TNF receptor gene therapy results in suppression of IgG2a anticollagen antibody in collagen induced arthritis. (Extended Report).(tumour necrosis factor )

Background: Therapeutic strategies to block tumour necrosis factor a (TNF[alpha]) activity in experimental autoimmune arthritis models and rheumatoid arthritis (RA) have proved highly successful, and provide sustained beneficial effects.

Objective: To examine whether TNF[alpha] inhibition has immunological activity beyond the reduction of inflammation in collagen induced arthritis (CIA), an established experimental model of RA.

Methods: Arthritic DBA/1 mice received single periarticular injections of retroviral constructs encoding human TNF receptor (TNF-R) into the affected arthritic paw, at the onset of arthritis. Severity of arthritis, antibodies to collagen type II (CII), and extent of pathological joint damage of arthritic paws were compared between TNF-R and media treated (control) animals 3, 7, 14, 21, and 49 days after disease onset.

Results: Severity of CIA was significantly decreased in TNF-R treated animals compared with controls, 14-34 days after disease onset. Joint destruction was reduced in TNF-R injected joints and in the uninjected contralateral and ipsilateral paws of TNF-R treated animals. Seven days after disease onset, TNF-R treated mice had lower levels of inflammatory Th1 driven IgG2a antibodies to CII (p<0.05) than controls. This altered the anticollagen IgG2a:IgG1 ratio towards Th2 driven IgG1.

Conclusions: Local TNF-R gene therapy in CIA appears to have systemic effects on the anti-CII antibodies. The overall influence of TNF-R gene therapy is that it inhibits the progression of CIA mainly by suppressing the inflammatory Th1 response rather than by stimulating a Th2 response. Therefore, periarticular TNF-R gene therapy may have excellent therapeutic potential in RA.

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Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease manifested by progressive synovial joint inflammation and erosion of the subchondral bone. Interactions between synovial hyperplasia, inflammation, and an altered humoral and cellular immune system characterise the pathology of RA. (1) Collagen induced arthritis (CIA) induced in DBA/1 mice by intradermal injection of collagen type II (CII), is an animal model of RA. Histological appearance of the erosions of cartilage and bone, inflammatory and immune cytokines, genetic and clinical features associated with both RA and CIA are similar. (2) Although the aetiopathogenesis of RA is still unclear, several studies suggest that activated CD4+ T cells have an important role in initiating and perpetuating the chronic inflammation characteristic of this disease. (3-5) It has been suggested that in RA, pathophysiological processes influencing the immune response may be driven by activated Th1 cells with insufficient Th2 cell differentiation to down modulate the ongoing inflammation. (6) Skapenko et al showed that CD4+ memory T cells from patients with untreated RA demonstrate an intrinsic abnormality towards differentiating into specific cytokine producing effector cells that might lead to the typical Th1 dominated chronic inflammation in RA. (6) Furthermore, high levels of circulating autoantibodies to CII are always associated with CIA, and seem to be essential for development and propagation of disease. Autoantibodies to CII belonging to the IgG2 subclass have been shown to be most efficient in binding complement, another crucial factor for initiation of joint inflammation in CIA. (7 8) Also, IgG2a production is associated with a Th1 response, whereas IgG1 production is associated with a Th2 response. (9)

Tumour necrosis factor [alpha] (TNF[alpha]) is a macrophage derived cytokine with multiple inflammatory and immunoregulatory properties. Raised levels of TNF[alpha] have been found in the sera and synovial fluid of patients with RA, suggesting it has an important role in the pathogenesis of RA. (2 10) Tissue expression of TNF[alpha] and its two receptors (p55 and p75 TNF-R) is seen at several sites within the synovial membrane and the cartilage/ pannus junction, indicating that a wide variety of cells may be targets for TNF activity in RA. (11-13) TNF[alpha] produced by synovial cells in RA seems to contribute to a cytokine cascade that subsequently leads to increases in interleukin (IL)1[beta] and granulocyte macrophage-colony stimulating factor (GM-CSF) levels. (2 12-14) Studies suggest that neutralisation of TNF[alpha] down regulates the production of IL1, IL6, IL8 and GM-CSF, thereby improving the arthritic disease process (11 15-17) Therefore, TNF[alpha] represents a suitable target for intervention of the ongoing inflammatory immune process in RA.

Several different treatment immune strategies have been examined for the management of RA. Protein based antiarthritic treatments have been shown to modulate the pathophysiology of the disease; however, they require frequent parenteral administration and are associated with several adverse side effects. (18 19) Gene therapy, requiring direct intra-articular or periarticular administration of vectors encoding for anti-inflammatory cytokines, avoids the delivery problems associated with protein administration. (19 20) In this study, retroviral vectors encoding for human p55 TNF-R were given periarticularly to paws of CII immunised mice on the day of arthritis onset to elucidate whether anti-inflammatory TNF-R gene therapy could ultimately influence the reactivity of autoimmune lymphocytes in CIA.

MATERIALS AND METHODS

Retroviral vector production

Retroviral construct--namely, MOIN-sTNF-Rc-Ig, encoded a fusion protein consisting of the extracellular domain of human 55 kDa TNF[alpha]-R covalently linked to the [C.sub.H]2 through [C.sub.H]3 domains of mouse IgG1 heavy chain. The soluble TNF receptor (sTNF-Rc-Ig) was amplified from sTNF-Rc-Ig plasmid using TNFsR5 and Ig3 oligomers. (21 22) The sTNF-Rc-Ig gene was inserted into the BamHI site of the retroviral construct MOIN, (23) resulting in MOIN-sTNF-Rc-Ig. To produce high titre virus, the vector was transfected into Phoenix cells, a 293-based amphotropic packaging cell line, the supernatant harvested 48 hours after transfection, and titres determined by a standard plaque assay. (24)

Induction and assessment of CIA …