Chemokines promote leukocyte recruitment during inflammation. The oxidative burst is an important effector mechanism, this leads to the generation of reactive nitrogen species (RNS), including peroxynitrite (ONOO). The current study was performed to determine the potential for nitration to alter the chemical and biological properties of the prototypical CC chemokine, CCL2. Immunofluorescence was performed to assess the presence of RNS in kidney biopsies. Co-localisation was observed between RNS-modified tyrosine residues and the chemokine CCL2 in diseased kidneys. Nitration reduced the potential of CCL2 to stimulate monocyte migration in diffusion gradient chemotaxis assays (p < 0.05). This was consistent with a trend towards reduced affinity of the nitrated chemokine for its cognate receptor CCR2b. The nitrated chemokine was unable to induce transendothelial monocyte migration in vitro and failed to promote leukocyte recruitment when added to murine air pouches (p < 0.05). This could potentially be attributed to reduced glycosaminoglycan binding ability, as surface plasmon resonance spectroscopy showed that nitration reduced heparan sulphate binding by CCL2. Importantly, intravenous administration of nitrated CCL2 also inhibited the normal recruitment of leukocytes to murine air pouches filled with unmodified CCL2. Together these data suggest that nitration of CCL2 during inflammation provides a mechanism to limit and resolve acute inflammation.

BACKGROUND AND AIMS:The αEβ7 integrin is crucial for retention of T lymphocytes at mucosal surfaces through its interaction with E-cadherin. Pathogenic or protective functions of these cells during human intestinal inflammation, such as ulcerative colitis [UC], have not previously been defined, with understanding largely derived from animal model data. Defining this phenotype in human samples is important for understanding UC pathogenesis and is of translational importance for therapeutic targeting of αEβ7-E-cadherin interactions.METHODS:αEβ7+ and αEβ7- colonic T cell localization, inflammatory cytokine production and expression of regulatory T cell-associated markers were evaluated in cohorts of control subjects and patients with active UC by immunohistochemistry, flow cytometry and real-time PCR of FACS-purified cell populations.RESULTS:CD4+αEβ7+ T lymphocytes from both healthy controls and UC patients had lower expression of regulatory T cell-associated genes, including FOXP3, IL-10, CTLA-4 and ICOS in comparison with CD4+αEβ7- T lymphocytes. In UC, CD4+αEβ7+ lymphocytes expressed higher levels of IFNγ and TNFα in comparison with CD4+αEβ7- lymphocytes. Additionally the CD4+αEβ7+ subset was enriched for Th17 cells and the recently described Th17/Th1 subset co-expressing both IL-17A and IFNγ, both of which were found at higher frequencies in UC compared to control.CONCLUSION:αEβ7 integrin expression on human colonic CD4+ T cells was associated with increased production of pro-inflammatory Th1, Th17 and Th17/Th1 cytokines, with reduced expression of regulatory T cell-associated markers. These data suggest colonic CD4+αEβ7+ T cells are pro-inflammatory and may play a role in UC pathobiology.

Indoleamine 2,3-dioxygenase (IDO)-induced immunosuppression can be clinically beneficial for autoimmune diseases. Primary biliary cirrhosis (PBC) is characterized by autoimmune lesions of intrahepatic bile duct epithelial cells that may lead to irreversible cirrhosis or hepatocellular carcinoma. The present study assessed the expression and function of IDO in a cell culture model and in PBC patients. IDO expression was monitored in a human immortalized but non-malignant biliary epithelial cell (iBEC) line. Increased expression of IDO1/2 was observed in the iBECs following stimulation with interferon-γ (IFN-γ). The induction of IDO was IFN-γ-dependent, but was independent of the transforming growth factor-β (TGF-β) pathway. IDO enzymatic activity was observed in the supernatant of iBECs following stimulation with IFN-γ using colorimetric assays. A total of 47 serum samples from PBC patients were used to examine IDO activity by high-performance liquid chromatography, with samples from 24 healthy volunteers used as controls. Patients with PBC exhibited an increased rate of tryptophan to kynurenine conversion (P>0.01). Liver sections from patients with PBC (n=5) and those of healthy controls (n=5) were used for immunohistochemical studies. IDO expression was observed in biliary epithelial cells and in hepatocytes of PBC patients. Finally, the effect of tryptophan metabolites on human cluster of differentiation (CD) 4+ T cells in inducing polarization towards a regulatory T cell phenotype was examined. 3-Hydroxykynurenine significantly upregulated the fraction of CD4+ cells expressing forkhead box p3 (Foxp3). The results of the present study suggest a therapeutic opportunity for the management of PBC and indicate that tryptophan catabolism could serve as a potential biomarker to monitor disease progression.

Purpose To investigate the clinical and functional aspects of MST1 ( STK4 ) deficiency in a profoundly CD4-lymphopenic kindred with a novel homozygous nonsense mutation in STK4. Although recent studies have described the cellular effects of murine Mst1 deficiency, the phenotype of MST1-deficient human lymphocytes has yet to be fully explored. Patient lymphocytes were therefore investigated in the context of current knowledge of murine Mst1 deficiency. Methods Genetic etiology was identified by whole exome sequencing of genomic DNA from two siblings, combined with linkage analysis in the wider family. MST1 protein expression was assessed by immunoblotting. The ability of patient lymphocytes to adhere to ICAM-1 under flow conditions was measured, and transwell assays were used to assess chemotaxis. Chemokine receptor expression was examined by flow cytometry and receptor signalling by immunoblotting. Results A homozygous nonsense mutation in STK4 (c.442C > T, p.Arg148Stop) was found in the patients, leading to a lack of MST1 protein expression. Patient leukocytes exhibited deficient chemotaxis after stimulation with CXCL11, despite preserved expression of CXCR3. Patient lymphocytes were also unable to bind effectively to immobilised ICAM-1 under flow conditions, in keeping with a failure to develop high affinity binding. Conclusion The observed abnormalities of adhesion and migration imply a profound trafficking defect among human MST1-deficient lymphocytes. By analogy with murine Mst1 deficiency and other defects of leucocyte trafficking, this is likely to contribute to immunodeficiency by impairing key aspects of T-cell development and function such as positive selection in the thymus, thymic egress and immune synapse formation in the periphery.

BACKGROUND:Expression of the chemokine receptor CXCR4 is known to regulate melanoma metastasis to distant sites with high expression of the CXCL12 ligand. However, the prognostic impact of CXCR4 expression and potential for autocrine-mediated activation of prosurvival mitogen-activated protein kinase signalling remains enigmatic. Furthermore, expression of the decoy receptor CXCR7 within the local cutaneous melanoma microenvironment remains undefined. OBJECTIVES:To define the contribution and prognostic impact of CXCR4-CXCR7-CXCL12 signalling in primary cutaneous melanomas and the immediate tumour microenvironment. METHODS:Immunohistochemical/immunofluorescent expression of CXCR4, CXCR7 or CXC12 was analysed in human metastatic melanoma cell lines, primary cutaneous cell types and a retrospective cohort of primary melanomas/benign naevi. CXCL12 secretion by melanoma/cutaneous cells was evaluated by enzyme-linked immunosorbent assay, and autocrine CXCR4-CXCL12 signalling was investigated by addition of a CXCL12-neutralizing antibody. RESULTS:CXCR4 expression was significantly higher in primary melanomas that subsequently metastasized after 7 years (P = 0·037). Stratification for American Joint Committee on Cancer (AJCC) stage II disease revealed significantly decreased disease-free survival in patients with > 50% CXCR4 expression (P = 0·036), while comparative analysis of CXCL12 expression in the adjacent epidermis of all AJCC stage melanomas revealed increased CXCL12 correlated with prolonged time to metastasis (P = 0·014). CXCR7 was expressed within the primary melanoma microenvironment but was absent on primary tumours. Addition of anti-CXCL12 to BRAF-mutant melanoma cells resulted in downregulation of phospho-CXCR4 and phospho-extracellular signal-related kinase, indicating autocrine CXCR4-CXCL12 signalling. CONCLUSIONS:CXCR4 expression defines a potential prognostic biomarker for AJCC stage II melanoma. Moreover, targeting the CXCR4-CXCR7-CXCL12 axis may represent a novel therapeutic strategy to prevent early melanoma progression.

Oxidative stress is a major and recurring cause of damage during inflammation, especially following organ transplantation. Initial ischaemia-reperfusion injury causes the production of many reactive oxygen and nitrogen species, and subsequent recruitment and activation of inflammatory cells can lead to further oxidative stress. This stress is well known to cause damage at the cellular level, for example by induction of senescence leading to the production of a characteristic senescence-associated secretory phenotype. Chemokines are an important component of the senescence-associated secretory phenotype, recruiting further leucocytes and reinforcing the stress and senescence responses. As well as inducing the production of proteins, including chemokines, oxidative stress can alter proteins themselves, both directly and by induction of enzymes capable of modification. These alterations can lead to important modifications to their biological activity and also alter detection by some antibodies, potentially limiting the biological relevance of some immunochemical and proteomic biomarkers. Peroxynitrite, a reactive nitrogen species generated during inflammation and ischaemia, can cause such modifications by nitrating chemokines. Matrix metalloproteinases, released by many stressed cells, can cleave chemokines, altering function, while peptidylarginine deiminases can inactivate certain chemokines by citrullination. This review discusses the relationship between inflammation and post-translational modification, focusing on the functional modulation of transplant-relevant pro-inflammatory chemokines.

Introduction T lymphocytes expressing the αEβ7 integrin are highly enriched within human intestinal epithelium and lamina propria. Studies exploring pathogenic or protective functions of αEβ7 expressing cells are lacking. Defining this phenotype is critical for our understanding of IBD pathogenesis and of translational importance with the development of etrolizumab, a humanised antibody specific to the β7 integrin that blocks α4β7:MAdCAM-1 and αEβ7:E-cadherin interactions. Methods Lymphocytes within colonic biopsies from a total of 43 UC and 35 non-disease control patients were studied. Multi-colour FACS was optimised to determine surface and intracellular protein expression (CD45, CD3, CD4, CD8, αE, β7, CD161, IL-17A, TNFα, IFNγ and IL-10). qPCR was performed on TCRαβ+ lymphocytes, FACS sorted into CD4+αEβ7+, CD4+αEβ7-, CD8+αEβ7+ and CD8+αEβ7- prior to gene expression assay. Dual stain IHC for αE, plus CD3, CD4, CD8 and FOXP3 was performed using a Ventana Benchmark XT autostainer. Severity of UC was stratified using the Mayo endoscopic score for ulcerative colitis. Results Ulcerative colitis was associated with a significantly increased frequency of T lymphocytes in the intestinal mucosa (p < 0.05). IHC revealed the highest expression of αE on CD4 and CD8 intraepithelial lymphocytes, although a substantial number of lamina propria lymphocytes also expressed this integrin. In UC, FACS demonstrated CD4+αEβ7+ lymphocytes had a higher potential to produce the pro-inflammatory cytokines IFNγ(p < 0.01), TNFα(p < 0.001) and IL-17A(p < 0.0001) than CD4+αEβ7- lymphocytes. In addition, a mean of 31.5% of the CD4+αEβ7+ lymphocytes produced both IL-17A and IFNγ compared to a mean of only 7.7% in the CD4+αEβ7- compartment (p < 0.001). IL-10 was not differentially expressed between CD4+αEβ7+ and CD4+αEβ7- lymphocytes in controls or UC, and a low frequency of αEβ7+FOXP3+ cells was observed by IHC. qPCR array confirmed higher mRNA levels of IFNγ(p < 0.001), TNFα(p < 0.01) and IL-17A(p < 0.01), and lower transcription of FOXP3 (p < 0.0001) in CD4+αEβ7+ cells compared to CD4+αEβ7- cells. Conclusion αEβ7 expression was associated with an enrichment of pro-inflammatory Th17, Th1 and Th17/Th1 T lymphocytes, and not associated with a regulatory phenotype. These data suggest therapeutic interventions targeting αE expressing T cells and the αEβ7 integrin itself may be viable approaches for reducing aberrant inflammatory responses in UC. Disclosure of Interest C. Lamb Grant/research support from: Genentech, J. Mansfield Grant/research support from: Genentech, G. Tew Employee of: Genentech, D. Gibbons Grant/research support from: Genentech, A. Long Grant/research support from: Genentech, P. Irving Grant/research support from: Genentech, L. Deihl Employee of: Genentech, J. Eastham Anderson Employee of: Genentech, G. O’Boyle Grant/research support from: Genentech, D. Jones Grant/research support from: Genentech, A. Hayday Grant/research support from: Genentech, M. Keir Employee of: Genentech, J. Egen Employee of: Genentech, J. Kirby Grant/research support from: Genentech.

Background: T lymphocytes expressing the αEβ7 integrin are highly enriched within human intestinal epithelium and lamina propria.While αEβ7 is critical for lymphocyte retention, studies exploring pathogenic or protective functions of αEβ7 expressing cells, particularly in the context of human diseases, such as ulcerative colitis (UC), are lacking.Defining this phenotype is critical for our understanding of IBD pathogenesis and of translational importance with the development of etrolizumab, a humanized antibody specific to the β7 integrin that blocks α4β7:MAdCAM-1 and αEβ7:E-cadherin interactions.Aim: To determine the differential phenotype of αEβ7+ and αEβ7colonic T lymphocytes in normal colon and UC.Methods: Lymphocytes within colonic biopsies from a total of 43 UC and 35 nondisease control patients were studied by flow cytometry (FACS), immunohistochemistry (IHC) and qPCR.Multi-colour FACS was optimised to determine surface and intracellular protein expression (CD45, CD3, CD4, CD8, αE, β7, CD161, IL-17A, TNFα, IFNγ & IL-10).qPCR was performed on TCRαβ+ lymphocytes, FACS sorted into CD4+αEβ7+, CD4+αEβ7-, CD8+αEβ7+ and CD8+αEβ7-prior to gene expression assay.Dual stain IHC for αE, plus CD3, CD4, CD8 and FOXP3 was performed using a Ventana Benchmark XT autostainer.Severity of UC was stratified using the Mayo endoscopic score for ulcerative colitis.Results: Ulcerative colitis was associated with a significantly increased frequency of T lymphocytes in the intestinal mucosa (p<0.05).IHC revealed the highest expression of αE on CD4 and CD8 intraepithelial lymphocytes, although a substantial number of lamina propria lymphocytes also expressed this integrin.In UC, FACS demonstrated CD4+αEβ7+ lymphocytes had a higher potential to produce the pro-inflammatory cytokines IFNγ (p<0.01),TNFα (p<0.001) and IL-17A (p<0.0001)than CD4+αEβ7-lymphocytes.In addition, a mean of 31.5% of the CD4+αEβ7+ lymphocytes produced both IL-17A and IFNγ compared to a mean of only 7.7% in the CD4+αEβ7-compartment (p<0.001).IL-10 was not differentially expressed between CD4+αEβ7+ and CD4+αEβ7-lymphocytes in UC or non-IBD controls, and a low frequency of αEβ7+FOXP3+ cells was observed by IHC.qPCR array confirmed higher mRNA levels of IFNγ (p<0.001),TNFα (p<0.01) and IL-17A (p<0.01), and lower transcription of FOXP3 (p<0.0001) in CD4+αEβ7+ cells compared to CD4+αEβ7cells.Conclusion: αEβ7 expression was associated with an enrichment of pro-inflammatory Th17, Th1 and Th17/Th1 T lymphocytes, and not associated with a regulatory phenotype.These data suggest therapeutic interventions targeting αE integrin expressing T cells and the αEβ7 integrin itself may be viable approaches for reducing aberrant inflammatory responses in UC.

Background: Despite intensive research and novel adjuvant therapies, there is currently no cure for metastatic melanoma. The chemokine receptor CXCR4 controls metastasis to sites such as the liver; however, the therapeutic blockade with the existing agents has proven difficult.Methods: AMD11070, a novel orally bioavailable inhibitor of CXCR4, was tested for its ability to inhibit the migration of melanoma cells compared with the commonly described antagonist AMD3100.Results: AMD11070 abrogated melanoma cell migration and was significantly more effective than AMD3100. Importantly for the clinical context, the expression of B-RAF-V600E did not the affect the sensitivity of AMD11070.Conclusion: Liver-resident myofibroblasts excrete CXCL12, which is able to promote the migration of CXCR4-expressing tumour cells from the blood into the liver. Blockade of this axis by AMD11070 thus represents a novel therapeutic strategy for both B-RAF wild-type and mutated melanomas.

The chemokine receptors CXCR3 and CXCR4 are primarily involved in memory Th1 cell–driven autoimmune diseases. Although recent studies in chronic inflammatory disease showed therapeutic success using combined blockade, details of CXCR3 and CXCR4 synergism are not understood. In this investigation, we intended to unravel the interaction of these chemokine receptors in static and dynamic cell-migration assays at both the cellular and molecular levels. Effects of combined stimulation by murine CXCL9 and CXCL12, ligands of CXCR3 and CXCR4, respectively, were analyzed using a murine central memory Th1 cell clone. Costimulation with CXCL9 desensitized the chemotaxis of Th1 cells toward CXCL12 by up to 54%. This effect was found in murine EL-4 cells, as well as in primary human T cells. Furthermore, under dynamic flow conditions CXCL12-induced crawling and endothelial transmigration of Th1 cells was desensitized by CXCL9. Subsequent experiments uncovered several molecular mechanisms underlying the heterologous cross-regulation of CXCR4 signaling by the CXCR3 ligand. CXCR4 surface expression was reduced, whereas CXCL12-induced Akt phosphorylation and intracellular Ca2+ signals were modulated. Moreover, blockade of Rac by NSC23766 revealed differential effects on CXCL12 and CXCL9 chemotaxis and abolished the desensitizing effect of CXCL9. The desensitization of CXCR4 via CXCR3 in memory Th1 cells suggests that their in vivo homeostasis, widely regulated by CXCL12, seemed to be significantly altered by CXCR3 ligands. Our data provide a more detailed understanding for the continuing extravasation and recruitment of Th1 lymphocytes into sites of persistent inflammation.

© PNAS, 2012. ‘The author(s) reserves the right to deposit his or her final manuscript in the author(s)’s funding body’s archive or designated repository on acceptance for publication by PNAS, provided that a link to the WORK in PNAS Online is included.’ This is the author's version of the work. It is posted here by permission of ACM for your personal use. Not for redistribution. The definitive version of this article is available at:

My talk was at “The Leading Edge of Leukocyte Migration”, the inaugural meeting of the British Society of Immunology Leukocyte Migration Group. It was in Glasgow on the 12-13th of September 2013.

Introduction The intestinal vascular microcirculation plays a pivotal role in the immune cell dysregulation that drives inflammatory bowel disease. T lymphocyte recruitment, adherence and migration are dependent on the integrin heterodimer α4β7, which binds with high affinity to vascular endothelium expressing MAdCAM-1, prior to lymphocyte diapedesis into the lamina propria. This integrin therefore provides a potential therapeutic target. Methods Using Cellix® technology, a dynamic in vitro model was created, testing the potential of β7 integrin blockade to impair lymphocyte adhesion to MAdCAM-1 under shear flow. The Cellix® system comprises a microfluidic platform and nano pump, with biochip channels that replicate the shear flow and stress found in post-capillary venules. Peripheral blood mononuclear cells (PBMC) were utilised, along with HuT78 T cells that constitutively express the α4β7 integrin and the chemokine receptor CXCR4, known to be upregulated in inflammatory bowel disease. Biochips were coated for 12 h at 4°C with 10 μg/ml MAdCAM-1 Fc, then blocked with 0.1% BSA for 30 min to prevent non-specific adherence to plastic. Adherence of T cells was quantitatively assessed by microscopy at a physiological flow rate of 1 dyne/cm3. The effect of an anti-human β7 integrin monoclonal antibody (clone Fib504, BD Pharmingen) on lymphocyte adherence was measured. 3 nM of the chemokine CXCL12 (ligand for CXCR4) was added to the system to model the pro-inflammatory environment present in inflammatory bowel disease. Experiments were performed at 37°C. Results HuT78 lymphocytes and PBMC (5×106/ml) provided consistent adherence to MAdCAM-1 under flow, mean ± SE adherent cells/hpf of 32.8±4.5 and 46.2±3 respectively. Adherence was significantly improved with the addition of 3 nM CXCL12 to 45.7±2.8 (p<0.05) and 78.5±1.5 (p<0.001). Incubating the cells with the Fib504 anti-β7 integrin antibody, led to a significant reduction in adherence of unstimulated cells to 17.8±2 (p<0.001) and 18±3.2 (p<0.0001). This reduction was maintained on stimulation with CXCL12, at 17.3±0.9 (p<0.001), and 5.3±0.9 (p<0.0001). Conclusion This novel in vitro model, demonstrated significant modulation of α4β7 lymphocyte adhesion to the ligand MAdCAM-1 with an anti-β7 antibody. This highly controlled model system provides a more physiological representation of chemokine driven responsiveness than previously published chemotaxis or adhesion assays, and thus the Cellix® platform may serve as a useful tool for the development and validation of future anti-lymphocyte adhesion therapeutics. This research supports the clinical investigation of therapeutics targeting the β7 subunit or α4β7 heterodimer in this disease setting. Competing interests None declared.

The recruitment of T lymphocytes during diseases such as rheumatoid arthritis is regulated by stimulation of the chemokine receptors expressed by these cells. This study was designed to assess the potential of a CXCR3-specific small-molecule agonist to inhibit the migration of activated human T cells toward multiple chemokines. Further experiments defined the molecular mechanism for this anti-inflammatory activity. Analysis in vitro demonstrated agonist induced internalization of both CXCR3 and other chemokine receptors coexpressed by CXCR3(+) T cells. Unlike chemokine receptor-specific antagonists, the CXCR3 agonist inhibited migration of activated T cells toward the chemokine mixture in synovial fluid from patients with active rheumatoid arthritis. A humanized mouse air-pouch model showed that intravenous treatment with the CXCR3 agonist prevented inflammatory migration of activated human T cells toward this synovial fluid. A potential mechanism for this action was defined by demonstration that the CXCR3 agonist induces receptor cross-phosphorylation within CXCR3-CCR5 heterodimers on the surface of activated T cells. This study shows that generalized chemokine receptor desensitization can be induced by specific stimulation of a single chemokine receptor on the surface of activated human T cells. A humanized mouse model was used to demonstrate that this receptor desensitization inhibits the inflammatory response that is normally produced by the chemokines present in synovial fluid from patients with active rheumatoid arthritis.

Chemokines represent a class of cytokines that control the migration of leucocytes. The human chemokine system comprises 44 ligands and 21 receptors that have evolved to control leucocyte migration. Although chemokines are an attractive therapeutic target for anti‐inflammatory intervention, clinical trials of small molecule receptor antagonists have failed to demonstrate efficacy. One often cited explanation for this is the apparent redundancy within the chemokine system, wherein several ligands bind and activate each receptor. The work of Scholten et al . and Nedjai et al . reported in this issue of the British Journal of Pharmacology demonstrates that this redundancy does not exist at the molecular level and provides a powerful insight into the complex nature of chemokine receptor activation. LINKED ARTICLES This article is a commentary on Scholten et al ., pp. 898–911 of this issue and Nedjai et al ., pp. 912–923 of this issue. To view Scholten et al . visit http://dx.doi.org/10.1111/j.1476‐5381.2011.01648.x and to view Nedjai et al . visit http://dx.doi.org/10.1111/j.1476‐5381.2011.01660.x