Helen M. McGettrick

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.

Fibroblasts from different sites may promote or inhibit recruitment of flowing lymphocytes by endothelial cells

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.

Identification and angiogenic role of the novel tumor endothelial marker CLEC14A

Tumor endothelial markers (TEMs) that are highly expressed in human tumor vasculature compared with vasculature in normal tissue hold clear therapeutic potential. We report that the C-type lectin CLEC14A is a novel TEM. Immunohistochemical and immunofluorescence staining of tissue arrays has shown that CLEC14A is strongly expressed in tumor vasculature when compared with vessels in normal tissue. CLEC14A overexpression in tumor vessels was seen in a wide range of solid tumor types. Functional studies showed that CLEC14A induces filopodia and facilitates endothelial migration, tube formation and vascular development in zebrafish that is, CLEC14A regulates pro-angiogenic phenotypes. CLEC14A antisera inhibited cell migration and tube formation, suggesting that anti-CLEC14A antibodies may have anti-angiogenic activity. Finally, in endothelial cultures, expression of CLEC14A increased at low shear stress, and we hypothesize that low shear stress due to poor blood flow in the disorganized tumor vasculature induces expression of CLEC14A on tumor vessels and pro-angiogenic phenotypes.

Shear Stress Regulated Gene Expression and Angiogenesis in Vascular Endothelium

The behavior of vascular EC is greatly altered in sites of pathological angiogenesis, such as a developing tumor or atherosclerotic plaque. Until recently it was thought that this was largely due to abnormal chemical signaling, i.e., endothelial cell chemo transduction, at these sites. However, we now demonstrate that the shear stress intensity encountered by EC can have a profound impact on their gene expression and behavior. We review the growing body of evidence suggesting that mechanotransduction, too, is a major regulator of pathological angiogenesis. This fits with the evolving story of physiological angiogenesis, where a combination of metabolic and mechanical signaling is emerging as the probable mechanism by which tight feedback regulation of angiogenesis is achieved in vivo.

Crosstalk Between Mesenchymal Stem Cells and Endothelial Cells Leads to Downregulation of Cytokine‐Induced Leukocyte Recruitment

Mesenchymal stem cells (MSC) have immunomodulatory properties, but their effects on endothelial cells (EC) and recruitment of leukocytes are unknown. We cocultured human bone marrow‐derived MSC with EC and found that MSC could downregulate adhesion of flowing neutrophils or lymphocytes and their subsequent transendothelial migration. This applied for EC treated with tumor necrosis factor‐α (TNF), interleukin‐1β (IL‐1), or TNF and interferon‐γ combined. Supernatant from cocultures also inhibited endothelial responses. This supernatant had much higher levels of IL‐6 than supernatant from cultures of the individual cells, which also lacked inhibitory functions. Addition of neutralizing antibody against IL‐6 removed the bioactivity of the supernatant and also the immunomodulatory effects of coculture. Studies using siRNA showed that IL‐6 came mainly from the MSC in coculture, and reduction in production in MSC alone was sufficient to impair the protective effects of coculture. Interestingly, siRNA knockdown of IL‐6‐receptor expression in MSC as well as EC inhibited anti‐inflammatory effects. This was explained when we detected soluble IL‐6R receptor in supernatants and showed that receptor removal reduced the potency of supernatant. Neutralization of transforming growth factor‐β indicated that activation of this factor in coculture contributed to IL‐6 production. Thus, crosstalk between MSC and EC caused upregulation of production of IL‐6 by MSC which in turn downregulated the response of EC to inflammatory cytokines, an effect potentiated by MSC release of soluble IL‐6R. These studies establish a novel mechanism by which MSC might have protective effects against inflammatory pathology and cardiovascular disease. Stem Cells 2013;31:2690–2702

Chemokine- and adhesion-dependent survival of neutrophils after transmigration through cytokine-stimulated endothelium

We examined the fate of neutrophils following transmigration through an endothelial monolayer cultured on “Transwell” membrane filters. Treatment of human umbilical vein endothelial cells (HUVEC) with increasing doses of tumor necrosis factor‐α increased the efficiency of transmigration and markedly reduced apoptosis among the transmigrated neutrophils in a dose‐dependent manner. Apoptosis was also inhibited after transmigration of neutrophils through HUVEC stimulated with interleukin (IL)‐1β but not so effectively after chemotaxis through unstimulated HUVEC driven by IL‐8 added below the filter. Inhibition of β2‐integrin binding after transmigration or coating the lower chamber with a nonadhesive polymer (polyhydroxyl‐ethyl‐methacrylate) abrogated neutrophil survival. Although integrin engagement during migration itself was not essential to inhibit apoptosis, activation of neutrophils through CXC chemokine receptors was necessary. Quite brief exposure to the HUVEC (30–120 min) was effective in reducing subsequent apoptosis, although if coincubation with the HUVEC were prolonged, neutrophil apoptosis was reduced further. Neutralization of granulocyte macrophage‐colony stimulating factor inhibited this additional effect. Thus, a complex interplay between migration‐ and activation‐dependent signals and adhesive interaction in tissue may combine to effectively prolong the survival of neutrophils recruited during inflammation.

Duffy antigen receptor for chemokines and CXCL5 are essential for the recruitment of neutrophils in a multicellular model of rheumatoid arthritis synovium

OBJECTIVE The role of chemokines and their transporters in rheumatoid arthritis (RA) is poorly described. Evidence suggests that CXCL5 plays an important role, because it is abundant in RA tissue, and its neutralization moderates joint damage in animal models of arthritis. Expression of the chemokine transporter Duffy antigen receptor for chemokines (DARC) is also up-regulated in early RA. The aim of this study was to investigate the role of CXCL5 and DARC in regulating neutrophil recruitment, using an in vitro model of RA synovium. METHODS To model RA synovium, RA synovial fibroblasts (RASFs) were cocultured with endothelial cells (ECs) for 24 hours. Gene expression in cocultured cells was investigated using TaqMan gene arrays. The roles of CXCL5 and DARC were determined by incorporating cocultures into a flow-based adhesion assay, in which their function was demonstrated by blocking neutrophil recruitment with neutralizing reagents. RESULTS EC-RASF coculture induced chemokine expression in both cell types. Although the expression of CXC chemokines was modestly up-regulated in ECs, the expression of CXCL1, CXCL5, and CXCL8 was greatly increased in RASFs. RASFs also promoted the recruitment of flowing neutrophils to ECs. Anti-CXCL5 antibody abolished neutrophil recruitment by neutralizing CXCL5 expressed on ECs or when used to immunodeplete coculture-conditioned medium. DARC was also induced on ECs by coculture, and anti-Fy6 antibody or small interfering RNA targeting of DARC expression effectively abolished neutrophil recruitment. CONCLUSION This study is the first to demonstrate, in a model of human disease, that the function of DARC is essential for editing the chemokine signals presented by ECs and for promoting unwanted leukocyte recruitment.

Direct observations of the kinetics of migrating T-cells suggest active retention by endothelial cells with continual bidirectional migration.

The kinetics and regulatory mechanisms of T cell migration through the endothelium have not been fully defined. In experimental, filter‐based assays in vitro, transmigration of lymphocytes takes hours, compared with minutes, in vivo. We cultured endothelial cell (EC) monolayers on filters, solid substrates, or collagen gels and treated them with TNF‐α, IFN‐γ, or both prior to analysis of lymphocyte migration in the presence or absence of flow. PBL, CD4+ cells, or CD8+ cells took many hours to migrate through EC‐filter constructs for all cytokine treatments. However, direct microscopic observations of EC filters, which had been mounted in a flow chamber, showed that PBL crossed the endothelial monolayer in minutes and were highly motile in the subendothelial space. Migration through EC was also observed on clear plastic, with or without flow. After a brief settling without flow, PBL and isolated CD3+ or CD4+ cells crossed EC in minutes, but the numbers of migrated cells varied little with time. Close observation revealed that lymphocytes migrated back and forth continuously across endothelium. Under flow, migration kinetics and the proportions migrating back and forth were altered little. On collagen gels, PBL again crossed EC in minutes and migrated back and forth but showed little penetration of the gel over hours. In contrast, neutrophils migrated efficiently through EC and into gels. These observations suggest a novel model for lymphoid migration in which EC support migration but retain lymphocytes (as opposed to neutrophils), and additional signal(s) are required for onward migration.

The autoimmune-associated genetic variant PTPN22 R620W enhances neutrophil activation and function in patients with rheumatoid arthritis and healthy individuals

Objectives A genetic variant of the leukocyte phosphatase PTPN22 (R620W) is strongly associated with autoimmune diseases including rheumatoid arthritis (RA). Functional studies on the variant have focussed on lymphocytes, but it is most highly expressed in neutrophils. We have investigated the effects of the variant on neutrophil function in health and in patients with RA. Methods Healthy individuals and patients with RA were genotyped for PTPN22 (R620W) and neutrophils isolated from peripheral blood. Neutrophil adhesion and migration across inflamed endothelium were measured. Calcium (Ca2+) release and reactive oxygen species (ROS) production in response to fMLP stimulation were also assessed. Results Expression of R620W enhanced neutrophil migration through cytokine activated endothelium (non-R620W=24%, R620W=45% migrating cells, p<0.001). Following fMLP stimulation, neutrophils that were heterozygous and homozygous for R620W released significantly more Ca2+ when compared to non-R620W neutrophils, in healthy individuals and patients with RA. fMLP stimulation, after TNF-α priming, provoked more ROS from neutrophils heterozygous for R620W in patients with RA (non-R620W vs R620W=∼1.75-fold increase) and healthy individuals (non-R620W vs R620W=fourfold increase) and this increase was statistically significant in healthy individuals (p<0.001) but not in patients with RA (p<0.25). Conclusions Expression of PTPN22 (R620W) enhanced neutrophil effector functions in health and RA, with migration, Ca2+ release and production of ROS increased. Neutrophils are found in large numbers in the RA joint, and this hyperactivity of R620W cells may directly contribute to the joint damage, as well as to the initiation and perpetuation of the chronic immune-mediated inflammatory processes driving the disease.

Stromal cells differentially regulate neutrophil and lymphocyte recruitment through the endothelium.

Stromal fibroblasts modify the initial recruitment of leucocytes by endothelial cells (EC), but their effects on subsequent transendothelial migration remain unclear. Here, EC and dermal or synovial fibroblasts were cultured on opposite surfaces of 3‐μm pore filters and incorporated in static or flow‐based migration assays. Fibroblasts had little effect on tumour necrosis factor‐α‐induced transendothelial migration of neutrophils, but tended to increase the efficiency of migration away from the endothelium. Surprisingly, similar close contact between EC and fibroblasts strongly reduced lymphocyte migration in static assays, and nearly abolished stable lymphocyte adhesion from flow. Fibroblasts did not alter endothelial surface expression of adhesion molecules or messenger RNA for chemokines. Inhibition of attachment did not occur when EC‐fibroblast contact was restricted by using 0·4‐μm pore filters, but under these conditions pre‐treatment with heparinase partially inhibited adhesion. In the 3‐μm pore co‐cultures, inhibition of metalloproteinase activity partially recovered lymphocyte adhesion, but addition of CXCL12 (SDF‐1α) to the endothelial surface did not. Hence, the ability of EC to present activating chemokines for lymphocytes may have been enzymatically inhibited by direct contact with fibroblasts. To avoid contact, we cultured EC and fibroblasts on separate 3‐μm pore filters one above the other. Here, fibroblasts promoted the transendothelial migration of lymphocytes. Fibroblasts generate CXCL12, but blockade of CXCL12 receptor had no effect on lymphocyte migration. While stromal cells can provide signal(s) promoting leucocyte migration away from the sub‐endothelial space, direct cell contact (which might occur in damaged tissue) may cause disruption of chemokine signalling, specifically inhibiting lymphocyte rather than neutrophil recruitment.