Biomedical subjects
Publications and source records attributed to C Pitzalis.
Advances in molecular genetics, cellular immunology and microbiology have offered promise in unravelling the aetiopathogenesis of inflammatory arthritides such as rheumatoid arthritis and reactive arthritis. Such insights are challenging the orthodox view that these diseases are primarily autoimmune in nature, and should lead to exciting and novel therapeutic approaches.
The accumulation of T cells within the joint is responsible for the perpetuation of synovitis. This process is partly regulated by selective binding to endothelium. However, adhesion to extra-cellular matrix proteins, like FN, may also be important. FN binding is mediated by certain members of the VLA (beta 1 integrin) family of proteins. To investigate the role of Tc-FN interactions in synovitis the binding of synovial fluid (SF) and peripheral blood (PB) T cells to FN-coated wells, and the expression of cell surface VLA molecules on these cells by double label immunofluorescence, were studied. SF T cells bound better to FN than PB T cells. VLA alpha 4 and VLA beta 1 but not VLA alpha 5 were up-regulated on SF compared with PB T cells. Anti-VLA alpha 4, VLA beta 1 and VLA alpha 5 MoAbs inhibited the binding of SF T cells to FN. The increased binding of SF T cells to FN could have been related to activation and/or to their predominantly memory phenotype. Purified resting memory or naive T cells bound poorly to FN. In contrast, compared with SF T cells, concanavalin A-activated T cells showed a very similar level of binding to FN, comparable expression of VLA molecules and the same pattern of inhibition of binding to FN by MoAbs. Thus, VLA molecules may play an important role in the retention of T cells in the joint and since T cells can be activated via VLA-FN interactions, this mechanism may perpetuate chronic inflammation.
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Murine monoclonal antibody (Mab) therapy in patients with rheumatoid arthritis (RA) produces an antimouse immunoglobulin response by the recipient. We studied a chimeric (human/mouse) CD7 Mab, in a dose ranging tolerability study in 10 patients with RA. Modest improvements in disease activity occurred with frequent acute adverse effects of malaise, fever and nausea. After treatment, peripheral blood T lymphocyte numbers fell by 50% and CD7 expression fell by 97% for less than 7 days. Our study demonstrates chimeric Mab function in vivo and illustrates the influence of antibody isotype and patient characteristics on adverse effects.
Natural killer (NK) cells and CD3+ large granular lymphocytes (LGL) were investigated in patients with Felty's syndrome (FS), rheumatoid arthritis (RA) and healthy controls. In most patients with FS, NK cell number and activity were decreased. CD3+ LGL were unchanged. However, in one patient a marked expansion of CD3- CD16+ CD56+ (NK) cells was seen and in a second, an expansion of CD3+ LGL. In FS there was also an increase in HLA- DR+ and CD8+ but not gamma delta+ T cells. Three of 11 patients with FS studied demonstrated a dominant rearrangement of the T cell receptor beta gene constant region consistent with oligoclonal T cell expansion.
We studied the lymphocyte proliferative response to Chlamydia trachomatis in Reiter's syndrome (RS) compared with that in other rheumatic diseases. RS patients showed significantly increased C trachomatis-specific synovial fluid (SF) T cell proliferation. Proliferating cells were found in both CD4+ and CD8+ T cell subsets. The SF lymphocyte proliferative response to C trachomatis in RS was inhibited by anti-class I and class II major histocompatibility complex monoclonal antibodies, while the response to tuberculin purified protein derivative was inhibited only by anti-class II monoclonal antibodies. T cell receptor gamma/delta-bearing T cells were not consistently increased in RS SF compared with peripheral blood, nor did such cells consistently expand upon in vitro culture with C trachomatis. Finally, there was no correlation between the cellular immune response and levels of antibody to C trachomatis antigens. Our results indicate that a specific T cell response to C trachomatis within the joint plays a role in the pathogenesis of RS.
The vast majority of T cells present in chronic inflammatory lesions are of the helper-inducer/memory (CD45RO+CD29+) phenotype; suppressor-inducer/naive cells (CD45RA+) are virtually absent. Furthermore, CD4+ T cells are found more frequently than CD8+ cells. Previous in vitro studies have suggested that this may be, in part, due to the increased capacity of CD45RO+CD29+ T cells to bind to endothelium and, thus, enter inflammatory foci but no in vivo evidence for preferential migration exists. To investigate this, suction blisters were generated over a purified protein derivative of tuberculin-induced delayed-type hypersensitivity lesions in humans and the phenotype of the blister cells was studied. At all time points, a preponderance of CD45RO+CD29+ cells over CD45RA+ cells and of CD4 over CD8 cells was demonstrated. Because of the rapid kinetics, this appears to represent preferential migration of these cell types rather than in situ proliferation or phenotype conversion. In addition the expression of the CD45RO but not the CD45RA antigen was up-regulated on blister T cells compared to blood T cells. Analysis of blister fluid showed high concentrations of interleukin 6 but not tumor necrosis factor-alpha or lymphotoxin. This study shows for the first time directly in vivo that CD45RO+ T cells migrate preferentially into inflammatory lesions. Furthermore, it illustrates the potential usefulness of this system in the analysis of the early phases of the immune inflammatory response.
T cell receptor (TCR) gamma/delta bearing lymphocytes in peripheral blood and synovial fluid from rheumatoid arthritis (RA) and seronegative spondyloarthritides (SSA) were evaluated by means of double label immunofluorescence and cytofluorographic analysis. Three monoclonal antibodies (MAb) were used. TCR delta-1 against a common delta chain epitope, BB3 directed against the T cell subset whose TCR is encoded by the V delta 2 gene, and A13 directed against the V delta 1 subset. Peripheral blood T gamma delta cells were significantly reduced compared to normal control blood in RA patients who had joint effusion but not in other RA patients. The decrease of T gamma delta in the RA PB was mainly confined to the A13+ subset. In RA synovial fluid (RA SF) T gamma delta cells were significantly increased compared to paired but not normal peripheral blood, the most significant being the increase of A13+ cells. In contrast, in patients with SSA, no change in T gamma delta cells was observed on PB or SF. These data suggest that in RA, but not SSA, T gamma delta cells migrate from PB to inflamed synovium and thus may be involved in the pathogenesis of RA.
Six patients with rheumatoid arthritis were treated with a CD7 mouse monoclonal antibody, RFT2, daily for 15 days. Only two patients had a significant improvement in clinical disease activity which lasted 7-14 days. No serious adverse effects occurred although all patients developed antibodies against mouse immunoglobulin. During treatment T-lymphocyte numbers decreased and T-lymphocyte CD7 expression was absent in all but one patient.
The effect of the monoclonal antibody RFT2, directed against CD7, on T cell proliferation in unseparated peripheral blood mononuclear cell populations induced by various stimulants was investigated. The addition of RFT2 to cell cultures inhibited the T cell proliferation induced by tuberculin PPD and OKT3 but not by phytohaemagglutinin, concanavalin A or phorbol myristic acid; RFT2 had to be present during the first 24 h of culture in order to elicit inhibition; inhibition of proliferation was not due to down regulation of interleukin-2 receptor on the surface of T cells; and suppressive effects could be transferred by mononuclear cells pre-treated with RFT2. These results are of particular relevance in view of the known in vivo suppressive effect of RFT2 in humans.
The alteration in the surface of endothelial cells (EC) in response to cytokines is likely to be of great importance to the regulation of cell migration and thereby to the evolution of inflammatory processes. We have generated three mAb against cytokine inducible Ag on EC. Whereas mAb 1.2B6 and 6.5B5 were found to react with ELAM-1 and ICAM-1, respectively, mAb 1.4C3 reacted with a novel molecule that showed a different pattern of expression from ELAM-1 or ICAM-1 after stimulation of EC by TNF, IL-1, or LPS. Like ELAM-1, the 1.4C3 Ag was minimally expressed on resting EC, whereas ICAM-1 was moderately expressed. After stimulation with IL-1, TNF, or LPS, ELAM-1 expression was maximal after 4 to 6 h, 1.4C3 Ag after 6 to 10 h, and ICAM-1 after 10 to 24 h. The duration of 1.4C3 expression was intermediate between ELAM-1 and ICAM-1, and was more prolonged in response to TNF than IL-1 or LPS. Whereas the expression of the three Ag showed a similar dose response to varying concentrations of IL-1 or LPS, EC required a 10-fold higher concentration of TNF for half maximal expression of ELAM-1 than for half maximal expression of 1.4C3 Ag or ICAM-1 (5 ng/ml compared to 0.5 ng/ml). Of the three Ag, only ICAM-1 was enhanced by IFN-gamma. SDS-PAGE under reducing conditions showed the 1.4C3 Ag to migrate as a single band with a relative molecular mass of approximately 95 kDa. mAb 1.4C3 adds to our understanding of the kinetics of the EC response to different cytokines and will be useful in studying the regulation of EC activation. Furthermore, the 1.4C3 molecule may have an important role in leukocyte-EC interactions.
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Monocytes, defined by staining with the Mo-2 and Leu-M3 monoclonal antibodies which both detect the CD14 antigen, were studied in peripheral blood of control subjects and patients with rheumatoid arthritis as well as in rheumatoid synovial fluid of the latter for expression of activation/differentiation markers. The monocytes in the rheumatoid synovial fluids showed increased expression of class II major histocompatibility antigens (HLA-DR and -DQ) and decreased positivity for the peanut agglutinin receptor as compared with those from patient and control peripheral blood. There were no differences in phenotype between the control and patient peripheral blood monocytes. These findings are consistent with the hypothesis that monocytes are activated and differentiate into more mature macrophage-like cells after entry into the rheumatoid joint.
The mononuclear infiltrate found in a variety of inflammatory dermatoses was characterized by a predominance of T helper-inducer lymphocytes (THi), CD4+/CD45RA-/CD45RO+, a population of cells responsible for maintaining and promoting immune reactions. Only small numbers of T-suppressor-inducer lymphocytes (TSi), CD4+/CD45RA+/CD45RO-, cells responsible for inducing CD8 suppressor-effector cells to 'down regulate' immune reactions, were seen. The predominance of CD4+ THi lymphocytes was common to all dermatoses studied and suggests a common final pathway in chronic cutaneous inflammation, irrespective of initial causative factors.
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A prospective study of 33 patients with polymyalgia rheumatica/giant cell arteritis (PMR/GCA) was undertaken, firstly, to monitor sequentially peripheral blood CD8+ lymphocyte levels and, secondly, to assess the expression of activation markers on T lymphocyte subsets. The results indicated that there was a significant decrease in absolute numbers and relative percentages of CD8+ T lymphocytes, which returned to normal ranges after approximately 24 months' treatment, and that there was an increased percentage of CD8+ lymphocytes in PMR/GCA which express HLA class II antigens.