Neutrophil migration into inflamed tissues is a fundamental component of innate immunity. A decisive step in this process is the polarised migration of blood neutrophils through endothelial cells (ECs) lining the venular lumen (transendothelial cell migration; TEM) in a luminal to abluminal direction. Using real-time confocal imaging we report that neutrophils can exhibit disrupted polarised TEM (“hesitant” and “reverse”) in vivo. These events were noted in inflammation following ischemia-reperfusion injury, characterised by reduced expression of junctional adhesion molecule C (JAM-C) from EC junctions, and were enhanced by EC JAM-C blockade or genetic deletion. The results identify JAM-C as a key regulator of polarised neutrophil TEM in vivo and suggest that reverse TEM neutrophils can contribute to dissemination of systemic inflammation.

Adhesion of parasitized erythrocytes to post-capillary venular endothelium or uninfected red cells is strongly implicated in the pathogenesis of severe Plasmodium falciparum malaria. Neoantigens at the infected red-cell surface adhere to a variety of host receptors, demonstrate serological diversity in field isolates and may also be a target of the host-protective immune response. Here we use sequential cloning of P. falciparum by micromanipulation to investigate the ability of a parasite to switch antigenic and cytoadherence phenotypes. Our data show that antigens at the parasitized cell surface undergo clonal variation in vitro in the absence of immune pressure at the rate of 2% per generation with concomitant modulations of the adhesive phenotype. A clone has the potential to switch at high frequency to a variety of antigenic and adhesive phenotypes, including a new type of cytoadherence behaviour, 'auto-agglutination' of infected erythrocytes. This rapid appearance of antigenic and functional heterogeneity has important implications for pathogenesis and acquired immunity.

Recent studies have demonstrated that neutrophils are not a homogenous population of cells. Here, we have identified a subset of human neutrophils with a distinct profile of cell-surface receptors [CD54(high), CXC chemokine receptor 1(low) (CXCR1(low))], which represent cells that have migrated through an endothelial monolayer and then re-emerged by reverse transmigration (RT). RT neutrophils, when in contact with endothelium, were rescued from apoptosis, demonstrate functional priming, and were rheologically distinct from neutrophils that had not undergone transendothelial migration. In vivo, 1-2% of peripheral blood neutrophils in patients with systemic inflammation exhibit a RT phenotype. A smaller population existed in healthy donors ( approximately 0.25%). RT neutrophils were distinct from naïve circulatory neutrophils (CD54(low), CXCR1(high)) and naïve cells after activation with formyl-Met-Leu-Phe (CD54(low), CXCR1(low)). It is important that the RT phenotype (CD54(high), CXCR1(low)) is also distinct from tissue-resident neutrophils (CD54(low), CXCR1(low)). Our results demonstrate that neutrophils can migrate in a retrograde direction across endothelial cells and suggest that a population of tissue-experienced neutrophils with a distinct phenotype and function are present in the peripheral circulation in humans in vivo.

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Copy number (CN) variation (CNV) has been shown to be common in regions of the genome coding for immune-related genes, and thus impacts upon polygenic autoimmunity. Low CN of FCGR3B has recently been associated with systemic lupus erythematosus (SLE). FcγRIIIb is a glycosylphosphatidylinositol-linked, low affinity receptor for IgG found predominantly on human neutrophils. We present novel data demonstrating that both in a family with FcγRIIIb-deficiency and in the normal population, FCGR3B CNV correlates with protein expression, with neutrophil uptake of and adherence to immune complexes, and with soluble serum FcγRIIIb. Reduced FcγRIIIb expression is thus likely to contribute to the impaired clearance of immune complexes, which is a feature of SLE, explaining the association between low FCGR3B CNV and SLE that we have confirmed in a Caucasian population. In contrast, antineutrophil cytoplasmic antibody–associated systemic vasculitis (AASV), a disease not associated with immune complex deposition, is associated with high FCGR3B CN. Thus, we define a role for FCGR3B CNV in immune complex clearance, a function that may explain why low FCGR3B CNV is associated with SLE, but not AASV. This is the first report of an association between disease-related gene CNV and variation in protein expression and function that may contribute to autoimmune disease susceptibility.

To navigate into and within tissues, leukocytes require guidance cues that enable them to recognize which tissues to enter and which to avoid. Such cues are partly provided at the time of extravasation from blood by an endothelial address code on the luminal surface of the vascular endothelium. Here, we review the evidence that fibroblasts help define an additional stromal address code that directs leukocyte behaviour within tissues. We examine how this stromal code regulates site-specific leukocyte accumulation, differentiation and survival in a variety of physiological stromal niches, and how the aberrant expression of components of this code in the wrong tissue at the wrong time contributes to the persistence of chronic inflammatory diseases.

We examined the hypothesis that stromal fibroblasts modulate the ability of endothelial cells (EC) to recruit lymphocytes in a site-specific manner. PBL were perfused over HUVEC that had been cultured with fibroblasts isolated from the inflamed synovium or the skin of patients with rheumatoid arthritis or osteoarthritis, or from normal synovium, with or without exposure to the inflammatory cytokines TNF-α+IFN-γ. Fibroblasts from inflamed synovium, but no others, caused unstimulated HUVEC to bind flowing lymphocytes. This adhesion was supported by α4β1-VCAM-1 interaction and stabilised by activation of PBL through CXCR4–CXCL12. Antibody neutralisation of IL-6 during co-culture effectively abolished the ability of EC to bind lymphocytes. Cytokine-stimulated EC supported high levels of lymphocyte adhesion, through the presentation of VCAM-1, E-selectin and chemokine(s) acting through CXCR3. Interestingly, co-culture with dermal fibroblasts caused a marked reduction in cytokine-induced adhesion, while synovial fibroblasts had variable effects depending on their source. In the dermal co-cultures, neutralisation of IL-6 or TGF-β caused partial recovery of cytokine-induced lymphocyte adhesion; this was complete when both were neutralised. Exogenous IL-6 was also found to inhibit response to TNF-α+IFN-γ. Normal stromal fibroblasts appear to regulate the cytokine-sensitivity of vascular endothelium, while fibroblasts associated with chronic inflammation bypass this and develop a directly inflammatory phenotype. Actions of IL-6 might be pro-inflammatory or anti-inflammatory, depending on the local milieu.

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