Regulation of Complement Receptor Immunoglobulin (CRIg) Expression by Cytokines

Author: Usma Munawara

Munawara, Usma, 2015 Regulation of Complement Receptor Immunoglobulin (CRIg) Expression by Cytokines, Flinders University, School of Biological Sciences

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The product of a B7 family-related protein V-set and Ig domain-containing 4 was recently identified to have complement binding properties and termed Complement Receptor Immunoglobulin (CRIg). Although it shares the phagocytosis promoting activities of the classical complement receptors, CR3 and CR4, it differs structurally and in biological properties, from these receptors. CRIg is expressed selectively on macrophages and has been found to display anti-inflammatory and immunosuppressive properties. Despite the importance of CRIg in infection, immunity and inflammation there is little known on the regulation of its expression by inflammatory mediators. This was the subject of the present thesis. The research addressed the hypothesis that cytokines differentially regulate CRIg expression in human macrophages, in a manner distinct from their effects on CR3 and CR4 expression. Furthermore, that expression on human dendritic cells is also regulated by cytokines.

The effects of cytokines on the regulation of CRIg expression on macrophages were examined when cytokines were added either to the monocytes or monocyte- derived macrophages (MDM) per se. The effects of these mediators were also examined on monocyte-derived dendritic cells (DC). Expression of complement receptors was evaluated by qRT-PCR as well as protein expression using Western blotting and flow cytometry. Macrophage phagocytosis activity was assessed by measuring the uptake of complement opsonised heat-killed Candida albicans and cytokine production by cytometric bead assay. MDM deficient in PKCα or PKCζ were generated by nucleofection with isozyme specific shRNA. The levels of PKC isozymes in macrophages were determined by Western blots.

The findings show that CRIg expression on macrophages is regulated by several cytokines which can be grouped into Th1, Th2, pyrogenic, immunosuppressive, regulatory and haematopoietic growth factor patterns. The data demonstrated that CRIg was absent in monocytes but the cells began to express the receptor during their culture as they differentiated into macrophages. While, LT-α, IL-1β, IL-6, IL-10, GM-CSF, M-CSF caused an increase, IFN-γ, TNF, TGF-β1, IL-4 and IL-13 induced a decrease in CRIg expression, showing that both Th1 and Th2 cytokines can similarly control expression of CRIg expression on macrophages. Cytokines acting directly on MDM expressing CRIg modulated this expression. However in this case, all of the cytokines apart from GM-CSF and LT-α caused a decrease in expression. Interestingly in both of the above scenarios the majority of cytokines had opposite effects on CR3 and CR4 expression. Cytokine induced modulation of complement receptor expression was not limited to MDM. The results showed that in monocyte-derived DC, CRIg, CR3 and CR4 expression could be either increased or decreased by cytokines but again the effects differed between CRIg and the classical complement receptors.

The mechanisms of regulation of CRIg expression in MDM were explored and the results revealed that PKCα is critical in the cytokine induced modulation of CRIg expression. MDM rendered PKCα deficient by nucleofection with shRNA showed increased expression of CRIg and showed significant reduction in the LPS-induced CRIg down regulation. The importance of TNF as a key regulatory cytokine was shown as the addition of TNF neutralisation antibody also increased CRIg expression. Although limited studies were conducted on functional aspects of CRIg expressing macrophages, the data revealed that increased and decreased CRIg expression correlated with the respective changes in phagocytosis of complement-opsonised C. albicans by macrophages and not with changes in CR3 and CR4. Furthermore MDM induced to express increased amounts of CRIg over CR3 while showing increased phagocytosis of complement opsonised fungi, released significantly less TNF. The effects of cytokines were evident when measuring CRIg protein e.g. by Western blotting as well as CRIg mRNA, suggesting that regulation is likely to be at a pre-transcriptional level. MDM expressed two forms of CRIg the long and short forms but interestingly DC expressed these as well as an additional intermediatory form. The different forms were similarly regulated by cytokines.

The thesis represents the first comprehensive study on how inflammatory mediators regulate CRIg expression in cells relevant to both innate and adaptive immunity. In the evolution of the inflammatory reaction macrophages encountering microbial pathogens release cytokines which in the main down regulate CRIg and enable the generation of a cascade of mediator/cytokine release which cause monocyte infiltration to the site which differentiate into macrophages which express CRIg levels dictated by the cytokines. Our data identified TNF and PKCα as a key intercellular and intracellular signalling molecules in this expression and that these may function by differentially regulating CRIg versus CR3/CR4 in the inflammatory sites. Our findings have significance in the adaptive immune response by showing that cytokines alter the expression of CRIg on DC. Accordingly cytokines (IL-10, TGF-β1) and agents (dexamethasone, PKC inhibitors) which upregulate CRIg expression promote the development of tolerogenic DC and those which decrease expression (TNF, IFN-γ) break the tolerance function of DC.

The findings not only increase our knowledge of the immune-biology of CRIg but are likely to lead to better interpretation of action of anti-inflammatory drugs including anti-TNF therapy in diseases such as rheumatoid arthritis (RA).

Keywords: Complement receptors, CRIg, CR3, CR4, cytokines, monocytes, macrophages, dendritic cells, inflammation, PKCα, phagocytosis.

Subject: Pathology thesis, Biological Sciences thesis

Thesis type: Doctor of Philosophy
Completed: 2015
School: School of Biological Sciences
Supervisor: Catherine A. Abbott