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AIMS: To determine the clinical, haematological, biochemical and rheological changes that occur in the asymptomatic steady state of sickle cell anaemia. METHODS: Patient self-assessment visual analogue scores (for wellbeing and tiredness), the blood concentration of acute phase proteins (C-reactive protein, orosomucoid, and fibrinogen), and blood rheology (percentage of dense cells and the number of sickled cells that occluded pores 5 microns in diameter) were studied longitudinally on 10 occasions in each of 20 outpatients with sickle cell anaemia. RESULTS: Patients in the steady state showed fluctuation in visual analogue scores, in concentration of acute phase proteins, and in rheological parameters consistent with minor episodes of tissue injury. Significantly more variation in acute phase proteins occurred in the steady state of 14 of the 20 patients who developed one or more vaso-occlusive crises during the 16 month study period. Rheological fluctuation in the steady state simulated rheological change during crisis, namely a transient rise and then fall in the number of dense and poorly filterable cells. CONCLUSIONS: The term "steady state" is a misnomer, being characterised by biochemical and rheological fluctuation consistent with minor episodes of microvascular occlusion that are insufficient to cause the overt tissue infarction of painful crisis.

Background and objectives Mesenchymal stem cells (MSC) act as endogenous regulators of the inflammatory infiltrate, communicating with blood vascular endothelial cells (EC) to limit leukocyte recruitment. Indeed exploiting these properties therapeutically is the principle underpinning clinical trials into chronic inflammatory diseases, including rheumatoid arthritis (RA). However, chronic inflammation is associated changes in the phenotype or function of MSC and their progeny, including ectopic fat deposits, which might represent damage-induced aberrant MSC differentiation. Here we examine how adipogenic differentiation of MSC influenced EC recruitment of leukocytes. Methods Co-cultures of EC with either MSC or MSC-derived adipocytes were formed on either side of porous filters and stimulated with tumour necrosis factor α (TNFα) for 4h. Neutrophil adhesion from flow was assessed by phase contrast microscopy. Microarray analysis was performed on EC and MSC following co-culture and assessed using BIOGRID and REACTOME. Metabolomic analysis was performed using mass spectrometry and analysed using PUTMEDID-LCMS workflows operating in the Taverna workflow environment. Results MSC were immunosuppressive, inhibiting neutrophil recruitment to TNFα-treated EC. Conditioned media from MSC-EC co-cultures, but not MSC mono-cultures, was able to recapitulate these effects and IL-6 was identified as the bioactive mediator. In co-culture, EC up-regulated 464 genes linked cytokine (eg, IL-6, IL-1) and chemokine (e.g. CXCL2) signalling pathways and leukocyte-endothelial interactions (eg, E-selectin). Moreover, 49 metabolites were up-regulated in EC following co-culture with MSC, including arachidonic acid metabolism, tryptophan and tyrosine metabolism, branched chain amino acids and sphingolipid metabolism. In contrast, MSC-derived adipocytes were no longer able to suppress PMN adhesion to inflamed EC. Neutralisation of IL-6 reversed the stimulatory effects of MSC-derived adipocytes in co-culture. Here we identified >40 extracellular metabolites differentially expressed between the EC-MSC and EC-MSC-derived adipocyte co-cultures. Conclusions Thus in the correct environment MSC act to endogenously dampen EC responses to cytokines, thereby limiting leukocyte recruitment in an IL-6 dependent manner. However, upon differentiation MSC lose this intrinsic immuno-protective capacity, adopting a more stimulatory, pro-inflammatory behaviour. Alterations in the phenotype and function of MSC may contribute to pathogenic inflammatory responses by altering the way in which IL-6 exerts its effects from anti-inflammatory to pro-inflammatory.

Objectives Fibroblasts actively regulate the recruitment of leukocytes by endothelial cells (EC), acting in a pro- or anti-inflammatory manner depending on their site of origin. The phenotype of the fibroblast may be a critical determinant of whether leukocyte recruitment, and therefore inflammation, resolves or persists. Here we examined how synovial fibroblasts from different stages of arthritis influenced the recruitment of peripheral blood lymphocytes (PBL) and their onward migration. Methods Fibroblasts were isolated from patients with resolving or persistent arthritis. Rheumatoid arthritis (RA) cohorts were categorised based on the stage of disease at the time of sample collection: very early (≤ 12wk duration); newly presented established (>12 wk duration) or long established undergoing replacement surgery. Endothelial-fibroblast co-cultures were formed on either side of porous filters, and PBL adhesion was assessed using phase-contrast microscopy. Results Rheumatoid fibroblasts increased PBL recruitment in a disease duration-dependent manner (early RA Conclusions Fibroblasts from acutely resolving and early persistent arthritis are distinct. The latter lose immunosuppressive capability and acquire modifications towards a pro-inflammatory phenotype early in the disease process, even before clinical criteria are fulfilled. They may thus induce recruitment or inappropriately support cytokine-driven recruitment, leading to an accumulation of PBL in the rheumatoid joint.

Introduction Heterozygous mutations in the gene encoding bone morphogenetic protein (BMP) receptor II (BMPR-II) are present in >70% of patients with heritable pulmonary arterial hypertension (hPAH) and 15–26% of idiopathic PAH (iPAH) cases. The genetic data strongly implicates the endothelium as the initiating cell type in PAH, and BMP9 is the major circulating ligand for BMPR-II on endothelial cells (EC). Since inflammation is emerging as a potential trigger for disease in BMPR-II mutation carriers, we hypothesised that BMP9 and BMPR-II mutation might impact on endothelial:leukocyte interaction. Methods We employed blood outgrowth endothelial cells (BOEC) isolated from healthy donors (control-BOEC) and BOEC derived from PAH patients with BMPR-II mutations (PAH-BOEC) in this study.BOEC can be generated from venous blood and are recognised as a non-invasive source of patient-derived EC. Endothelial:neutrophil interaction was studied using an in vitro flow-based model. Neutrophil recruitment to LPS (100 ng/ml; 4h)-stimulated BOECs was quantified in the presence or absence of BMP9 (5 ng/ml; 16h) under physiologically relevant sheer stress (0.1 Pa). Surface expression of E-selectin was quantified using flow cytometry, ELISA was used to assess the secretion of IL-8 and mRNA levels were quantified by qPCR. Results LPS stimulation induced robust neutrophil recruitment to control-BOEC, whereas BMP9 had no effect.Co-stimulation with LPS and BMP9 resulted in a synergistic increase in neutrophil recruitment and control-BOEC E-selectin and IL-8 expression, compared to stimulation with LPS alone. Moreover, BMP9 stimulated the upregulation of TLR4 on control-BOEC (the receptor for LPS), thus providing a potential mechanism for the enhanced inflammatory response seen with LPS and BMP9 co-stimulation. In contrast to the findings in control-BOEC, LPS stimulation failed to induce neutrophil recruitment in PAH-BOEC. However, co-stimulation with LPS and BMP9 restored neutrophil recruitment in PAH-BOEC. Conclusions BMP9 markedly enhances neutrophil recruitment to the endothelium in response to LPS. Deficiency in BMP9 signalling (as a consequence of BMPR-II mutations in PAH-BOEC) impairs this process, and BMP9 restores normal endothelial:neutrophil interactions in PAH-BOECs. These findings identify a hitherto unknown role for BMP9 in endothelial:neutrophil interaction and implicate this process in the pathobiology of PAH.

T-cells are recruited from the blood into extra-vascular tissues during acute inflammation. However, in chronic inflammatory diseases, including atherosclerosis, an inappropriate accumulation of T-cells in the diseased tissue contributes to pathogenesis. Very little is known about the mechanisms by which T-cell trafficking is regulated during inflammation, and it is thus difficult to target this aspect of pathology for the development of new anti-atherogenic therapies. Here we describe a unique immune regulatory peptide that imposes a tonic inhibition of T-cell trafficking during inflammation. PEPtide Inhibitor of Trans-Endothelial Migration (PEPITEM) introduces a new paradigm into the pathways that regulate the inflammatory response. We propose that loss of this regulatory pathway makes the immune system ‘leaky’, allowing inappropriate access of T-cells to vulnerable tissues in chronic diseases. Lymphocyte trafficking was assessed in vitro using videomicroscopy on TNF-α/IFN-γ activated endothelial cells (EC) and lymphocytes isolated from healthy donors or patients with chronic inflammatory disease. In vivo , lymphocyte recruitment was assessed in a model of zymosan-driven peritoneal inflammation. PEPITEM was identified using mass spectrometry. Our studies began with an interest in adiponectin, an anti-inflammatory adipose tissue-derived cytokine. Using an in vitro migration assay, we observed that the migration of human lymphocytes was dose-dependently blocked by adiponectin. Adiponectin achieves its effects on T-cell migration by the induction of a novel mediator released from B-cells. Thus, the effect of adiponectin was lost when B cells are absent, but could be regained by the addition of supernatants from adiponectin stimulated B-cells. Interestingly, the B-cell derived product did not act directly on T-cells; rather, it stimulated EC to release the lipid mediator sphingosine-1-phosphate, which in turn inhibited the migration of T-cells. We used mass spectrometry to isolate a B-cell derived peptide, corresponding uniquely in the human genome to a proteolytic excision product of the 14.3.3ζδ protein. Synthetic PEPITEM could also effectively inhibit T-cell migration. In zymosan-induced peritonitis in the mouse, T-cell recruitment was significantly increased in a strain lacking B cells when compared to wild-type animals. This excess of T-cell recruitment was ameliorated by treatment with PEPITEM. Lymphocytes isolated from patients with chronic inflammatory disease (type-1-diabetes) were released from the inhibitory effects of adiponectin, but this regulatory pathway could be re-established by the addition of exogenous PEPITEM. We believe that PEPITEM and its associated pathway may have therapeutic efficacy in a number of disease scenarios including atherosclerosis.

Background and Objectives Fibroblasts actively regulate the recruitment of leukocytes by endothelial cells (EC), acting in a pro- or anti-inflammatory manner depending on their site of origin. The phenotype of the fibroblast may be a critical determinant of whether leukocyte recruitment, and therefore inflammation, resolves or persists. Here we examined how synovial fibroblasts from different stages of arthritis influenced the recruitment of peripheral blood lymphocytes (PBL) and their onward migration. Materials and Methods Fibroblasts were isolated from patients with resolving or persistent arthritis. Rheumatoid arthritis (RA) cohorts were categorised based on the stage of the disease at the time of sample collection: very early; newly presented but established or long established undergoing replacement surgery. Two forms of co-cultures were developed: (1) To assess effects on recruitment, EC and fibroblasts were cultured on opposite sides of porous philtres and incorporated into a novel flow chamber. PBL were perfused and observed as they bind to the EC surface. (2) To examine effects on migration, EC monolayers were formed on a philtre above a collagen gel in which fibroblasts were incorporated. PBL migration through the construct and their location within the gel were assessed. Conditioned media from co-cultures were collected and analysed by Luminex. Results Fibroblasts from patients with RA increased the ability of EC to support PBL recruitment from flow in a disease duration-specific manner, with binding increasing from very early Conclusions Collectively these initial data indicate that changes in the ability of fibroblasts to influence endothelial and lymphocyte behaviours may occur very early in the development of RA. Moreover, some of these changes are distinct from the phenotype exhibited by fibroblasts taken from non-inflamed tissue and acutely resolving arthritis.

Background and objectives Fibroblasts actively regulate the recruitment of leucocytes by endothelial cells, acting in a pro- or anti-inflammatory manner depending on their site of origin.1 Effects of fibroblasts on leucocyte migration through tissue and their subsequent fate remain unclear. Here, the authors explored the concept that fibroblasts from different tissues modulate the migration patterns of peripheral blood lymphocyte (PBL) in a site specific manner. Materials and methods Fibroblasts were isolated from the skin, bone marrow and synovium of patients with rheumatoid arthritis (RA) undergoing joint replacement surgery. Two forms of co-cultures were developed, appropriate to answer specific questions: (1) endothelial cells and fibroblasts cultured on separate 3.0 µm porous filters, one above the other; (2) endothelial monolayers formed on a filter above a collagen gel in which fibroblasts were incorporated. In the multi-filter model, PBL adhesion and migration was assessed at 24 h by flow cytometry. In the collagen construct, PBL migration into the gel and their depth of penetration was assessed at intervals over 48 h. Results The authors used the multi-filter model to assess the ability of fibroblasts to influence PBL migration through resting endothelial monolayers. Fibroblasts, synovial and dermal, enhanced the ability of endothelial cells to support T cell adhesion, and significantly promoted their onward transendothelial migration. Although fibroblasts generate CXCL12 and IL-6, blockade of either protein had no effect on T cell migration. Interestingly, fibroblasts appeared to restrain onward motion of recruited T cells. This indicated that the process of migration changed the migratory phenotype of recruited T cells and/or that fibroblasts were providing ‘stop’ signals resulting in T cell retention within the fibroblast layer. Next, the authors employed the multi-cellular collagen gel construct to visually observe the effects of fibroblasts on PBL migration into and retention within tissue. Once again, all fibroblast types promoted PBL transendothelial migration, while reducing their onward penetration of the collagen gel. Notably, migrating PBL tracked closely to fibroblast rich regions of the gel, indicating that fibroblasts are capable of retaining recruited cells. Conclusions Collectively these data suggest a novel regulated step in lymphocyte migration, where stromal-derived ‘go’ and ‘stop’ signals control lymphocyte migration into and within tissues, respectively.

Mesenchymal stem cells (MSC) have been suggested to have protective effects through modulation of immune responses. We examined the hypothesis that MSC can modify the ability of endothelial cells (EC) to recruit neutrophils and lymphocytes. Using flow-based assays, leukocytes were perfused over HUVEC that had been cultured with MSC, either on opposite sides of porous filers or in direct contact for 24 h, and then stimulated with tumour necrosis factor-α (TNF-α) for 4 h or TNF-α and interferon-γ for 24 h. Cytokine-stimulated EC supported neutrophil and lymphocyte recruitment, and a combination of rolling, firm adhesion and transmigration was observed. EC cultured with MSC supported significantly lower total adhesion of both types of leukocytes, and the proportion of adherent leukocytes that migrated was reduced compared to EC monocultures. The effects were dependent on the number of MSC added, but were evident for ratios of MSC to EC

The possible role of white blood cells in tissue ischaemia has attracted recent interest. White blood cells can block narrow vessels, particularly if perfusion pressure is reduced or if the cells become activated. To investigate the role of white blood cells in ischaemia microfiltration techniques were used to measure the flow properties of these cells in patients with severe leg ischaemia before and after amputation. Compared with controls white blood cells from patients showed impaired ability to flow through 8 micron and 5 micron pore filters. This applied to fractionated granulocytes and mononuclear cells as well as to unfractionated mixed white blood cells. White blood cells from blood draining the ischaemic leg had worse filterability than those from arm blood of the same patients. After amputation of the ischaemic leg there was definite improvement, the flow properties of the cells being no longer significantly different from controls. These abnormalities detected in white blood cells probably reflect activation of the cells by factors released in the ischaemic tissue. As activation alters the mechanical and adhesive properties of white blood cells, a vicious cycle of microcirculatory trapping at low perfusion pressure, activation, tissue damage, and further activation and trapping might contribute to the progressive worsening of tissue ischaemia.