPS-induced TNFα production and that LPS rapidly induces Btk kinase activity in normal monocytes. In addition, adenoviral overexpression of Btk in normal human monocytes enhanced TNFα production. We examined the role of Btk in TNFα production using luciferase reporter adenoviral constructs and have established that overexpression of Btk results in the stabilization of TNFα mRNA via the 3′ untranslated region. Stimulation with LPS also induced the activation of related tyrosine kinase, Tec, suggesting that the Tec family kinases are important components for LPS-induced TNFα production. This study provides the first clear evidence that tyrosine kinases of the Tec family, in particular Btk, are key elements of LPS-induced TNFα production and consequently may provide valuable therapeutic targets for intervention in inflammatory conditions

Apremilast, a novel PDE4 inhibitor, inhibits spontaneous production of tumour necrosis factor-alpha from human rheumatoid synovial cells and ameliorates experimental arthritis

Introduction: Type 4 phosphodiesterases (PDE4) play an important role in immune cells through the hydrolysis of the second messenger, cAMP. Inhibition of PDE4 has previously been shown to suppress immune and inflammatory responses, demonstrating PDE4 to be a valid therapeutic target for immune-mediated pathologies. We assessed the anti-inflammatory effects of a novel PDE4 inhibitor, apremilast, in human synovial cells from rheumatoid arthritis (RA) patients, as well as two murine models of arthritis

The role of CXCR4/SDF-1 and VLA-4/VCAM-1 in migration of bystander-activated T cells : implications for rheumatoid arthritis

Bystander lymphocytes generated in vitro by cytokine stimulation (IL-2, IL-6, TNFα) have previously been shown to demonstrate significant identity in phenotype and activation of monocyte signalling pathways to T cells isolated from rheumatoid arthritis (RA) synovial tissue. Tck are a therefore relevant surrogate model for studying the RA T cell. The results presented in this thesis extend previous studies of cytokine- activated T cells (Tck) through phenotypic analysis and determination of migratory responsiveness. Cytokine stimulation upregulated expression of chemokine receptors and integrins on Tck, including CXCR4, VLA-4 and LFA-1. Results of in vitro chemotaxis and extravasation studies revealed that increased expression of CXCR4 and VLA-4 integrin resulted in concentration-dependent Tck migration to their ligands, CXCL12 and VCAM-1, which could be blocked through specific inhibitors and neutralizing antibodies. Increased expression of VLA-4 and LFA-1 also resulted in increased Tck extravasation, inhibited through blockade of VLA-1, LFA-1 or their ligands. Moreover, Tck demonstrated an increased chemotactic response to RA fibroblast- like synoviocytes cultured in vitro, which could be decreased using inhibitors against VLA-4 and CXCR4. The RA/ SCID mouse model was established to assess Tck migratory responsiveness in vivo; however the model did not prove sufficiently robust to definitively determine if the in vitro results reflected the situation in vivo. The activated phenotype of Tck results in increased migratory responsiveness to chemokines and proteins found in the RA synovial joint environment. Tcks elicit proinflammatory cytokine production from monocytes, which was further increased in the migrated Tck subset. Tck also exhibit a phenotype similar to RA synovial tissue T cells, indicating that cytokine activation of T cells could contribute to RA pathogenicity by migration and subsequent cell-cell interactions, perpetuating the inflammatory cascade in RA. The CXCR4/ CXCL12 and VLA-4/ VCAM-1 axes could also potentially be exploited for therapeutic gain in treatment of RA.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Defining therapeutic targets by using adenovirus: blocking NF-kB inhibits both inflammatory and destructive mechanisms in rheumatoid synovium but spares anti-inflammatory mediators

The role of the transcription factor NF-κB in the pathogenesis of rheumatoid arthritis has long been a subject of controversy. We used an adenoviral technique of blocking NF-κB through overexpression of the inhibitory subunit IκBα, which has the advantage that it can be used in the diseased tissue itself, with >90% of the synovial macrophages, fibroblasts, and T cells infected. We found that the spontaneous production of tumor necrosis factor α and other pro-inflammatory cytokines is NF-κB-dependent in rheumatoid synovial tissue, in contrast to the main anti-inflammatory mediators, like IL-10 and -11, and the IL-1 receptor antagonist. Of even more interest, IκBα overexpression inhibited the production of matrix metalloproteinases 1 and 3 while not affecting their tissue inhibitor. Blocking NF-κB in the rheumatoid joint thus has a very beneficial profile, reducing both the inflammatory response and the tissue destruction. The adenoviral technique described here has widespread applicability, allowing rapid testing of the effects of blocking a potential therapeutic target in either cultures of normal cells or in the diseased tissue itself