Abstract
The phagocytosis-promoting complement receptor, Complement Receptor Immunoglobulin (CRIg), is exclusively expressed on macrophages. It has been demonstrated that expression in macrophages could be modulated by inflammatory mediators, including cytokines. This raised the possibility that a major phagocyte, the neutrophil, may also express CRIg following activation with inflammatory mediators. Here we show that resting peripheral blood neutrophil lysates subjected to protein analysis by Western blot revealed a 35 kDa CRIg isoform, consistent with the expression of CRIg mRNA by RT-PCR. By flow cytometry, CRIg was detected intracellularly and in very minor amounts on the cell surface. Interestingly, expression on the cell surface was significantly increased to functional levels after activation with inflammatory mediators/neutrophil activators; N-Formylmethionine-leucyl-phenylalanine, tumor necrosis factor (TNF), Granulocyte-Macrophage Colony stimulating Factor (GM-CSF), bacterial lipopolysaccharide, leukotriene B4 and phorbol myristate acetate. The increase in expression required p38 MAP kinase and protein kinase C activation, as well as intracellular calcium. Neutrophils which were defective in actin microfilament reorganization due to a mutation in ARPC1B or inhibition of its upstream regulator, Rac2 lose their ability to upregulate CRIg expression. Inhibition of another small GTPase, Rab27a, with pharmacological inhibitors prevented the increase in CRIg expression, suggesting a requirement for the actin cytoskeleton and exocytosis. Engagement of CRIg on TNF-primed neutrophils with an anti-CRIg monoclonal antibody increased the release of superoxide and promoted the activation of p38 but not ERK1/ERK2 or JNK MAP kinases. The TNF-induced increase in killing of Staphylococcus aureus was blocked by the anti-CRIg antibody. Adding to the anti-microbial role of CRIg, it was found that GM-CSF priming lead to the release of neutrophil extracellular traps. Interestingly in contrast to the above mediators the anti-inflammatory cytokine IL-10 caused a decrease in basal expression and GM-CSF induced increase in CRIg expression. The data demonstrate that neutrophils also express CRIg which is regulated by inflammatory mediators and cytokines. The findings show that the neutrophil antimicrobial function involving CRIg requires priming as a means of arming the cell strategically with microbial invasion of tissues and the bloodstream.
Highlights
CRIg or V-set and immunoglobulin domain–containing 4 (VSIG4) has distinct structural and biological properties from the classical complement receptors, CR3 and CR4 [1, 2]
A significant finding from our work was that expression on the cell surface required activation with inflammatory mediators and this led to the activation of neutrophil functional responses through CRIg engagement with the anti-CRIg monoclonal antibody
CRIg expression by neutrophils may have been previously missed as in our study, we found that expression on the cell surface is poor, unless the neutrophils have been stimulated with inflammatory mediators or agonists
Summary
CRIg or V-set and immunoglobulin domain–containing 4 (VSIG4) has distinct structural and biological properties from the classical complement receptors, CR3 and CR4 [1, 2]. CRIg has been found to suppress the immune response [5, 6] and inhibit the activation of the alternative pathway [7, 8] These properties have further led to the findings that CRIg is protective in a number of autoimmune/chronic inflammation disease models [7, 8]. Our previous work examining CRIg on human monocyte-derived macrophages demonstrated that CRIg expression could be substantially depressed as well as increased by treating macrophages with inflammatory mediators and drugs such as dexamethasone [10, 11]. This raised the possibility that other phagocytes such as neutrophils may express CRIg following activation by inflammatory mediators and cytokines, and was the subject of this study
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