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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.)

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.)

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.)

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.

The goal of this study was to determine the role of integrin-mediated adhesion of bone-marrow-derived progenitor cells (BMPCs) as a requirement for the endothelial barrier protection in a lung injury model. C57BL mice were used as the source for BMPCs, which were characterized as CD34(+) and fetal liver kinase-1 (Flk1)(+) and also an expression of a repertoire of integrins. We used a mouse model of bacterial lipopolysaccharide (LPS)-induced lung vascular injury and edema formation to test the effects of BMPC integrin expression in preventing endothelial barrier injury. Adhesion of BMPCs to purified extracellular matrix proteins induced focal adhesion kinase (Fak) phosphorylation and formation of branching point structures in a alpha(4) and alpha(5) integrin-dependent manner. BMPCs expressing red fluorescent protein (RFP) were administered via the retro-orbital venous route in mice treated intraperitonially with LPS (7.5 mg/kg body weight). We observed increased retention of RFP-labeled Flk1(+) and CD34(+) BMPCs for up to 8 weeks in mice injured with LPS. BMPC transplantation increased survival by 50% (at 72-96 hours after LPS) and reduced lung vascular injury and extravascular water content induced by LPS. However, blocking with anti-alpha(4) or anti-alpha(5) integrin antibody or shRNA-mediated silencing of alpha(4) or alpha(5) integrins in donor BMPCs failed to prevent the vascular injury or edema formation and mortality. Thus, alpha(4) and alpha(5) integrin-dependent adhesion of BMPCs in lung tissue plays a critical role in preventing lung vascular injury and increasing survival in a mouse model of LPS-induced acute lung injury.

Mononuclear leukocytes infiltrate the artery wall via integrin-mediated mechanisms and play an integral role in intimal formation after stenting. We sought to determine if acute antagonism of the alpha4 subunit of very late antigen-4 is sufficient for the late attenuation of stent intimal area (IA). Twenty-four hypercholesterolemic rabbits underwent iliac artery balloon injury, followed 2 weeks later by stent implantation, and the animals were randomized to receive an anti-alpha4 antibody (HP1/2) or a nonspecific isotypic control immunoglobulin (1E6) intravenously 1 h before stenting. Compared with controls, HP1/2-treated rabbits showed 50%, 51%, and 44% reductions in the percentage on intimal cells that were macrophages on days 3, 7, and 28 after stenting and a 59% reduction in intimal proliferation on day 3. Although stent IA was reduced by 63% and 48% in the antibody-treated group compared with the control group on days 3 and 7, this difference was not present on day 28. These data highlight the need for sustained, anti-inflammatory therapies for the prevention of stent intimal formation.

Adhesion molecules and stromal cell-derived factor-1 (SDF-1)/CXCR4 signaling play key roles in homing and mobilization of hematopoietic stem cells (HSC). Active signaling through SDF-1/CXCR4 and upregulation of adhesion molecules are required for homing, whereas downregulation of adhesion molecules and disruption of SDF-1/CXCR4 signaling are required for mobilization of HSC. We studied the surface expression of CXCR4 very late activation antigen (VLA)-4 and VLA-5 on myeloma cells mobilized with cyclophosphamide and GM-CSF in 12 multiple myeloma patients undergoing HSC mobilization for autologous transplantation. We also studied the plasma levels of SDF-1 in apheresis collection of these patients. We observed a statistically significant decrease in the levels of SDF-1 and surface expression of CXCR4 on myeloma cells in four consecutive apheresis collections compared with premobilization bone marrow specimens. We also observed a statistically significant decrease in surface expression of VLA-4 in myeloma cells in the apheresis collections compared with premobilization bone marrow samples. Furthermore, myeloma cells derived from apheresis collections had decreased adhesion and trans-stromal migration in response to SDF-1, which could be reversed by short incubation with interleukin-6. Hence, mobilization of myeloma cells involves SDF-1/CXCR4 signaling and downregulation of VLA-4.