Soluble CD40L activates soluble and cell-surface integrin αvß3, α5ß1, and α4ß1 by binding to the allosteric ligand-binding site (site 2).

CD40L is a member of the TNF superfamily that participates in immune cell activation. It binds to and signals through several integrins, including αvß3 and α5ß1, 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 α4ß1, which is prevalently expressed on the same CD4+ T cells as CD40L, is another receptor for CD40L. Soluble (s)CD40L activated soluble integrins αvß3, α5ß1, and α4ß1 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.

The tellurium-based immunomodulator, AS101 ameliorates adjuvant-induced arthritis in rats.

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-1ß 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-1ß (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.

Personalized extended interval dosing of natalizumab in MS: A prospective multicenter trial.

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.

Blockade of VLA4 sensitizes leukemic and myeloma tumor cells to CD3 redirection in the bone marrow microenvironment.

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.

Neutrophils promote VLA-4-dependent B cell antigen presentation and accumulation within the meninges during neuroinflammation.

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.

VLA-4 phosphorylation during tumor and immune cell migration relies on its coupling to VEGFR2 and CXCR4 by syndecan-1.

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 Gαißγ, AC7 and PKA, which phosphorylates S988 on the integrin. This mechanism is blocked by a syndecan-mimetic peptide (SSTNVEGFR2), 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.

Dissecting Integrin Expression and Function on Memory B Cells in Mice and Humans in Autoimmunity.

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.

Age-related tumor growth in mice is related to integrin α 4 in CD8+ T cells.

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.

Anti-α4ß1 Integrin Antibodies Attenuated Brain Inflammatory Changes in a Mouse Model of Alzheimer's Disease.

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 Aß 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 α4ß1 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-ß 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.

PI3Kγ Activates Integrin α4 and Promotes Immune Suppressive Myeloid Cell Polarization during Tumor Progression.

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 α4-dependent MDSC recruitment to tumors and by stimulating the immunosuppressive polarization of MDSCs and TAMs. Here, we show that integrin α4 promotes immunosuppressive polarization of MDSCs and TAMs downstream of PI3Kγ, thereby inhibiting antitumor immunity. Genetic or pharmacological suppression of either PI3Kγ or integrin α4 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 α4 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 α4 prevents accumulation of MDSC and reduces myeloid cell expression of immunosuppressive factors that stimulate tumor immune escape, these results highlight PI3Kγ and integrin α4 as targets for the design of cancer therapeutics. Cancer Immunol Res; 5(11); 957-68. ©2017 AACR.

Increased Mobilization of CD45+CD34+VLA-4+ Cells in Acute Viral Myocarditis Induced by Coxsackievirus B3.

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-1ß, 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.

Wnt/ß-Catenin Signaling Induces Integrin α4ß1 in T Cells and Promotes a Progressive Neuroinflammatory Disease in Mice.

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 ß-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 ß-catenin expression in T cells leads to aberrant and Th1-biased T cell activation, enhanced expression of integrin α4ß1, and infiltration of activated T cells into the spinal cord, without affecting regulatory T cell function. Importantly, expression of ß-catenin in mature naive T cells was sufficient to drive integrin α4ß1 expression and CNS migration, whereas pharmacologic inhibition of integrin α4ß1 reduced the abnormal T cell presence in the CNS of ß-catenin-expressing mice. Together, these results implicate deregulation of the Wnt/ß-catenin pathway in CNS inflammation and suggest novel therapeutic strategies for neuroinflammatory disorders.

Impaired Transmigration of Myeloid-Derived Suppressor Cells across Human Sinusoidal Endothelium Is Associated with Decreased Expression of CD13.

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.

Technical Advance: New in vitro method for assaying the migration of primary B cells using an endothelial monolayer as substrate.

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.

Positron Emission Tomography Imaging of Macaques with Tuberculosis Identifies Temporal Changes in Granuloma Glucose Metabolism and Integrin α4ß1-Expressing Immune Cells.

Positron emission tomography and computed tomography imaging (PET/CT) is an increasingly valuable tool for diagnosing tuberculosis (TB). The glucose analog [18F]fluoro-2-deoxy-2-d-glucose ([18F]-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 [64Cu]-labeled CB-TE1A1P-PEG4-LLP2A ([64Cu]-LLP2A), a high affinity peptidomimetic ligand for very late Ag-4 (VLA-4; also called integrin α4ß1) binding cells in granulomas, and compared [64Cu]-LLP2A with [18F]-FDG over the course of infection. We found that [64Cu]-LLP2A retention was driven by macrophages and T cells, with less contribution from neutrophils and B cells. In macaques, granulomas had higher [64Cu]-LLP2A uptake than uninfected tissues, and immunohistochemical analysis of granulomas with known [64Cu]-LLP2A uptake identified significant correlations between LLP2A signal and macrophage and T cell numbers. The same cells coexpressed integrin α4 and ß1, further supporting that macrophages and T cells drive [64Cu]-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 [64Cu]-LLP2A is a PET probe for VLA-4, which when used in conjunction with [18F]-FDG, may be a useful tool for understanding granuloma biology in TB.

The α4ß1 Homing Pathway Is Essential for Ileal Homing of Crohn's Disease Effector T Cells In Vivo.

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-α4ß7 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-α4ß7 treatment did not result in reduced Teff cell homing to the colon in vivo. However, the integrin α4ß1 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 α4ß1 in vitro. Moreover, in vivo homing of CD Teffs to the ileum was reduced by inhibition of α4 and α4ß1 integrins, but not α4ß7 integrins. CONCLUSIONS: Our findings suggest that Teff cell homing to the ileum through the axis α4ß1-VCAM-1 is an essential and nonredundant pathway in CD in vivo, possibly affecting efficacy of clinical treatment with antiadhesion compounds.

VHL promotes immune response against renal cell carcinoma via NF-κB-dependent regulation of VCAM-1.

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 α4ß1 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.

MFG-E8-derived peptide attenuates adhesion and migration of immune cells to endothelial cells.

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 α4ß1 (receptor for VCAM-1) at the dissociation constant (KD) 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 α4ß1 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.

Secreted Phospholipase A2 Type IIA (sPLA2-IIA) Activates Integrins in an Allosteric Manner.

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 αvß3 and α4ß1 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.

Leukocyte adhesion molecule dynamics after Natalizumab withdrawal in Multiple Sclerosis.

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.