The investigation of citrullinated protein, B-cells and their survival niches in atherosclerosis with coexisting rheumatoid arthritis.
PhD thesis, University of Glasgow.
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Rheumatoid arthritis (RA) is an autoimmune disease that primarily affects thejoints and is characterized by bone and cartilage erosion. These patients have an
increased risk of developing co-morbid cardiovascular disease (CVD). In this study the vascular adventitia from patients with coexisting RA and CVD was investigated to identify factors in the vascular environment that could affect the immune component of RA. This included the histological evaluation of aortic adventitial sections for known RA-associated autoantigens; B cells their survival
factors and associated cytokines. In addition, these human studies were the impetus for the initiation of a study to investigate the effect of B cell depletion
Citrullinated proteins (CP), RA associated antigens, are an inflammation specific entity. These modified proteins are processed by (PADI), a calcium dependent enzyme expressed by inflammatory cells and some microbial species. The presence of RA specific autoantibodies (ACPA) suggests a role for citrullinated proteins in RA pathogenesis. The aim of the present study was to determine whether the inflammatory milieu in the vascular adventitia of RA patients had any association with CP. The expression of CP was confirmed in the vascular adventitia, and CVD patients with coexisting RA were found to have increased levels of CP compared to non-RA CVD patients. In addition, ACPA levels were detectable in serum samples of only RA patients. When ACPA positive RA patients were sub grouped (CP>5.5), a strong association was observed between ACPA levels and C-reactive protein (CRP) in this subgroup (CP>5.5). This is suggestive of a CP and ACPA link in vascular inflammation in patients with coexisting RA.
The presence of B-lymphocytes has been shown in both murine and human vascular adventitia. In this study we demonstrated that the presence of B cell aggregates is tissue specific, since matched internal mammary artery (IMA) had no detectable B cells in the adventitia. Furthermore, BAFF and APRIL are present in the aortic adventitia and support the conclusion that a survival niche is present in the aortic adventitia; cytokine expression was demonstrated in lymphocytes, adipocytes and vascular endothelial cells. Interestingly, smoking resulted in higher levels of BAFF expression in the aortic adventitia of CVD patients. Previous studies have shown that smoking induces vasoconstriction of small vessels (vasa vasorum), and hence hypoxia of the vascular endothelial cells. We hypothesised that hypoxia would induce the increased expression of BAFF and APRIL in vascular endothelial cells. Using murine vascular endothelial cells we investigated the effect of hypoxia on BAFF and APRIL mRNA transcript
levels. However, hypoxia did not have any effect on transcript levels. In addition, significant correlations were observed in RA patients only, between B
cells and APRIL, B cells and CP, APRIL and CP. This part of my thesis demonstrates that the aortic adventitia of RA patients with coronary heart disease is associated with an environment that is conducive to B cell survival and
harbours a known RA-associated self-antigen. Importantly, these insights provide a rationale for the perpetuation of B cell auto-reactivity outside of both lymphoid tissue and the joint in RA.
Previous studies have shown that atherosclerosis is a T cell mediated disease with an imbalance in Th1 and Th2 responses. Macrophages and interleukin 18 (IL-
18, proatherogenic) cytokine have been demonstrated in aortic adventitia of humans in previous studies. However, their expression in the vascular adventitia of RA patients has not been investigated. The present study confirmed
macrophage IL-18 expression in vascular adventitia, which is similar among the Subgroups (RA vs. non RA) and (smokers vs. non smokers). Furthermore, a Th2 phenotype has been proposed as atheroprotective, mediated by anti oxLDL antibody producing B cells. Interestingly, recent studies have demonstrated interleukin 33 (IL-33) a Th2 cytokine to reduce the atherosclerotic lesion in an experimental model of atherosclerosis. However, it’s expression and
significance in human vessels has not been extensively investigated. In this study, using immunohistochemistry, IL-33 was localised to vascular endothelial cells of vasa vasorum in aortic adventitia and lining layer of IMA. Importantly, IL-33 was down regulated in smoker’s AA suggestive of failed protective mechanism
of this Th2 cytokine. In contrast, IL-33 expression was not affected by smoking in the matched IMA tissue.
In the past B-lymphocytes have been shown as an atheroprotective pathway; whereas the use of B cells depletion therapy (Rituximab) suggests a proinflammatory role of these cells in RA. Could B cell depletion therefore
mediate unexpected deficit on vascular lesions in RA? My thesis revealed expression of B cells and their survival factors in the aortic adventitia. Therefore
to investigate the impact of B cell depletion therapy on atherosclerosis, a mouse model (huCD20/ApoE-/-) was generated. The preliminary data are suggestive of
reduction in atherosclerotic plaques with B cells depletion. These studies will be extended in future to formally test the above hypothesis.
In summary I have shown for the first time the cytokine and cellular niche that exists in human aorta in RA patients. Such data will in due course inform the mechanism of accelerated vascular pathogenesis in RA.
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