Complement Receptor Immunoglobulin (CRIg): expression and function in human phagocytes

Abstract

The fifth human complement receptor, complement receptor immunoglobulin (CRIg; VSIG4; Z39Ig), was first documented in the year 2000. Since this initial discovery, CRIg has been described as a highly efficient phagocytosis promoting complement receptor, a player in the regulation of homeostasis, a regulator of the alternative pathway of complement, and also as a regulator of T cell activation. However, despite this broad range of attributed functions, CRIg remains to be the lesser known receptor of the complement receptor family; this is likely owing to the current lack of clarification as to its cellular expression in humans, mystery surrounding the mechanisms by which expression is controlled, a lack of commercially available antibodies, along with an inability to directly relate findings in murine models with human disease. Thus, before we can begin to investigate the role of CRIg in immune disease and how CRIg may potentially be used therapeutically, we must first come to understand the role of CRIg in the human state of health. In this thesis, a comprehensive study into the immune cellular expression of CRIg in healthy human phagocytes will be presented as we attempt to unravel the uncertainties which cloud the field of CRIg biology. Currently, there are limited antibodies available which detect human and murine CRIg, and thus, the investigation presented herein will begin with the development and screening of multiple new, cross-reactive rat anti-murine CRIg monoclonal antibodies suitable for use in a range of methodologies, including Western blot, flow cytometry, and immunohistochemistry. These antibodies stand to be the first of their kind. Next, we performed an in-depth investigation into the influence of cytokines/inflammatory mediators on CRIg expression by human monocyte-derived macrophages and dendritic cells. We observed a differential effect of cytokines/mediators on macrophage CRIg expression which was dependent on the developmental state of the cells, and demonstrated that those agents which induced an upregulation of CRIg protein expression also enhanced the ability of the cells to phagocytose the fungal pathogen, Candida albicans, identifying a control point through which these mediators act. In dendritic cells, we observed a similar influence of cytokines/mediators over CRIg expression, and found that cells stimulated to express high protein levels of CRIg negatively regulated the T cell proliferative response to phytohaemagglutinin and allogeneic stimulation, and reduced the cytokine production of T cells in response to allogeneic stimulation. This negative regulation could be inhibited by the addition of an anti-CRIg antibody to the cultures, and thus identifies CRIg as a key effector molecule in dendritic cell-control over the T cell response. The steroid hormone vitamin D has vitally important roles in many processes, including in the modulation of the immune response. With this in consideration, along with the growing global concern that is vitamin D deficiency, we next assessed the relationship between vitamin D and CRIg expression by human monocyte-derived macrophages. We observed that cells matured in the presence of the active form of vitamin D showed a marked increase in CRIg protein and mRNA expression, and that this increase correlated with the ability of the cells to phagocytose the pathogens Candida albicans and Staphylococcus aureus. Interestingly, treatment had no effect on the expression of the ‘classical’ complement receptors, CR3 or CR4, indicating that vitamin D promotes innate immune defence through promoting CRIg expression by macrophages. Finally, in our endeavour to recharacterise CRIg in the state of human health, we demonstrate that CRIg expression in humans is not restricted to tissue-resident macrophages as previously described. For the first time, we present evidence of the expression and regulation of CRIg on the cell surface of human circulating phagocytes; monocytes and neutrophils. We additionally assessed this expression in comparison with cells from a case of immunodeficiency caused by a novel mutation in ARPC1B. We found that healthy human neutrophils express functional CRIg protein within intracellular stores, and upon stimulation with endogenous and exogenous inflammatory mediators, export this protein to the cell surface. This was in comparison with neutrophils which have an inability to polymerise microfilaments due to a mutation in ARPC1B, which failed to upregulate CRIg expression. Similarly, inhibition of the upstream regulator of Arp2/3, Rac-2 inhibited the ability of healthy neutrophils to upregulate surface CRIg protein, indicating that CRIg upregulation on the cell surface of neutrophils is a result of exocytosis. Together, the findings to be presented herein represent a substantial increase in our knowledge of the biology of CRIg in healthy human systems, and with the information gained, it will form a backbone for future comprehensive studies into the role of CRIg in immunity and in inflammatory diseased states, such as in rheumatoid arthritis and type 1 diabetes.