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Despite undeniable improvement in the management of rheumatoid arthritis (RA), the discovery of more effective, less toxic and, ideally, less immune suppressive drugs are much needed. In the current study, we set to explore the potential anti-rheumatic activity of the non-toxic, tellurium-based immunomodulator, AS101 in an experimental animal model of RA. The effect of AS101 was assessed on adjuvant-induced arthritis (AIA) rats. Clinical signs of arthritis were assessed. Histopathological examination was used to assess inflammation, synovial changes and tissue lesions. Very late antigen-4 (VLA-4) + cellular infiltration was detected using immunohistochemical staining. Enzyme-linked immunosorbent assay (ELISA) was used to measure circulating anti-cyclic citrullinated-peptide autoantibody (ACPA) and real-time polymerase chain reaction (PCR) was used to measure the in-vitro effect of AS101 on interleukin (IL)-6 and IL-1β expression in activated primary human fibroblasts. Prophylactic treatment with intraperitoneal AS101 reduced clinical arthritis scores in AIA rats (P < 0·01). AS101 abrogated the migration of active chronic inflammatory immune cells, particularly VLA-4 + cells, into joint cartilage and synovium, reduced the extent of joint damage and preserved joint architecture. Compared to phosphate-buffered saline (PBS)-treated AIA rats, histopathological inflammatory scores were significantly reduced (P < 0·05). Furthermore, AS101 resulted in a marked reduction of circulating ACPA in comparison to PBS-treated rats (P < 0·05). Importantly, AS101 significantly reduced mRNA levels of proinflammatory mediators such as IL-6 (P < 0·05) and IL-1β (P < 0·01) in activated primary human fibroblasts. Taken together, we report the first demonstration of the anti-rheumatic/inflammatory activity of AS101 in experimental RA model, thereby supporting an alternative early therapeutic intervention and identifying a promising agent for therapeutic intervention., (© 2020 British Society for Immunology.)

CD40L is a member of the TNF superfamily that participates in immune cell activation. It binds to and signals through several integrins, including αvβ3 and α5β1, which bind to the trimeric interface of CD40L. We previously showed that several integrin ligands can bind to the allosteric site (site 2), which is distinct from the classical ligand-binding site (site 1), raising the question of if CD40L activates integrins. In our explorations of this question, we determined that integrin α4β1, which is prevalently expressed on the same CD4+ T cells as CD40L, is another receptor for CD40L. Soluble (s)CD40L activated soluble integrins αvβ3, α5β1, and α4β1 in cell-free conditions, indicating that this activation does not require inside-out signaling. Moreover, sCD40L activated cell-surface integrins in CHO cells that do not express CD40. To learn more about the mechanism of binding, we determined that sCD40L bound to a cyclic peptide from site 2. Docking simulations predicted that the residues of CD40L that bind to site 2 are located outside of the CD40L trimer interface, at a site where four HIGM1 (hyper-IgM syndrome type 1) mutations are clustered. We tested the effect of these mutations, finding that the K143T and G144E mutants were the most defective in integrin activation, providing support that this region interacts with site 2. We propose that allosteric integrin activation by CD40L also plays a role in CD40L signaling, and defective site 2 binding may be related to the impaired CD40L signaling functions of these HIGM1 mutants., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Objective: To determine whether natalizumab efficacy is maintained when switching to personalized extended interval dosing based on individual natalizumab trough concentrations in patients with relapsing-remitting multiple sclerosis (RRMS)., Methods: This was a prospective multicenter single-arm trial with 1 year follow-up and a 1-year extension phase. Participants were adult persons with RRMS treated with natalizumab without disease activity in the year prior to enrollment. The natalizumab treatment interval was based on longitudinal natalizumab trough concentrations. Patients received 3 monthly MRI scans, relapse assessments, and disability scoring during follow-up. The primary endpoint was the occurrence of gadolinium-enhancing lesions on MRI. Secondary endpoints were new/enlarging T2 lesions on MRI and relapses and progression on the Expanded Disability Status Scale (EDSS) during follow-up and extension phase., Results: Sixty-one patients were included. Eighty-four percent extended the interval from a 4-week interval to a 5- to 7-week interval. No patient developed gadolinium-enhancing lesions (95% confidence interval [CI] 0%-7.4%) during follow-up. No new/enlarging T2 lesions (95% CI 0%-7.4%) or relapses (95% CI 0%-7.4%) were reported during follow-up and in the extension phase. Median EDSS was comparable at baseline (3.0, interquartile range [IQR] 2.0-5.0) and after follow-up (3.0, IQR 2.0-5.0)., Conclusion: Personalized extended interval dosing did not induce recurrence of MS disease activity. Natalizumab efficacy was maintained in stable patients with RRMS receiving personalized extended interval dosing based on individual natalizumab concentrations., Classification of Evidence: This study provides Class IV evidence that personalized extended interval dosing of natalizumab does not result in recurrence of disease activity in stable patients with RRMS., (© 2020 American Academy of Neurology.)

Redirecting T cells to specifically kill malignant cells has been validated as an effective anti-cancer strategy in the clinic with the approval of blinatumomab for acute lymphoblastic leukemia. However, the immunosuppressive nature of the tumor microenvironment potentially poses a significant hurdle to T cell therapies. In hematological malignancies, the bone marrow (BM) niche is protective to leukemic stem cells and has minimized the efficacy of several anti-cancer drugs. In this study, we investigated the impact of the BM microenvironment on T cell redirection. Using bispecific antibodies targeting specific tumor antigens (CD123 and BCMA) and CD3, we observed that co-culture of acute myeloid leukemia or multiple myeloma cells with BM stromal cells protected tumor cells from bispecific antibody-T cell-mediated lysis in vitro and in vivo. Impaired CD3 redirection cytotoxicity was correlated with reduced T cell effector responses and cell-cell contact with stromal cells was implicated in reducing T cell activation and conferring protection of cancer cells. Finally, blocking the VLA4 adhesion pathway in combination with CD3 redirection reduced the stromal-mediated inhibition of cytotoxicity and T cell activation. Our results lend support to inhibiting VLA4 interactions along with administering CD3 redirection therapeutics as a novel combinatorial regimen for robust anti-cancer responses.

The success of B cell depletion therapies and identification of leptomeningeal ectopic lymphoid tissue (ELT) in patients with multiple sclerosis (MS) has renewed interest in the antibody-independent pathogenic functions of B cells during neuroinflammation. The timing and location of B cell antigen presentation during MS and its animal model experimental autoimmune encephalomyelitis (EAE) remain undefined. Using a new EAE system that incorporates temporal regulation of MHCII expression by myelin-specific B cells, we observed the rapid formation of large B cell clusters in the spinal cord subarachnoid space. Neutrophils preceded the accumulation of meningeal B cell clusters, and inhibition of CXCR2-mediated granulocyte trafficking to the central nervous system reduced pathogenic B cell clusters and disease severity. Further, B cell-restricted very late antigen-4 (VLA-4) deficiency abrogated EAE dependent on B cell antigen presentation. Together, our findings demonstrate that neutrophils coordinate VLA-4-dependent B cell accumulation within the meninges during neuroinflammation, a key early step in the formation of ELT observed in MS., Competing Interests: The authors declare no competing interest., (Copyright © 2019 the Author(s). Published by PNAS.)

When targeted by the tumor-promoting enzyme heparanase, cleaved and shed syndecan-1 (Sdc1) then couples VEGFR2 (also known as KDR) to VLA-4, activating VEGFR2 and the directed migration of myeloma cells. But how VEGFR2 activates VLA-4-mediated motility has remained unknown. We now report that VEGFR2 causes PKA-mediated phosphorylation of VLA-4 on S988, an event known to stimulate tumor metastasis while suppressing cytotoxic immune cells. A key partner in this mechanism is the chemokine receptor CXCR4, a well-known mediator of cell motility in response to gradients of the chemokine SDF-1 (also known as CXCL12). The entire machinery necessary to phosphorylate VLA-4, consisting of CXCR4, AC7 (also known as ADCY7) and PKA, is constitutively associated with VEGFR2 and is localized to the integrin by Sdc1. VEGFR2 carries out the novel phosphorylation of Y135 within the DRY microswitch of CXCR4, sequentially activating Gα i βγ, AC7 and PKA, which phosphorylates S988 on the integrin. This mechanism is blocked by a syndecan-mimetic peptide (SSTN VEGFR2 ), which, by preventing VEGFR2 linkage to VLA-4, arrests tumor cell migration that depends on VLA-4 phosphorylation and stimulates the LFA-1-mediated migration of cytotoxic leukocytes., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Immunological memory ensures life-long protection against previously encountered pathogens, and in mice and humans the spleen is an important reservoir for long-lived memory B cells (MBCs). It is well-established that integrins play several crucial roles in lymphocyte survival and trafficking, but their involvement in the retention of MBCs in secondary lymphoid organs, and differences between B cell subsets in their adhesion capacity to ICAM-1 and/or VCAM-1 have not yet been confirmed. Here, we use an autoimmune mouse model, where MBCs are abundant, to show that the highest levels of LFA-1 and VLA-4 amongst B cells are found on MBCs. In vivo blockade of VLA-4 alone or in combination with LFA-1, but not LFA-1 alone, causes a release of MBCs from the spleen into the blood stream. In humans, we find that in peripheral blood, spleens, and tonsils from healthy donors the highest expression levels of the integrins LFA-1 and VLA-4 are also found on MBCs. Consistent with this, we found MBCs to have a higher capacity to adhere to ICAM-1 and VCAM-1 than naïve B cells. In patients with the autoimmune disease rheumatoid arthritis, it is the MBCs that have the highest levels of LFA-1 and VLA-4; moreover, compared with healthy donors, naïve B and MBCs of patients receiving anti-TNF medication have enhanced levels of the active form of LFA-1. Commensurate levels of the active αL subunit can be induced on B cells from healthy donors by exposure to the integrin ligands. Thus, our findings establish the selective use of the integrins LFA-1 and VLA-4 in the localization and adhesion of MBCs in both mice and humans.

Cancer incidence increases with age, but paradoxically, cancers have been found to grow more quickly in young mice compared with aged ones. The cause of differential tumor growth has been debated and, over time, attributed to faster tumor cell proliferation, decreased tumor cell apoptosis, and/or increased angiogenesis in young animals. Despite major advances in our understanding of tumor immunity over the past 2 decades, little attention has been paid to comparing immune cell populations in young and aged mice. Using mouse colon adenocarcinoma model MC38 implanted in young and mature mice, we show that age substantially influences the number of tumor-infiltrating cytotoxic CD8+ T cells, which control cancer progression. The different tumor growth pace in young and mature mice was abrogated in RAG1null mice, which lack mature T and B lymphocytes, and upon selective depletion of endogenous CD8+ cells. Transcriptome analysis further indicated that young mice have decreased levels of the Itga4 gene (CD49d, VLA-4) in tumor-infiltrating lymphocytes when compared with mature mice. Hypothesizing that VLA-4 can have a tumor-protective effect, we depleted the protein, which resulted in accelerated tumor growth in mature mice. These observations may explain the paradoxical growth rates observed in murine cancers, point to the central role of VLA-4 in controlling tumor growth, and open new venues to therapeutic manipulation.

Background: Alzheimer's disease (AD) is associated with age-associated central nervous system degeneration and dementia. This decline in the function correlates with deposition of Aβ peptide containing plaques and associated reactive gliosis. The inflammatory phenotype of microglia, in particular, is often considered detrimental to cognitive function in AD. In addition to the changes in the CNS, altered immune changes in the periphery have recently been observed in AD suggesting a critical immune- related communication between the periphery and the brain., Objective: We hypothesized that modulating the peripheral immune system may alter the proinflammatory gliosis associated with AD. Therapeutic antibodies against the α4β1 integrin receptor have been used clinically to attenuate the ability of various immune cells to adhere to endothelium and migrate into target tissues such as the intestines (Crohn's disease) or brain (multiple sclerosis). We hypothesized that a similar peripheral antibody-based therapy would attenuate gliosis by altering immune cell infiltration or phenotype in peripheral organs and the brain using an APP/PS1 mouse model of Alzheimer's disease., Method: Littermate control wild-type and APP/PS1 mice were tail vein injected with either saline, isotype control (IgG2b), or an antibody recognizing α4-integrin, anti-CD49d, once a week for 4 consecutive weeks. To understand CNS and peripheral immune changes, brains and spleen were used., Results/conclusion: Our data suggests that the antibody therapy was able to reduce microgliosis, astrogliosis, and synaptic changes in the APP/PS1 mice compared to isotype control injections without changing amyloid-β plaque load. Interestingly, both isotype control and antibody therapy also reduced the number of proinflammatory cytokines in the spleen although changes in the brain were less robust. The anti-CD49d and isotype control treatments also reduced CD4 immunoreactivity in the brains, suggesting a possible mechanism for attenuation of inflammation in the brain. This data suggests that it is indeed feasible to alter the immune component of AD brain changes using a clinically feasible strategy of delivering a particular subtype of IgG or epitope selective antibodies that target infiltration of the peripheral immune system., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Immunosuppressive myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) accumulate in tumors where they inhibit T cell-mediated antitumor immune responses and promote tumor progression. Myeloid cell PI3Kγ plays a role in regulating tumor immune suppression by promoting integrin α 4 -dependent MDSC recruitment to tumors and by stimulating the immunosuppressive polarization of MDSCs and TAMs. Here, we show that integrin α 4 promotes immunosuppressive polarization of MDSCs and TAMs downstream of PI3Kγ, thereby inhibiting antitumor immunity. Genetic or pharmacological suppression of either PI3Kγ or integrin α 4 blocked MDSC recruitment to tumors and also inhibited immune suppressive myeloid cell polarization, thereby reducing expression of IL10 and increasing expression of IL12 and IFNγ within tumors. Inhibition of PI3Kγ or integrin α 4 within tumors stimulated dendritic cell and CD8 + T-cell recruitment and maturation, as well as tumor cell cytotoxicity in vivo , thereby inhibiting tumor growth. As blockade of PI3Kγ or integrin α 4 prevents accumulation of MDSC and reduces myeloid cell expression of immunosuppressive factors that stimulate tumor immune escape, these results highlight PI3Kγ and integrin α 4 as targets for the design of cancer therapeutics. Cancer Immunol Res; 5(11); 957-68. ©2017 AACR ., (©2017 American Association for Cancer Research.)

The mechanisms leading to autoimmune and inflammatory diseases in the CNS have not been elucidated. The environmental triggers of the aberrant presence of CD4 + T cells in the CNS are not known. In this article, we report that abnormal β-catenin expression in T cells drives a fatal neuroinflammatory disease in mice that is characterized by CNS infiltration of T cells, glial activation, and progressive loss of motor function. We show that enhanced β-catenin expression in T cells leads to aberrant and Th1-biased T cell activation, enhanced expression of integrin α4β1, and infiltration of activated T cells into the spinal cord, without affecting regulatory T cell function. Importantly, expression of β-catenin in mature naive T cells was sufficient to drive integrin α4β1 expression and CNS migration, whereas pharmacologic inhibition of integrin α4β1 reduced the abnormal T cell presence in the CNS of β-catenin-expressing mice. Together, these results implicate deregulation of the Wnt/β-catenin pathway in CNS inflammation and suggest novel therapeutic strategies for neuroinflammatory disorders., (Copyright © 2017 by The American Association of Immunologists, Inc.)

Human monocytic myeloid-derived suppressor cells (MO-MDSCs) within the hepatic compartment suppress inflammation and impair immune surveillance in liver cancer. It is currently not known whether recruitment of MO-MDSCs from blood via hepatic sinusoidal endothelium (HSEC) contributes to their enrichment within the hepatic compartment. We compared the transmigratory potential of MO-MDSCs and monocytes after adhesion to hepatic endothelial monolayers in flow-based assays that mimic in vivo shear stress in the sinusoids. Despite comparable binding to HSEC monolayers, proportionally fewer MO-MDSCs underwent transendothelial migration, indicating that the final steps of extravasation, where actin polymerization plays an important role, are impaired in MO-MDSCs. In this article, we found reduced levels of CD13 on MO-MDSCs, which has recently been reported to control cell motility in monocytes, alongside reduced VLA-4 expression, an integrin predominantly involved in adherence to the apical side of the endothelium. CD13 and VLA-4 blocking and activating Abs were used in flow-based adhesion assays, live-cell imaging of motility, and actin polymerization studies to confirm a role for CD13 in impaired MO-MDSC transmigration. These findings indicate that CD13 significantly contributes to tissue infiltration by MO-MDSCs and monocytes, thereby contributing to the pathogenesis of hepatic inflammation., (Copyright © 2017 by The American Association of Immunologists, Inc.)

Migration of B cells supports their development and recruitment into functional niches. Therefore, defining factors that control B cell migration will lead to a better understanding of adaptive immunity. In vitro cell migration assays with B cells have been limited by poor adhesion of cells to glass coated with adhesion molecules. We have developed a technique using monolayers of endothelial cells as the substrate for B cell migration and used this technique to establish a robust in vitro assay for B cell migration. We use TNF-α to up-regulate surface expression of the adhesion molecule VCAM-1 on endothelial cells. The ligand VLA-4 is expressed on B cells, allowing them to interact with the endothelial monolayer and migrate on its surface. We tested our new method by examining the role of L-plastin (LPL), an F-actin-bundling protein, in B cell migration. LPL-deficient (LPL -/- ) B cells displayed decreased speed and increased arrest coefficient compared with wild-type (WT) B cells, following chemokine stimulation. However, the confinement ratios for WT and LPL -/- B cells were similar. Thus, we demonstrate how the use of endothelial monolayers as a substrate will support future interrogation of molecular pathways essential to B cell migration., (© Society for Leukocyte Biology.)

Positron emission tomography and computed tomography imaging (PET/CT) is an increasingly valuable tool for diagnosing tuberculosis (TB). The glucose analog [ 18 F]fluoro-2-deoxy-2-d-glucose ([ 18 F]-FDG) is commonly used in PET/CT that is retained by metabolically active inflammatory cells in granulomas, but lacks specificity for particular cell types. A PET probe that could identify recruitment and differentiation of different cell populations in granulomas would be a useful research tool and could improve TB diagnosis and treatment. We used the Mycobacterium -antigen murine inflammation model and macaques with TB to identify [ 64 Cu]-labeled CB-TE1A1P-PEG 4 -LLP2A ([ 64 Cu]-LLP2A), a high affinity peptidomimetic ligand for very late Ag-4 (VLA-4; also called integrin α4β1) binding cells in granulomas, and compared [ 64 Cu]-LLP2A with [ 18 F]-FDG over the course of infection. We found that [ 64 Cu]-LLP2A retention was driven by macrophages and T cells, with less contribution from neutrophils and B cells. In macaques, granulomas had higher [ 64 Cu]-LLP2A uptake than uninfected tissues, and immunohistochemical analysis of granulomas with known [ 64 Cu]-LLP2A uptake identified significant correlations between LLP2A signal and macrophage and T cell numbers. The same cells coexpressed integrin α4 and β1, further supporting that macrophages and T cells drive [ 64 Cu]-LLP2A avidity in granulomas. Over the course of infection, granulomas and thoracic lymph nodes experienced dynamic changes in affinity for both probes, suggesting metabolic changes and cell differentiation or recruitment occurs throughout granuloma development. These results indicate [ 64 Cu]-LLP2A is a PET probe for VLA-4, which when used in conjunction with [ 18 F]-FDG, may be a useful tool for understanding granuloma biology in TB., (Copyright © 2017 by The American Association of Immunologists, Inc.)

Milk fat globule-epidermal growth factor-factor 8 (MFG-E8) plays an immunomodulatory role in inflammatory diseases. MFG-E8-derived short peptide (MSP68) greatly reduces neutrophil infiltration and injury in the lung during sepsis. In this study, we examined the effect of MSP68 on chemotaxis of various immune cells and its regulatory mechanism. Bone marrow-derived neutrophils (BMDNs) from C57BL/6 mice, human monocyte THP-1 cell line, and human T lymphocyte Jurkat cell line were used for adhesion and migration assays using a Transwell method in the presence of MSP68. Treatment with MSP68 significantly inhibited the BMDN and THP-1 cell but not Jurkat cell adhesion on the TNF-α-stimulated pulmonary artery endothelial cell (PAEC) monolayer dose-dependently. MSP68 also significantly reduced BMDN adhesion on VCAM-1-coated wells dose dependently. Surface plasmon resonance (SPR) analysis revealed that MSP68 efficiently recognized integrin α 4 β 1 (receptor for VCAM-1) at the dissociation constant (K D ) of 1.53 × 10 -7 M. These findings implicate that MSP68 prevents neutrophil adhesion to the activated endothelial cells by interfering with the binding between integrin α 4 β 1 on neutrophils and VCAM-1 on endothelial cells. Moreover, MSP68 significantly attenuated the migration of BMDN and THP-1 cells but not Jurkat cells to their chemoattractants. Pretreatment with MSP68 inhibited the transmigration of BMDNs across the PAECs toward chemoattractants, fMLP, MIP-2, and complement fragment 5a (C5a) dose-dependently. Finally, we identified that the activation of p38 MAPK in BMDNs by fMLP was inhibited by MSP68. Thus, MSP68 attenuates extravasation of immune cells through the endothelial cell lining into inflamed tissue, implicating MSP68 to be a novel, therapeutic agent for inflammatory diseases caused by excessive immune cell infiltration., (© Society for Leukocyte Biology.)

Vascular cell adhesion molecule 1 (VCAM-1) is an adhesion molecule assigned to the activated endothelium mediating immune cells adhesion and extravasation. However, its expression in renal carcinomas inversely correlates with tumor malignancy. Our experiments in clear cell renal cell carcinoma (ccRCC) cell lines demonstrated that von Hippel Lindau (VHL) loss, hypoxia, or PHD (for prolyl hydroxylase domain-containing proteins) inactivation decreased VCAM-1 levels through a transcriptional mechanism that was independent of the hypoxia-inducible factor and dependent on the nuclear factor κB signaling pathway. Conversely, VHL expression leads to high VCAM-1 levels in ccRCC, which in turn leads to better outcomes, possibly by favoring antitumor immunity through VCAM-1 interaction with the α4β1 integrin expressed in immune cells. Remarkably, in ccRCC human samples with VHL nonmissense mutations, we observed a negative correlation between VCAM-1 levels and ccRCC stage, microvascular invasion, and symptom presentation, pointing out the clinical value of VCAM-1 levels as a marker of ccRCC progression., (© 2017 Labrousse-Arias et al.)

Background: The precise mechanisms controlling homing of T effector (Teff) cells to the inflamed gut in Crohn's disease (CD) are still unclear, and clinical outcome data from patients with inflammatory bowel disease treated with the anti-α4β7 integrin antibody vedolizumab suggest differences between ulcerative colitis and CD., Methods: Expression of homing molecules was studied with flow cytometry and immunohistochemistry. Their functional role was investigated in in vitro adhesion assays and in a humanized mouse model of T cell homing to the inflamed gut in vivo., Results: Despite in vitro blockade of CD Teff adhesion to mucosal vascular addressin cell adhesion molecule-1 (MadCAM-1) and in contrast to previous observations in ulcerative colitis, anti-α4β7 treatment did not result in reduced Teff cell homing to the colon in vivo. However, the integrin α4β1 was expressed in higher levels on Teffs from patients with CD compared with controls, while its expression in the peripheral blood declined, and its expression in the intestine increased during the course of clinical vedolizumab treatment. Consistently, adhesion of CD Teffs to vascular cell adhesion molecule-1 (VCAM-1) was blocked by inhibition of α4 and α4β1 in vitro. Moreover, in vivo homing of CD Teffs to the ileum was reduced by inhibition of α4 and α4β1 integrins, but not α4β7 integrins., Conclusions: Our findings suggest that Teff cell homing to the ileum through the axis α4β1-VCAM-1 is an essential and nonredundant pathway in CD in vivo, possibly affecting efficacy of clinical treatment with antiadhesion compounds.

Secreted phospholipase A2 type IIA (sPLA2-IIA) is a well-established pro-inflammatory protein and has been a major target for drug discovery. However, the mechanism of its signaling action has not been fully understood. We previously found that sPLA2-IIA binds to integrins αvβ3 and α4β1 in human and that this interaction plays a role in sPLA2-IIA's signaling action. Our recent studies found that sPLA2-IIA activates integrins in an allosteric manner through direct binding to a newly identified binding site of integrins (site 2), which is distinct from the classical RGD-binding site (site 1). The sPLA2-IIA-induced integrin activation may be related to the signaling action of sPLA2-IIA. Since sPLA2-IIA is present in normal human tears in addition to rheumatoid synovial fluid at high concentrations the sPLA2-IIA-mediated integrin activation on leukocytes may be involved in immune responses in normal and pathological conditions.

Objectives: Bone marrow-derived cells (BMCs) have recently been identified to play a vital role in repairing damaged myocardium; however, it is not known whether or not mobilization of BMCs is involved in the pathogenesis of acute viral myocarditis (VMC). Thus, we analyzed the expression of CD45+CD34+VLA-4+ cells and vascular cell adhesion protein-1 (VCAM-1) in a murine model of acute VMC., Methods: Male BALB/c mice were intraperitoneally infected with coxsackievirus B3 to establish acute VMC. The frequency of CD45+CD34+VLA-4+ cells in the heart, peripheral blood, and bone marrow was examined by flow cytometry 3, 7, 14, and 28 days after injection. Cardiac VCAM-1 and pathology scores were determined by immunohistochemistry, and myocardial VCAM-1, IL-1β, and TNF-α were analyzed by RT-PCR and Western blot., Results: In mice with acute VMC, the CD45+CD34+VLA-4+ cell population in the heart was significantly increased by day 7 and then decreased; in contrast, the CD45+CD34+VLA-4+ cell population decreased in the bone marrow and peripheral blood by day 3 and then increased. High expression of VCAM-1 was detected in the heart in parallel with CD45+CD34+VLA-4+ cell expression., Conclusions: In mice with acute VMC, VCAM-1-induced CD45+CD34+VLA-4+ cell mobilization into the injured heart is involved in the repair of injured myocardium., (© 2017 S. Karger AG, Basel.)

Cell-adhesion molecules (CAMs) dynamics in Multiple Sclerosis (MS) patients have been widely studied after Natalizumab (NTZ) introduction. However, their temporal dynamics after NTZ withdrawal (NTZ-W) has not been described. We prospectively evaluate changes in the expression levels of CAMs (CD49d, CD29, L-Selectin and CD11a) involved in T cell migration of 22 MS patients after NTZ-W. CD49d, CD29 and CD11a expression experienced a continuous increase expression two months after NTZ-W and Cd49d expression at month six after NTZ-W correlated to NTZ treatment duration, both in CD45 + CD4 + and CD45 + CD8 + . CD49d expression up to month three after NTZ-W was related to MS activity in CD45 + CD8 + at the end of the study. Results from this study suggest that patients with a longer NTZ treatment are more susceptible to present a "molecular rebound" after NTZ-W. CD49d determination may be a useful tool to closely monitor MS activity in patients who interrupt NTZ., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Binding of intercellular adhesion molecule-1 to the β2-integrin leukocyte function associated antigen-1 (LFA-1) is known to induce cross-talk to the α4β1 integrin. Using different LFA-1 monoclonal antibodies, we have been able to study the requirement and mechanism of action for the cross-talk in considerable detail. LFA-1-activating antibodies and those inhibitory antibodies that signal to α4β1 induce phosphorylation of Thr-758 on the β2-chain, which is followed by binding of 14-3-3 proteins and signaling through the G protein exchange factor Tiam1. This results in dephosphorylation of Thr-788/789 on the β1-chain of α4β1 and loss of binding to its ligand vascular cell adhesion molecule-1. The results show that with LFA-1 antibodies, we can activate LFA-1 and inhibit α4β1, inhibit both LFA-1 and α4β1, inhibit LFA-1 but not α4β1, or not affect LFA-1 or α4β1 These findings are important for the understanding of integrin regulation and for the interpretation of the effect of integrin antibodies and their use in clinical applications., (© 2016 by The American Society of Hematology.)

The skin is an important barrier organ and frequent target of autoimmunity and allergy. In this study, we found innate-like B cells that expressed the anti-inflammatory cytokine IL-10 in the skin of humans and mice. Unexpectedly, innate-like B1 and conventional B2 cells showed differential homing capacities with peritoneal B1 cells preferentially migrating into the inflamed skin of mice. Importantly, the skin-homing B1 cells included IL-10-secreting cells. B1 cell homing into the skin was independent of typical skin-homing trafficking receptors and instead required α4β1-integrin. Moreover, B1 cells constitutively expressed activated β1 integrin and relocated from the peritoneum to the inflamed skin and intestine upon innate stimulation, indicating an inherent propensity to extravasate into inflamed and barrier sites. We conclude that innate-like B cells migrate from central reservoirs into skin, adding an important cell type with regulatory and protective functions to the skin immune system., (Copyright © 2016 by The American Association of Immunologists, Inc.)

A better understanding of the cellular targets of HIV infection in the female genital tract may inform HIV prevention efforts. Proposed correlates of cellular susceptibility include the HIV co-receptor CCR5, peripheral homing integrins, and immune activation. We used a CCR5-tropic pseudovirus to quantify HIV entry into unstimulated endocervical CD4(+) T cells collected by cytobrush. Virus entry was threefold higher into cervix-derived CD4(+) T cells than blood, but was strongly correlated between these two compartments. Cervix-derived CD4(+) T cells expressing CD69, α(4)β(7), or α(4)β(1) were preferential HIV targets; this enhanced susceptibility was strongly correlated with increased CCR5 expression in α(4)β(7)(+) and CD69(+) CD4(+) T cells, and to a lesser extent in α(4)β(1)(+) CD4(+) T cells. Direct binding of gp140 to integrins was not observed, integrin inhibitors had no effect on virus entry, and pseudotypes with an env that preferentially binds α(4)β(7) still demonstrated enhanced entry into α(4)β(1)(+) cells. In summary, a rapid and sensitive HIV entry assay demonstrated enhanced susceptibility of activated endocervical CD4(+) T cells, and those expressing α(4)β(7) or α(4)β(1). This may relate to increased CCR5 expression by these cell subsets, but did not appear to be due to direct interaction of α(4)β(7) or α(4)β(1) with HIV envelope.

The Th cells that regulate peritoneal B-1 cell functions have not yet been well characterized. To address this question, we investigated peritoneal CD4(+) T cells, observed a high frequency of the conjugates of B-CD4(+) T cells in the peritoneal cavity, and identified a population of CD49d(high)CD4(+) T cells that constituted about half of all CD4(+) T cells in the peritoneal cavity, but were rarely found in other compartments. Peritoneal CD49d(high)CD4(+) T cells were CD44(high)CD62L(low); expressed integrin α4β1 and CXCR3; and rapidly secreted IFN-γ, TNF-α, and IL-2, showing features of proinflammatory Th1 cells. Peritoneal CD49d(high)CD4(+) T cells developed spontaneously, were detected at the age of 12 d, and showed stem cell-like properties. Their development was observed in mice deficient for signaling lymphocytic activation molecule-associated protein, but not in athymic nude mice and mice lacking in expression of MHC class II on thymic epithelial cells. Peritoneal CD49d(high)CD4(+) T cells were more resistant to irradiation and more sensitive to NAD-induced cell death than CD49d(low)CD4(+) T cells. Notably, peritoneal CD49d(high)CD4(+) T cells also showed some characteristics of follicular Th cells, such as the expression of programmed cell death 1, ICOS, IL-21, and CXCR5. Moreover, peritoneal CD49d(high)CD4(+) T cells enhanced the secretion of IgM Abs by B-1a cells and IgG Abs by splenic B cells. These data suggest that peritoneal CD49d(high)CD4(+) T cells may be innate-like CD4(+) T cells, which develop early and have a dual capacity to support both humoral and cellular immunity., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Mucosal-associated invariant T (MAIT) cells have a semi-invariant TCR Vα-chain, and their optimal development is dependent upon commensal flora and expression of the nonpolymorphic MHC class I-like molecule MR1. MAIT cells are activated in an MR1-restricted manner by diverse strains of bacteria and yeast, suggesting a widely shared Ag. Recently, human and mouse MR1 were found to bind bacterial riboflavin metabolites (ribityllumazine [RL] Ags) capable of activating MAIT cells. In this study, we used MR1/RL tetramers to study MR1 dependency, subset heterogeneity, and protective effector functions important for tuberculosis immunity. Although tetramer(+) cells were detected in both MR1(+/+) and MR1(-/-) TCR Vα19i-transgenic (Tg) mice, MR1 expression resulted in significantly increased tetramer(+) cells coexpressing TCR Vβ6/8, NK1.1, CD44, and CD69 that displayed more robust in vitro responses to IL-12 plus IL-18 and RL Ag, indicating that MR1 is necessary for the optimal development of the classic murine MAIT cell memory/effector subset. In addition, tetramer(+) MAIT cells expressing CD4, CD8, or neither developing in MR1(+/+) Vα19i-Tg mice had disparate cytokine profiles in response to RL Ag. Therefore, murine MAIT cells are considerably more heterogeneous than previously thought. Most notably, after mycobacterial pulmonary infection, heterogeneous subsets of tetramer(+) Vα19i-Tg MAIT cells expressing CXCR3 and α4β1 were recruited into the lungs and afforded early protection. In addition, Vα19iCα(-/-)MR(+/+) mice were significantly better protected than were Vα19iCα(-/-)MR1(-/-), wild-type, and MR1(-/-) non-Tg mice. Overall, we demonstrate considerable functional diversity of MAIT cell responses, as well as that MR1-restricted MAIT cells are important for tuberculosis protective immunity., (Copyright © 2015 by The American Association of Immunologists, Inc.)

IRF8 (interferon-regulatory factor-8) plays a critical role in regulating myeloid cell differentiation. However, the role of this transcription factor in the development of Ly6C+ inflammatory monocytes and their migration to the infected brain has not been examined. We have previously shown that West Nile virus (WNV) infection of wild-type (WT) mice triggers a significant increase in numbers of Ly6C+ monocytes in the bone marrow. These cells traffic via the blood to the infected brain, where they give rise to proinflammatory macrophages. Here, we show that WNV-infected IRF8-deficient (IRF8-/-) mice had significantly reduced numbers of Ly6C+ monocytes in the periphery, with few of these cells found in the blood. Furthermore, low numbers of inflammatory monocyte-derived macrophages were observed in the brains of IRF8-/- mice throughout infection. Adoptive transfer of IRF8-/- Ly6C+ monocytes demonstrated that these cells were intrinsically unable to traffic to the inflamed brain. Low expression of the chemokine receptor CCR2 and integrin VLA-4 by IRF8-/- monocytes likely contributed to this defect, as the interactions between these proteins and their ligands are critical for monocyte egress and migration to inflammatory foci. These data highlight a critical role for IRF8 in inflammatory monocyte differentiation and migration during WNV infection., (© 2014 S. Karger AG, Basel.)

Multiple sclerosis (MS) is a disorder of putative autoimmune origin, where immune cells invade the central nervous system and cause damage by attacking the myelin sheath of nerve cells. The blockade of the integrin very late antigen-4 (VLA-4) with the monoclonal antibody natalizumab has become the most effective therapy against MS since its approval in 2004. It is assumed that the inhibition of VLA-4-mediated immune cell adhesion to the endothelium of the blood-brain barrier (BBB) alleviates pathogenic processes of MS and, therefore, reduces disease severity and burden. Not all approaches to treat additional immune-mediated disorders (e.g. Rasmussen encephalitis and neuromyelitis optica) with natalizumab have been successful, but allowed researchers to gain additional insight into mechanisms of specific immune cell subsets' migration through the BBB in the human system. While the long-term efficacy and general tolerability of natalizumab in MS are clear, the over 400 cases of natalizumab-associated progressive multifocal leukoencephalopathy (PML) have been of great concern and methods of risk stratification in patients have become a major area of research. Modern risk stratification includes established factors such as treatment duration, previous immune-suppressive therapy, and anti-John Cunningham virus (JCV) antibody seropositivity, but also experimental factors such as anti-JCV antibody titers and levels of L-selectin. Today, anti-VLA-4 therapy is reserved for patients with highly active relapsing-remitting MS and patients are monitored closely for early signs of potential PML., (© The Japanese Society for Immunology. 2014. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Anti-adhesion therapies that target α(4) integrins (e.g., natalizumab) are thought to work by blocking T-cell recruitment to the intestinal tissues in patients with Crohn's disease (CD); however, little direct evidence is available to confirm this contention. We wished to evaluate the importance of T cell-associated α(4) integrins in a chronic colitis model in mice and to determine the effect of natalizumab treatment on intestinal tissue T-cell accumulation in human CD. Adoptive transfer of T cells lacking α(4) (α(4)(-/-)) but not β(1) integrin into immunodeficient mice produced significantly attenuated disease. This was correlated with reduced numbers of colon CD4 T cells compared with the control mice; however, tissue distribution of T helper type 1 (Th1) and T helper type 17 (Th17) cells and regulatory T cells (Tregs) was not affected by the lack of α(4). Furthermore, α(4)(-/-) T cells demonstrated defective homing to the chronically inflamed small intestines and colons. Finally, patients treated with natalizumab showed significant reduction in mucosal CD4 T cells and no skewing in the foxp3(+) Treg or T-bet(+)Th1 fractions thereof. These results demonstrate a direct role for T cell-associated α(4)β(7) but not α(4)β(1) integrins during initiation and perpetuation of chronic colitis. Moreover, our data demonstrated that natalizumab treatment reduced mucosal CD4 T-cell accumulation in CD patients.

To explore the TCR sensitivity of recent thymic emigrants (RTEs), we triggered T cells with altered peptide ligands (APLs). Upon peptide stimulation in vitro, RTEs exhibited increased TCR signal transduction, and following infection in vivo with APL-expressing bacteria, CD8 RTEs expanded to a greater extent in response to low-affinity Ags than did their mature T cell counterparts. RTEs skewed to short-lived effector cells in response to all APLs but also were characterized by diminished cytokine production. RTEs responding to infection expressed increased levels of VLA-4, with consequent improved entry into inflamed tissue and pathogen clearance. These positive outcomes were offset by the capacity of RTEs to elicit autoimmunity. Overall, salient features of CD8 RTE biology should inform strategies to improve neonatal vaccination and therapies for cancer and HIV, because RTEs make up a large proportion of the T cells in lymphodepleted environments., (Copyright © 2014 by The American Association of Immunologists, Inc.)

The focus of this study is the characterization of human T cell blood-brain barrier migration and corresponding molecular trafficking signatures. We examined peripheral blood and cerebrospinal fluid immune cells from patients under long-term anti-very late antigen-4 (VLA-4)/natalizumab therapy (LTNT) and from CNS specimens. LTNT patients' cerebrospinal fluid T cells exhibited healthy central-/effector-memory ratios, but lacked CD49d and showed enhanced myeloma cell adhesion molecule (MCAM) expression. LTNT led to an increase of PSGL-1 expression on peripheral T cells. Although vascular cell adhesion molecule-1 (VLA-4 receptor) was expressed at all CNS barriers, P-selectin (PSGL-1-receptor) was mainly detected at the choroid plexus. Accordingly, in vitro experiments under physiological flow conditions using primary human endothelial cells and LTNT patients' T cells showed increased PSGL-1-mediated rolling and residual adhesion, even under VLA-4 blockade. Adhesion of MCAM(+)/TH17 cells was not affected by VLA-4 blocking alone, but was abrogated when both VLA-4 and MCAM were inhibited. Consistent with these data, MCAM(+) cells were detected in white matter lesions, and in gray matter of multiple sclerosis patients. Our data indicate that lymphocyte trafficking into the CNS under VLA-4 blockade can occur by using the alternative adhesion molecules, PSGL-1 and MCAM, the latter representing an exclusive pathway for TH17 cells to migrate over the blood-brain barrier., (© 2014 Schneider-Hohendorf et al.)

The presence of erythroblasts in the peripheral blood is generally associated with severe underlying disorders. The anti-very late antigen-4 (anti-VLA-4) antibody natalizumab, which is approved for treatment of multiple sclerosis, mediates an increase in circulating haematopoietic stem cells and may also trigger erythroblastaemia. We investigated the prevalence of erythroblastaemia in sequential blood smears of 14 natalizumab-treated and 14 interferon-treated patients with multiple sclerosis. Erythroblastaemia was found in 13 natalizumab-treated subjects (93%), whereas all controls were negative (p<0.0001). Knowledge of this frequent side effect is crucial for the correct interpretation of blood smears in natalizumab-treated patients and to avoid unnecessary diagnostic procedures., (© The Author(s) 2014.)

B10.RIII mice were immunized with interphotoreceptor retinoid binding protein peptide to induce uveitis. Mice were injected intraperitoneally with anti-very late antigen-4 (VLA-4), anti-leukocyte function-associated antigen-1 (LFA-1), or a control Ab every other day from Day 5 to Day 13 post-immunization. The eyes and spleens were harvested on Day 14 or 28. The eyes were used for histologic/cytokine mRNA expression analyses. The spleens were used for Ag-recall cytokine production assays and intracellular cytokine assays. Treatment with both Abs led to a profoundly significant reduction in severity of uveitis and cytokine mRNA expression in the eye. However, cytokine production by splenocytes was significantly upregulated. Discontinuation of Ab treatment led to an increase in uveitis severity and cytokine mRNA expression in the eye, but led to a decrease in cytokine production and intracellular IFN-γ(+) and IL-17A(+)cytokine profile by splenocytes. Thus, blockade of these molecules using specific Abs may be a therapeutic option for patients with uveitis; however, such treatment must be continued., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Natalizumab is a humanized monoclonal antibody against the leukocyte adhesion molecule very late antigen (VLA)-4, and is currently an approved therapy for patients with relapsing-remitting multiple sclerosis (RRMS). However, it is unknown whether natalizumab is beneficial for progressive forms of MS. Therefore, we assessed the effects of anti-VLA-4 monoclonal antibody (mAb) therapy in a progressive experimental autoimmune encephalomyelitis (EAE) mouse model. Notably, we found that early therapy could significantly reduce the severity of progressive EAE, while treatment initiated at an advanced stage was less efficient. Furthermore, we observed the accumulation of a novel subset of GM-CSF-producing CD11b+CD4+ T cells in the CNS throughout disease progression. Importantly, early therapeutic anti-VLA-4 mAb treatment suppressed the accumulation of these GM-CSF-producing CD11b+CD4+ T cells in the CNS along with activated microglia/macrophages populations, and also conferred a protective effect against inflammation-mediated neurodegeneration, including demyelination and axonal loss. Collectively, our data suggest that early treatment with anti-VLA-4 mAb can provide neuroprotection against progressive CNS autoimmune disease by preventing the accumulation of pathogenic GM-CSF-producing CD11b+CD4+ T cells in the CNS.

T cells express multiple integrin molecules. The significance of signaling through these molecules on acquisition of T-cell effector functions and memory formation capacity remains largely unknown. Moreover, the impact of stimulation through these signals on the generation of T cells for adoptive immunotherapy has not been elucidated. In this study, using a recombinant fragment of fibronectin, CH-296, we demonstrated that stimulation via very late Ag (VLA)-4 and VLA-5 in human and BALB/c mouse CD8(+) T cells, in combination with TCR stimulation, enhances effector multifunctionality and in vivo memory formation. Using TCR-transgenic mouse-derived CD8(+) T cells expressing TCR specific for the syngeneic CMS5 fibrosarcoma-derived tumor Ag, we showed that stimulation by CH-296 improved the ability of tumor-specific CD8(+) T cells to inhibit CMS5 tumor growth when adoptively transferred into hosts with progressing tumors. Improved antitumor effects were associated with decreased infiltration of Foxp3(+) CD4(+) Treg cells in tumors. These results suggest that stimulation via VLA-4 and VLA-5 modulates the qualities of effector T cells and could potentially increase the efficacy of adoptive therapy against cancer., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Objective: To determine the long-term effect of natalizumab (NTZ) treatment on the expression of integrins and chemokine receptors involved in the migration of T cells towards the central nervous system (CNS)., Methods: We drew the blood of 23 patients just before starting NTZ therapy and every 12 months thereafter, for up to 48 months of treatment. We assessed the ex-vivo expression of phenotype markers (CCR7 and CD45RA), CNS-addressing integrins (CD11a, CD49d and CD29) and chemokine receptors (CXCR3 and CCR6) in CD4+ or CD8+ T-cell subsets by flow cytometry., Results: As compared to the pre-NTZ values, there was a marked increase in central memory (CCR7+/CD45RA-) CD4+ T cells and in effector memory (CCR7-/CD45RA-) CD8+ T cells at 12 and 24 months. In addition to an expected downregulation of both VLA-4 subunits (CD49d/CD29), we also found decreased T-cell expression of CXCR3 at 12 months, and of CD11a (LFA-1 αL subunit) at 12 months, but mostly at 24 months of NTZ treatment., Conclusion: Our data show a nadir of CD11a expression at 2 years of NTZ treatment, at the peak of incidence of progressive multifocal leukoencephalopathy (PML), indirectly suggesting that a lack of these molecules may play a role in the onset of PML in NTZ-treated patients., (© The Author(s) 2013.)

Integrin-ligand interactions between germinal center (GC) B cells and Ag-presenting follicular dendritic cells (FDCs) have been suggested to play central roles during GC responses, but their in vivo requirement has not been directly tested. In this study, we show that, whereas integrins αLβ2 and α4β1 are highly expressed and functional on mouse GC B cells, removal of single integrins or their ligands had little effect on B cell participation in the GC response. Combined β2 integrin deficiency and α4 integrin blockade also did not affect the GC response against a particulate Ag. However, the combined integrin deficiency did cause B cells to be outcompeted in splenic GC responses against a soluble protein Ag and in mesenteric lymph node GC responses against gut-derived Ags. Similar findings were made for β2-deficient B cells in mice lacking VCAM1 on FDCs. The reduced fitness of the GC B cells did not appear to be due to decreased Ag acquisition, proliferation rates, or pAKT levels. In summary, our findings provide evidence that αLβ2 and α4β1 play overlapping and context-dependent roles in supporting interactions with FDCs that can augment the fitness of responding GC B cells. We also find that mouse GC B cells upregulate αvβ3 and adhere to vitronectin and milk-fat globule epidermal growth factor VIII protein. Integrin β3-deficient B cells contributed in a slightly exaggerated manner to GC responses, suggesting this integrin has a regulatory function in GC B cells.

Chlamydia trachomatis infection is the most common sexually transmitted bacterial infection in the United States and a significant health burden worldwide. Protection from Chlamydia infection in the genital mucosa is dependent on IFN-γ derived from CD4(+) Th1 cells. These CD4(+) T cells must home successfully to the genital tract to exert their effector function and decrease C. trachomatis burden. Although adhesion receptors expressed by CD4(+) T cells in the genital tract have been characterized, the integrin receptor required for Chlamydia-specific CD4(+) T cell-mediated protection has not been explored. In this study, we demonstrate that C. trachomatis infection of the upper genital tract results in recruitment of Chlamydia-specific CD4(+) T cells robustly expressing the integrin α4β1. Interfering with α4β1, but not α4β7, function resulted in defective CD4(+) T cell trafficking to the uterus and high bacterial load. We conclude that integrin α4β1 is necessary for CD4(+) T cell-mediated protection against C. trachomatis infection in the genital mucosa. By identifying homing molecules required for successful CD4(+) T cell trafficking to C. trachomatis-infected tissues, we will be better equipped to design vaccines that elicit sterilizing, long-lasting immunity without inducing immune pathologies in the upper genital tract.

Introduction: Natalizumab blocks α4-integrins and is a prototypic agent for a series of anti-inflammatory drugs that impair trafficking of immune cells into the CNS. However, modulation of the access of immune cells to the CNS is associated with impaired immune surveillance and detrimental viral infections of the CNS. Here, we explored the potency of cellular immune responses within the CNS to protect against viral encephalitis in mice with T cell conditional disruption of VLA-4 integrin (α4β1) expression., Results: While VLA-4 expression in virus specific Th1 cells is non-redundant for their ability to access the CNS, α4-integrin deficient Th17 cells enter the CNS compartment and generate an inflammatory milieu upon intrathecal vaccinia virus (VV) infection. However, in contrast to Th1 cells that can adopt direct cytotoxic properties, Th17 cells fail to clear the virus due to insufficient Eomes induced perforin-1 expression., Conclusion: The quality of the intrathecal cellular antiviral response under conditions of impaired VLA-4 function jeopardizes host protection. Our functional in vivo data extend our mechanistic understanding of anti-viral immunity in the CNS and help to estimate the risk potential of upcoming therapeutic agents that target the trafficking of immune cells into distinct anatomical compartments.

Objective: The inability to successfully treat women with ovarian cancer is due to the presence of metastatic disease at diagnosis and the development of platinum resistance. Ovarian cancer metastasizes throughout the peritoneal cavity by attaching to and invading through the mesothelium lining the peritoneum using a mechanism that involves α4β1 integrin and its ligand (vascular cell adhesion molecule) VCAM-1. Integrin α4β1 expression on tumor cells is known to confer protection from therapy in other cancers, notably multiple myeloma. We evaluated the role of α4β1 integrin in response to platinum-based therapy in a mouse model of peritoneal ovarian cancer metastasis by treatment with a humanized anti-α4β1 integrin function-blocking antibody., Methods: Integrin α4β1 expression on primary human ovarian cancer cells, fallopian tube and ovarian surface epithelia and fresh tumor was assessed by flow-cytometry. The therapeutic impact of anti-α4β1 treatment was assessed in murine models of platinum-resistant peritoneal disease and in vitro using the platinum resistant ovarian cancer cell lines., Results: Treatment of tumor-bearing mice with human-specific α4β1 integrin function-blocking antibodies, anti-VCAM-1 antibody or carboplatin alone had no effect on tumor burden compared to the IgG control group. However, the combined treatment of anti-α4β1 integrin or anti-VCAM-1 with carboplatin significantly reduced tumor burden. In vitro, the combination of carboplatin and anti-α4β1 integrin antibodies resulted in increased cell death and doubling time., Conclusions: Our findings support a role for α4β1 integrin in regulating treatment response to carboplatin, implicating α4β1 integrin as a potential therapeutic target to influence platinum responsiveness in otherwise resistant disease., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Very late antigen-4 (VLA-4) is frequently overexpressed on melanoma cells contributing to inflammation-dependent metastasis. Melanoma cell adhesion to endothelium via VLA-4-vascular cell adhesion molecule-1 (VCAM-1) interaction was used to study VLA-4 activation during melanoma cell response to inflammation. Cooperation among major inflammatory mediators was analyzed in melanoma cells exposed to single inflammatory factors in the presence of inhibitors for other assayed mediators. A stepwise cascade of hierarchized molecules heterogeneously made and used during melanoma response to IL-18, induced hydrogen peroxide (H2O2), in turn activating VLA-4 and melanoma cell adhesion to endothelium. The cascade involved prostaglandin E2 (PGE2) production from melanoma induced by IL-18-dependent tumor necrosis factor-α (TNFα); next, PGE2-induced IL-1β via vascular endothelial growth factor (VEGF) secretion, which in turn induced VLA-4 activation via cyclooxygenase 2-dependent H2O2. This sequence operated in IL-18R/VLA-4/VEGF-expressing murine (B16) and human (A375 and 883) melanomas, but not in those without this phenotype. Separation of active VLA-4-expressing B16 melanoma cells through immobilized VCAM-1 verified their higher IL-18R/TNFR1/VEGFR2 expression and metastatic growth than inactive VLA-4-expressing cells. However, cooperation among melanoma cell sub-populations with heterogeneous cytokine receptor levels may occur through VLA-4-stimulating factors, leading to intratumoral amplification of metastatic potential. Therefore, expression of the VLA-4-stimulating factor sequence may help to predict melanoma prometastatic risk, and offers therapeutic targets for metastatic melanoma deactivation through VLA-4 activation blockade.

We have previously described a microarray platform combining live basophils with protein arrays suitable for high-throughput detection of sensitisation against allergens. During optimisation of this technique, we observed severe losses of adhering cells during the washing steps, particularly after activation. In order to preserve cell binding, we tested the cell adhesion characteristics of different extracellular matrix proteins: human collagen I, fibronectin (FN) from bovine plasma and laminin (LN). FN was more effective than LN and collagen. Cell detachment after activation was in part due to reduced surface expression of VLA-4, the main ligand for FN, which was significantly decreased within 15 min of stimulation with 1 μg/mL calcium ionophore A23187, reaching a minimum after 2 h then slowly recovering. These optimised conditions were used for testing of well-characterised sera from allergic patients using two newly developed rat basophil leukaemia stable reporter cell lines (RBL NF-AT/GFP and RBL NF-AT/DsRed), which both express the human high-affinity IgE receptor alpha chain (FcεRIα). Both cell lines were able to detect sensitisation to specific allergens showing the expected bell-shaped dose-response curve, and correlated (R² = 0.75) with the standard beta-hexosaminidase assay, which is not suitable for an array format.

CD47 plays an important but incompletely understood role in the innate and adaptive immune responses. CD47, also called integrin-associated protein, has been demonstrated to associate in cis with β1 and β3 integrins. Here we test the hypothesis that CD47 regulates adhesive functions of T-cell α4β1 (VLA-4) and αLβ2 (LFA-1) in in vivo and in vitro models of inflammation. Intravital microscopy studies reveal that CD47(-/-) Th1 cells exhibit reduced interactions with wild-type (WT) inflamed cremaster muscle microvessels. Similarly, murine CD47(-/-) Th1 cells, as compared with WT, showed defects in adhesion and transmigration across tumor necrosis factor-α (TNF-α)-activated murine endothelium and in adhesion to immobilized intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion protein 1 (VCAM-1) under flow conditions. Human Jurkat T-cells lacking CD47 also showed reduced adhesion to TNF-α-activated endothelium and ICAM-1 and VCAM-1. In cis interactions between Jurkat T-cell β2 integrins and CD47 were detected by fluorescence lifetime imaging microscopy. Unexpectedly, Jurkat CD47 null cells exhibited a striking defect in β1 and β2 integrin activation in response to Mn(2+) or Mg(2+)/ethylene glycol tetraacetic acid treatment. Our results demonstrate that CD47 associates with β2 integrins and is necessary to induce high-affinity conformations of LFA-1 and VLA-4 that recognize their endothelial cell ligands and support leukocyte adhesion and transendothelial migration.

Activated T cells use very late antigen-4/α4β1 integrin for capture, rolling on, and firm adhesion to endothelial cells, and use leukocyte function-associated antigen-1/αLβ2 integrin for subsequent crawling and extravasation. Inhibition of α4β1 is sufficient to prevent extravasation of activated T cells and is successfully used to combat autoimmune diseases, such as multiple sclerosis. Here we show that effector T cells lacking the integrin activator Kindlin-3 extravasate and induce experimental autoimmune encephalomyelitis in mice immunized with autoantigen. In sharp contrast, adoptively transferred autoreactive T cells from Kindlin-3-deficient mice fail to extravasate into the naïve CNS. Mechanistically, autoreactive Kindlin-3-null T cells extravasate when the CNS is inflamed and the brain microvasculature expresses high levels of integrin ligands. Flow chamber assays under physiological shear conditions confirmed that Kindlin-3-null effector T cells adhere to high concentrations of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1, albeit less efficiently than WT T cells. Although these arrested T cells polarize and start crawling, only few remain firmly adherent over time. Our data demonstrate that the requirement of Kindlin-3 for effector T cells to induce α4β1 and αLβ2 integrin ligand binding and stabilization of integrin-ligand bonds is critical when integrin ligand levels are low, but of less importance when integrin ligand levels are high.

Kindlin-3 is an integrin-binding focal adhesion adaptor absent in patients with leukocyte and platelet adhesion deficiency syndrome and is critical for firm integrin-dependent leukocyte adhesion. The role of this adaptor in leukocyte diapedesis has never been investigated. In the present study, the functions of Kindlin-3 in this process were investigated in effector T lymphocytes trafficking to various lymphoid and nonlymphoid tissues. In vitro, Kindlin-3-deficient T cells displayed severely impaired lymphocyte function antigen-1-dependent lymphocyte adhesion but partially conserved very late antigen-4 adhesiveness. In vivo, the number of adoptively transferred Kindlin-3-deficient T effectors was dramatically elevated in the circulating pool compared with normal effectors, and the Kindlin-3 mutant effectors failed to enter inflamed skin lesions. The frequency of Kindlin-3-deficient T effectors arrested on vessel walls within inflamed skin-draining lymph nodes was also reduced. Strikingly, however, Kindlin-3-deficient effector T cells accumulated inside these vessels at significantly higher numbers than their wild-type lymphocyte counterparts and successfully extravasated into inflamed lymph nodes. Nevertheless, on entering these organs, the interstitial motility of these lymphocytes was impaired. This is the first in vivo demonstration that Kindlin-3-stabilized integrin adhesions, although essential for lymphocyte arrest on blood vessels and interstitial motility, are not obligatory for leukocyte diapedesis.

Inflammatory responses in the brain after cerebral ischemia have been studied extensively in male mice, but not female mice, thus potentially giving a less-than-accurate view of gender associated pathological processes. In humans, cerebral infarcts are typically smaller in premenopausal females than in age-matched males. In the current study, we confirmed smaller infarcts in female vs. male mice after middle cerebral artery occlusion and 96 h of reperfusion. Moreover, we explored immunological alterations related to this difference and found that the percentage of CD4+ T lymphocytes was significantly higher in spleens in males than females, with increased expression of the activation markers, CD69 and CD44. In contrast, the percentage of CD8+ T lymphocytes was significantly higher in spleens of females than males, leading to the identification of a small but distinct population of IL-10-secreting CD8+CD122+ suppressor T cells that were also increased in females. Finally, we observed that males have a greater percentage of activated macrophages/microglia in the brain than females, as well as increased expression of the VLA-4 adhesion molecule in both brain and spleen. This new information suggesting gender-dependent immunological mechanisms in stroke implies that effective treatments for human stroke may also be gender specific.

Bone marrow (BM) eosinopoiesis is a common feature during allergen exposure in atopic individuals. Airway exposure to staphylococcal superantigens aggravates allergic airway disease and increases the output of BM eosinophils. However, the exact mechanisms regulating eosinophil mobilization and trafficking to the peripheral circulation and airways remain to be elucidated. Therefore, this study aimed to investigate the mechanisms determining the BM eosinopoiesis in allergic mice under exposure to staphylococcal enterotoxin A (SEA). Ovalbumin (OVA)-sensitized male BALB/C mice were intranasally exposed to SEA (1 μg), and at 4, 12, 24, and 48 h later animals were challenged with OVA (10 μg, twice a day). Measurement of IL-5, eotaxin, and granulocyte-macrophage colony-stimulating factor (GM-CSF) levels, flow cytometry for CCR3(+), VLA4(+), and CCR3(+)VLA4(+), as well as adhesion assays to VCAM-1 were performed in BM. Prior airway exposure to SEA time dependently increased the BM eosinophil number in OVA-challenged mice. Eosinophils gradually disappear from peripheral blood, being recruited over time to the airways, where they achieve a maximal infiltration at 24 h. SEA exposure increased the levels of IL-5 and eotaxin (but not GM-CSF) in BM of OVA-challenged mice. Marked increases in CCR3(+) and CCR3(+)VLA4(+) expressions in BM eosinophils of OVA-challenged mice were observed, an effect largely reduced by prior exposure to SEA. Adhesion of BM eosinophils to VCAM-1 was increased in OVA-challenged mice, but prior SEA exposure abrogated this enhanced cell adhesion. Accumulation of BM eosinophils by airway SEA exposure takes place through IL-5- and CCR3-dependent mechanisms, along with downregulation of CCR3/VL4 and impaired cell adhesion to VCAM-1.

Monocyte-derived macrophages are essential for recovery after spinal cord injury, but their homing mechanism is poorly understood. Here, we show that although of common origin, the homing of proinflammatory (M1) and the "alternatively activated" anti-inflammatory (M2) macrophages to traumatized spinal cord (SC) was distinctly regulated, neither being through breached blood-brain barrier. The M1 macrophages (Ly6c(hi)CX3CR1(lo)) derived from monocytes homed in a CCL2 chemokine-dependent manner through the adjacent SC leptomeninges. The resolving M2 macrophages (Ly6c(lo)CX3CR1(hi)) derived from monocytes trafficked through a remote blood-cerebrospinal-fluid (CSF) barrier, the brain-ventricular choroid plexus (CP), via VCAM-1-VLA-4 adhesion molecules and epithelial CD73 enzyme for extravasation and epithelial transmigration. Blockage of these determinants, or mechanical CSF flow obstruction, inhibited M2 macrophage recruitment and impaired motor-function recovery. The CP, along with the CSF and the central canal, provided an anti-inflammatory supporting milieu, potentially priming the trafficking monocytes. Overall, our finding demonstrates that the route of monocyte entry to central nervous system provides an instructional environment to shape their function., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Integrin very late antigen-4 (VLA4) is induced during inflammation and can regulate monocyte migration. It has been implicated in atherogenesis, a significant concern in systemic lupus erythematosus (SLE). The aim of this study was to define VLA4 expression in SLE monocytes. Flow cytometry, reverse transcription polymerase chain reaction, Western blotting, and immunohistochemistry staining with confocal microscopy were used to evaluate VLA4 expression in SLE patients and controls. We found elevated expression of VLA4 in SLE patients with significantly increased VLA4 staining intracellularly compared to control. Exposure of control monocytes to SLE sera or immune complexes led to increased intracellular expression, and immune complexes were capable of driving redistribution of surface VLA4 to the cytoplasm. Therefore, VLA4 was found to be subject to complex regulation with SLE sera driving both RNA expression and redistribution of protein. Stimulation of SLE monocytes with a VLA4 ligand induced significant TNFα expression, confirming a functional effect. This behavior may contribute to increased atherosclerosis and monocyte infiltrates in end organs.