Role of Th1 and Th2 cytokines in the pathogenesis of systemic autoimmune diseases

Systemic lupus erythematosus (SLE), is a prototypic systemic autoimmune disease characterised by multi-system involvement, female preference, activation of T-cells, B-cell hyper-activity, autoantibody production and immune complex deposition. The origin of the defects leading to pathogenicity in systemic lupus erythematosus, is still controversial. The therapeutic strategies today for treatment of lupus disease are mainly based on a general suppression of the immune system with uncertainty about their long-term effects. The underlying mechanism for the development of the disease is yet to be clarified. Cytokines play a critical role in regulating the quantitative and qualitative responses of T cells, B cells, macrophages, and other cell types. Many cytokine disorders have been reported in both SLE patients and the animal models but findings are often difficult to reconcile especially differences between data from the in vitro and in vivo studies. In the murine model, it was suggested that the balance of Th1/Th2 cytokines related to the pathogenesis of SLE. Recent evidence clearly demonstrates that Th1 cytokines are involved in the immuno-pathogenesis of SLE. Several factors are required for optimal induction of Th1 activity, chief among them are IL-12 and IL-18. IL-12 promoted IFN-gamma dependent renal injury in MRL/lpr mice, which develop spontaneous lupus-like autoimmune disease. In order to understand the mechanism of immune regulation in SLE, I carried out detailed analysis of the nature and pathological relevance of Th1 and Th2 cytokines, IL-12 and IL-18 in particular, in the pathogenesis of SLE. I found that serum fi-om patients with SLE contained significantly higher concentrations of IL-18 than normal individuals. To investigate the potential role of IL-18 in SLE, I studied the effect of recombinant-IL-18 on MRL/lpr mice, which develop spontaneous lupus-like autoimmune disease. MRL/lpr mice produced significantly more IL-18 as disease progressed compared with the wild-type MRL/++ mice. MRL/lpr mice injected daily with IL-18 or IL-18 + IL-12 resulted in accelerated proteinuria, glomerulonephritis, and vasculitis. In contrast, the treatment had no effect on the control MRL/++ mice. IL-18 and IL-18 + IL-12-treated MRL/lpr mice produced more inflammatory cytokines (LFN-gamma, TNF-alpha and IL-6) compared with untreated MRL/lpr mice. IL-18-treated MRL/r mice also exhibited the butterfly facial rashes characteristic of clinical SLE. In contrast, MRL/lpr mice treated with a combination of IL-18 and IL-12, while showing more severe vasculitis than those treated with IL-18 alone, did not present any facial rash. Histological analysis of the facial lesion revealed extensive epidermal thickening with intense inflammatory cell infiltrate and immunoglobulin deposition accompanied by extensive apoptosis in the IL-18-treated mice compared with control or IL-12 + IL-18 treated mice. IL-18 may thus be a novel target for therapeutic intervention of spontaneous autoimmune diseases. Elevated levels of IL-12 (p40/p70) have been reported in MRL/lpr serum and have been linked to increased nitric oxide production and disease activity. Therefore, studies were also performed to determine whether IL-12 and nitric oxide (NO) play a significant role (similar to MRL/lpr mice) in induction of the disease in NZB/W mice a lupus-like model with different genetic backgrounds from MRL/lpr mice and with intact Fas. The results demonstrate that serum of NZB/W FI mice contains higher level of total IL-12 (p40/p70) than control mice and IL-12 is increased in correlation with disease of this lupus-like strain. In himians, the serum level of total IL-12 is significantly higher in SLE patients than control individuals. Whole blood culture from SLE patients also showed higher IL-12 production, when cultured with LPS and IFN-gamma, compared with control individuals. Results presented in this thesis demonstrate that IL-18 and IL-12 play important roles in the induction of SLE through the activation of Th1 cells

Effect of therapeutic plasma exchange on immunoglobulins in myasthenia gravis

An integrated understanding of therapeutic plasma exchange (TPE) effects on immunoglobulins, autoantibodies, and natural or acquired (vaccine) protective antibodies in patients with autoimmune myasthenia gravis (MG) is lacking. Prior studies measured TPE effects in healthy volunteers or heterogeneous autoimmune diseases populations. We prospectively profiled plasma IgA, IgM, IgG, IgG subclasses (IgG1-4), acetylcholine receptor autoantibodies (AChR+), and protective antibodies in patients with AChR+ MG receiving TPE for an exacerbation. TPE was performed according to institutional practice and patients were profiled for up to 12 weeks. Ten patients were enrolled (median age=72.9 years; baseline MG-Composite=21; median TPE treatments=6 during their first course) and all improved. The maximum decrease in all immunoglobulins, including AChR autoantibodies, was achieved on the final day of the first TPE course (approximately 60–70% reduction). Three weeks post-TPE mean AChR autoantibody, total IgG, IgG1 and IgG2 titers were below the reference range and had not recovered to within 20% of baseline, whereas other measured immunoglobulins approached baseline values. We did not generally observe an “overshoot” of immunoglobulins above pre-TPE levels or accelerated recovery of pathologic AChR autoantibodies. Protective antibody profiles showed similar patterns as other IgGs and were detectable at levels associated with protection from infection. A slow return to baseline for IgGs (except IgG3) was observed, and we did not observe any obvious effect of concomitant medications on this recovery. Collectively, these findings enhance our understanding of the immunological effects of TPE and further supports the concept of rapid immunoglobulin depletion for the treatment of patients with MG

A Phase I, Randomized, Double‐Blind, Placebo‐Controlled, Single‐Dose and Multiple‐Rising‐Dose Study of the BTK Inhibitor TAK‐020 in Healthy Subjects

Bruton’s tyrosine kinase (BTK) is a target for treatment of hematologic malignancies and autoimmune diseases. TAK‐020 is a highly selective covalent BTK inhibitor that inhibits both B cell receptor and fragment crystallizable receptor signaling. We assessed the safety/tolerability and pharmacokinetics/pharmacodynamics (PDs) of TAK‐020 in healthy subjects. Each cohort of the single‐rising dose (n = 72; 9 cohorts) and the multiple‐rising dose (n = 48; 6 cohorts) portions of the study comprised six TAK‐020‐treated and two placebo‐treated, subjects aged 18–55 years (inclusive). The PD effects were assessed by measuring BTK occupancy and the inhibition of fragment crystallizable epsilon receptor 1 (FcεRI)‐mediated activation of basophils. Overall, treatment‐emergent adverse events (TEAEs) were similar to placebo; there were no serious TEAEs or no TEAEs leading to discontinuation. TAK‐020 was rapidly absorbed (median time to maximum plasma concentration (Tmax) 45–60 minutes) with a half‐life of ~ 3–9 hours at doses ≥ 2.5 mg. TAK‐020 exposure was generally dose proportional for single doses ≤ 70 mg and after multiple doses of ≤ 60 mg once daily. Target occupancy was dose dependent, with doses ≥ 2.5 mg yielding maximum and sustained occupancy > 70% for > 96 hours. Single doses ≥ 4.4 mg reduced FcεRI‐mediated activation of basophils by > 80% and comparable inhibition was observed with daily dosing ≥3.75 mg for 9 days. Inhibition persisted for 24–72 hours postdose and the duration generally increased with dose. TAK‐020 was generally well‐tolerated in healthy subjects after single and multiple doses and demonstrated target engagement and pathway modulation. The PD effects outlasted drug exposures, as expected for covalent inhibition of BTK

A proinflammatory role of IL-18 in the development of spontaneous autoimmune disease

Serum from patients with systemic lupus erythematosus (SLE) contained significantly higher concentrations of IL-18 than normal individuals. MRL/lpr mice, which develop spontaneous lupus-like autoimmune disease, also had higher serum levels of IL-18 than wild-type MRL/�� mice. Daily injections of IL-18 or IL-18 plus IL-12 resulted in accelerated proteinuria, glomerulonephritis, vasculitis, and raised levels of proinflammatory cytokines in MRL/lpr mice. IL-18-treated MRL/lpr mice also developed a “butterfly” facial rash resembling clinical SLE. In contrast, MRL/lpr mice treated with IL-18 plus IL-12 did not develop a facial rash. The facial lesion in the IL-18-treated mice showed epidermal thickening with intense chronic inflammation accompanied by increased apoptosis, Ig deposition, and early systemic Th2 response compared with control or IL-12 plus IL-18-treated mice. These data therefore show that IL-18 is an important mediator of lupus-like disease and may thus be a novel target for therapeutic intervention of spontaneous autoimmune diseases

Induction of cartilage integration by a chondrocyte/collagen-scaffold implant

The integration of implanted cartilage is a major challenge for the success of tissue engineering protocols. We hypothesize that in order for effective cartilage integration to take place, matrix-free chondrocytes must be induced to migrate between the two tissue surfaces. A chondrocyte/collagen-scaffold implant system was developed as a method of delivering dividing cells at the interface between two cartilage surfaces. Chondrocytes were isolated from bovine nasal septum and seeded onto both surfaces of a collagen membrane to create the chondrocyte/collagen-scaffold implant. A model of two cartilage discs and the chondrocyte/collagen-scaffold sandwiched in between was used to effect integration in vitro. The resulting tissue was analysed histologically and biomechanically. The cartilage–implant–cartilage sandwich appeared macroscopically as one continuous piece of tissue at the end of 40 day cultures. Histological analysis showed tissue continuum across the cartilage–scaffold interface. The integration was dependent on both cells and scaffold. Fluorescent labeling of implanted chondrocytes demonstrated that these cells invade the surrounding mature tissue and drive a remodelling of the extracellular matrix. Using cell-free scaffolds we also demonstrated that some chondrocytes migrated from the natural cartilage into the collagen scaffold. Quantification of integration levels using a histomorphometric repair index showed that the chondrocyte/collagen-scaffold implant achieved the highest repair index compared to controls, reflected functionally through increased tensile strength. In conclusion, cartilage integration can be achieved using a chondrocyte/collagen-scaffold implant that permits controlled delivery of chondrocytes to both host and graft mature cartilage tissues. This approach has the potential to be used therapeutically for implantation of engineered tissue

TRACP Influences Th1 Pathways by Affecting Dendritic Cell Function

Introduction: TRACP is highly expressed by osteoclasts, activated macrophages, and dendritic cells (DCs). Knockout mice lacking TRACP have an intrinsic defect in osteoclastic resorption and macrophages that display abnormal immunomodulatory responses and cytokine secretion profiles. Our aim in this study was to investigate the significance of TRACP in the inductive phase of the immune response by examining dendritic cells from TRACP?/? mice. Materials and Methods: Maturational state and function of leukocyte subsets in mice was assessed by flow cytometry. The ability of the immune system to respond to nonspecific activation and to specific antigen was assessed by delayed type hypersensitivity and the presence of isotype-specific serum antibody in vivo and T-cell proliferation and cytokine production in vitro. Results: The ability of lipopolysaccharide (LPS) to upregulate MHC II and CD80 in DCs from TRACP?/? mice was reduced compared with wildtype mice, although production of IL-10 by DCs from TRACP-deficient animals was increased. T- and B-cell responses not involving antigen presentation (anti-CD3, TNP-ficoll) were normal in TRACP?/? mice, but responses to T-dependent antigens were impaired. Specifically, TRACP?/? mice had defective delayed hypersensitivity responses to picryl chloride and reduced proliferative responses to ovalbumin compared with wildtype mice. In response to ovalbumin, but not anti-CD3, T cells from TRACP?/? mice produced less interferon-? (IFN-?), but there was no difference in IL-4 production: TRACP?/? mice also produced less ovalbumin (OVA)-specific IgG2a after immunization. Conclusions: The finding that DCs from TRACP?/? mice have impaired maturation and defective Th1 responses shows that TRACP is important for polarizing responses in n