Oliver Haworth

Identification of a phenotypically and functionally distinct population of long-lived neutrophils in a model of reverse endothelial migration

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 [CD54high, CXC chemokine receptor 1low (CXCR1low)], 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 (≈0.25%). RT neutrophils were distinct from naïve circulatory neutrophils (CD54low, CXCR1high) and naïve cells after activation with formyl‐Met‐Leu‐Phe (CD54low, CXCR1low). It is important that the RT phenotype (CD54high, CXCR1low) is also distinct from tissue‐resident neutrophils (CD54low, CXCR1low). 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.

Endosialin (TEM1, CD248) is a marker of stromal fibroblasts and is not selectively expressed on tumour endothelium

Fibroblasts are a diverse cell type and display clear topographic differentiation and positional memory. In a screen for fibroblast specific markers we have characterized four monoclonal antibodies to endosialin (TEM1/CD248). Previous studies have reported that endosialin is a tumour endothelium marker and is localized intracellularly. We demonstrate conclusively that endosialin is a cell surface glycoprotein and is predominantly expressed by fibroblasts and a subset of pericytes associated with tumour vessels but not by tumour endothelium. These novel antibodies will facilitate the isolation and classification of fibroblast and pericyte lineages as well as the further functional analysis of endosialin.

Monocytes/macrophages express chemokine receptor CCR9 in rheumatoid arthritis and CCL25 stimulates their differentiation

IntroductionMonocytes/macrophages accumulate in the rheumatoid (RA) synovium where they play a central role in inflammation and joint destruction. Identification of molecules involved in their accumulation and differentiation is important to inform therapeutic strategies. This study investigated the expression and function of chemokine receptor CCR9 in the peripheral blood (PB) and synovium of RA, non-RA patients and healthy volunteers.MethodsCCR9 expression on PB monocytes/macrophages was analysed by flow cytometry and in synovium by immunofluorescence. Chemokine receptor CCR9 mRNA expression was examined in RA and non-RA synovium, monocytes/macrophages from PB and synovial fluid (SF) of RA patients and PB of healthy donors using the reverse transcription polymerase chain reaction (RT-PCR). Monocyte differentiation and chemotaxis to chemokine ligand 25 (CCL25)/TECK were used to study CCR9 function.ResultsCCR9 was expressed by PB monocytes/macrophages in RA and healthy donors, and increased in RA. In RA and non-RA synovia, CCR9 co-localised with cluster of differentiation 14+ (CD14+) and cluster of differentiation 68+ (CD68+) macrophages, and was more abundant in RA synovium. CCR9 mRNA was detected in the synovia of all RA patients and in some non-RA controls, and monocytes/macrophages from PB and SF of RA and healthy controls. CCL25 was detected in RA and non-RA synovia where it co-localised with CD14+ and CD68+ cells. Tumour necrosis factor alpha (TNFα) increased CCR9 expression on human acute monocytic leukemia cell line THP-1 monocytic cells. CCL25 induced a stronger monocyte differentiation in RA compared to healthy donors. CCL25 induced significant chemotaxis of PB monocytes but not consistently among individuals.ConclusionsCCR9 expression by monocytes is increased in RA. CCL25 may be involved in the differentiation of monocytes to macrophages particularly in RA.

Resolving the problem of persistence in the switch from acute to chronic inflammation

The rapid induction of an inflammatory response is crucial for effective host defense. A wide range of proinflammatory agents ensure that this response occurs. Prominent among these are members of the prostaglandin and leukotriene family that are enzymatically derived from arachidonic acid (AA, C20:4) (1). At the onset of inflammation, AA is metabolized via either cyclooxygenases (COX) to produce prostaglandins (PGs) and thromboxanes or lipoxygenases to generate leukotrienes (LTs) (1). These ecicosanoids induce and help establish the inflammatory infiltrate. However, at some point during the inflammatory cascade, inhibition and ultimately resolution of the inflammatory response must occur; otherwise, tissue homeostasis cannot occur and inflammation persists.

A role for the integrin α6β1 in the differential distribution of CD4 and CD8 T-cell subsets within the rheumatoid synovium

OBJECTIVEnCD4 and CD8 T-cell subsets accumulate in distinct microdomains within the inflamed rheumatoid synovium. The molecular basis for their differential distribution remains unclear. Since chemokines and adhesion molecules play an important role in the positioning of leucocytes at sites of inflammation, we tested the hypothesis that the differential expression and function of chemokine and/or adhesion molecules explains why CD4(+) T cells accumulate within perivascular cuffs, whereas CD8(+) T cells distribute diffusely within the tissue.nnnMETHODSnExpression of an extensive panel of chemokine receptors and adhesion molecules on matched CD4(+) and CD8(+) T cells from peripheral blood (PB) and synovial fluid (SF) was analysed by multicolour flow cytometry. Migration assays and flow-based adhesion assays were used to assess the functional consequences of any differences in the expression of chemokine and adhesion receptors.nnnRESULTSnCD4(+) and CD8(+) T cells from PB and SF expressed unique yet consistent patterns of chemokine and adhesion receptors. SF CD8(+) T cells were much less promiscuous in their expression of chemokine receptors than SF CD4(+) T cells. The alpha(6)beta(1) integrin was highly expressed on PB CD4(+) T cells, but not on PB CD8(+) T cells. Laminin, the ligand for alpha(6)beta(1), retained CD4(+) T cells, but less so CD8(+) T cells, within inflamed synovial tissue.nnnCONCLUSIONnInfiltrating PB CD4(+) T cells, but not CD8(+) T cells, express functional levels of the alpha(6)beta(1) integrin. We propose that this leads to their retention within the rheumatoid synovium in perivascular cuffs, which are defined and delineated by the expression of laminin.

Differential expression of CD148 on leukocyte subsets in inflammatory arthritis

IntroductionMonocytic cells play a central role in the aetiology of rheumatoid arthritis, and manipulation of the activation of these cells is an approach currently under investigation to discover new therapies for this and associated diseases. CD148 is a transmembrane tyrosine phosphatase that is highly expressed in monocytes and macrophages and, since this family of molecules plays an important role in the regulation of cell activity, CD148 is a potential target for the manipulation of macrophage activation. For any molecule to be considered a therapeutic target, it is important for it to be increased in activity or expression during disease.MethodsWe have investigated the expression of CD148 in two murine models of arthritis and in joints from rheumatoid arthritis (RA) patients using real-time PCR, immunohistochemistry, and studied the effects of proinflammatory stimuli on CD148 activity using biochemical assays.ResultsWe report that CD148 mRNA is upregulated in diseased joints of mice with collagen-induced arthritis. Furthermore, we report that in mice CD148 protein is highly expressed in infiltrating monocytes of diseased joints, with a small fraction of T cells also expressing CD148. In human arthritic joints both T cells and monocytes expressed high levels of CD148, however, we show differential expression of CD148 in T cells and monocytes from normal human peripheral blood compared to peripheral blood from RA and both normal and RA synovial fluid. Finally, we show that synovial fluid from rheumatoid arthritis patients suppresses CD148 phosphatase activity.ConclusionsCD148 is upregulated in macrophages and T cells in human RA samples, and its activity is enhanced by treatment with tumour necrosis factor alpha (TNFα), and reduced by synovial fluid or oxidising conditions. A greater understanding of the role of CD148 in chronic inflammation may lead to alternative therapeutic approaches to these diseases.

Stromal cells as new therapeutic targets in rheumatoid arthritis

Rheumatoid arthritis is a painful, debilitating disease in which inflammation is localized mainly to synovial joints. Despite a plethora of therapies targeted at inflammatory cells such as lymphocytes, there has been little impact made on affecting a cure. Even with the most effective current treatments (antitumor necrosis factor-α agents), only about 50% of patients gain a 70% response as measured by conventional disease activity criteria (ARCxa070). In recent years, stromal cells that define the microenvironment in which inflammation occurs have been shown to play an important role in the pathogenesis of rheumatoid arthritis, especially during the switch from acute to chronic persistent disease. In this review, the role that stromal cells, such as endothelial cells, fibroblasts and macrophages play in the pathogenesis of rheumatoid arthritis are examined, and it is suggested that these cells in addition to inflammatory immune cells provide an important and underappreciated therapeutic target.