The movement of leukocytes from the blood into peripheral tissues is a central feature of immune surveillance, but also contributes to the pathogenesis of inflammatory and autoimmune diseases. Integrins are a family of adhesion and signaling molecules made up of paired alpha and beta subunits, and the integrin alpha4beta1 plays a prominent role in the trafficking of mononuclear leukocytes. We have previously described the direct interaction of the signaling adaptor molecule paxillin with the cytoplasmic domain of the alpha4 integrin subunit. This interaction is critical for alpha4beta1 integrin dependent cell adhesion under shear flow conditions as it provides a needed connection to the actin cytoskeleton. Furthermore, the alpha4-paxillin interaction is required for effective alpha4beta1 dependent leukocyte migration and does so through the temporal and spatial regulation of the small GTPase Rac. These findings make the alpha4-paxillin interaction a potentially attractive therapeutic target in controlling leukocyte trafficking.
Protein Binding ; Paxillin/*metabolism/physiology ; Models, Biological ; Leukocytes/cytology/*metabolism ; Integrin alpha4beta1/metabolism/physiology ; Integrin alpha4/*metabolism/physiology ; Humans ; Cell Movement/*physiology ; Cell Adhesion/physiology

There is growing evidence that crosstalk between mantle cell lymphoma (MCL) cells and stromal microenvironments, such as bone marrow and secondary lymphoid tissues, promotes tumor progression by enhancing survival and growth as well as drug resistance of MCL cells. Recent advances in the understanding of lymphoma microenvironment have led to the identification of crucial factors involved in the crosstalk and subsequent generation of their targeted agents. In the present study, we evaluated the combinatory effect of blocking antibodies (Ab) targeting CXCR4 and VLA-4, both of which were known to play significant roles in the induction of environment-mediated drug resistance (EMDR) in MCL cell line, Jeko-1. Simultaneous treatment with anti-CXCR4 and anti-VLA-4 Ab not only reduced the migration of Jeko-1 cells into the protective stromal cells, but also enhanced sensitivity of Jeko-1 to a chemotherapeutic agent to a greater degree than with either Ab alone. These combinatorial effects were associated with decreased phosphorylation of ERK1/2, AKT and NF-kappaB. Importantly, drug resistance could not be overcome once the adhesion of Jeko-1 to the stromal occurred despite the combined use of Abs, suggesting that the efforts to mitigate migration of MCLs should be attempted as much as possible. Our results provide a basis for a future development of therapeutic strategies targeting both CXCR4 and VLA-4, such as Ab combinations or bispecific antibodies, to improve treatment outcomes of MCL with grave prognosis.
Antibodies, Bispecific ; Antibodies, Blocking ; Bone Marrow ; Cell Line ; Drug Resistance ; Drug Therapy* ; Integrin alpha4beta1* ; Lymphoid Tissue ; Lymphoma ; Lymphoma, Mantle-Cell* ; NF-kappa B ; Phosphorylation ; Prognosis ; Stromal Cells

Macrophages have two major roles in regulating the dynamic equilibrium in erythropoiesis, promoting the differentiation and maturation of nucleated red blood cells into reticulocytes and removing old red blood cells. A recent mouse study has demonstrated that the phenotype of macrophages in erythroblastic islands is CD169+ VCAM-1+ ER-HR3+ CD11b+ F4/80+ Ly-6G+. Molecular connections between erythroid progenitor cells and central macrophages help to maintain the function and integrity of erythroblastic islands. New research advances in Kruppel-like factor 1 (KLF1) provide new evidence for the important role of macrophages in erythroblastic islands. Macrophages play an important role in erythropoiesis both in sickness and in health, and provide a potential targeted therapy for diseases such as polycythemia vera and beta-thalassemia in the future.
Animals ; Erythropoiesis ; Humans ; Integrin alpha4beta1 ; physiology ; Kruppel-Like Transcription Factors ; physiology ; Macrophages ; physiology ; Mice ; Phenotype ; Vascular Cell Adhesion Molecule-1 ; physiology

OBJECTIVE: To observe the means of fibronectin(FN) alternative splicing and the expression of EIIIA-FN variant in rat skin after bruise, for the sake of providing some help for forensic estimation of wound interval. METHODS: Total RNA was isolated from wounded skin, and reverse transcription polymerase chain reaction was conducted to amplify target segments. RESULTS: Detectable EIIIA+(526 bp) segments, lacked in normal organize, was amplified at 1 h after experimental wound, and the levels were increased within 24 h. CONCLUSION The alternative splicing EIIIA-fibronectin variant would be a satisfied criterion for research of skin injury.
Alternative Splicing ; Animals ; Epithelium/metabolism* ; Fibronectins/genetics* ; Forensic Medicine ; Integrin alpha4beta1/biosynthesis* ; RNA, Messenger/metabolism* ; Rats ; Reverse Transcriptase Polymerase Chain Reaction ; Skin/metabolism* ; Time Factors

To investigate the effect of vascular endothelial growth factor (VEGF) on beta1 integrin (VLA-4 and VLA-5) activation ability in K562 cells transfected with antisense VEGF121 cDNA, K562 cells were transfected with antisense (As), sense (S) and vector (V, pcDNA(3)). Flow cytometry was used to evaluate the expression rate of VLA-4 (CD49d/CD29) and VLA-5 (CD49e/CD29) and beta1 integrin activation ability in the transfected K562 cells. The results showed that the expression rates of VLA-4 and VLA-5 were more than 92% in the transfected K562 cells and there was no difference among the K562/V, K562/S and K562/As cells. However, beta1 integrin activated 9EG7 expression rate in K562/As cells was higher than that in K562/V cells [(75.6 +/- 10.5)% vs (41.2 +/- 2.1)%, P < 0.01)] after activation with beta1 integrin activator 8A2. It is concluded that function of beta1 integrin to be activated is restored in K562 cells transfected with antisense VEGF121 cDNA.
DNA ; genetics ; DNA, Antisense ; genetics ; Endothelial Growth Factors ; genetics ; metabolism ; Flow Cytometry ; Humans ; Integrin alpha4beta1 ; biosynthesis ; Integrin alpha5beta1 ; biosynthesis ; Intercellular Signaling Peptides and Proteins ; genetics ; metabolism ; K562 Cells ; Lymphokines ; genetics ; metabolism ; Transfection ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factors

Endothelial cell have been shown to play an active role in many phases of immunologic process, including binding of T lymphocytes, neutrophils, platelets, and monocytes to the endothelium, as well as phagocytosis. Endothelial cells can also serve as targets that undergo cell injury. The most prominent mediators of endothelial cell activation are IL-1beta and TNF-alpha. VLA-4 was first identified as an endothelial cell surface receptor. We performed the culture of endothelial cells derived from human glomerular capillaries and studied the cytokine-regulated expression of VCAM-1, and the effect of dexamethasone and TGF-beta on the cytokine induced VCAM-1 expression using ELISA method. Expression of VCAM-1 was not detectable above background level in the basal unstimulated state. However, VCAM-1 was rapidly induced after exposure to IL-1beta (5ng/ml) or TNF-alpha (1, 10ng/ml) (O.D.=1.76+/-0.15, 1.95+/-0.35, 1.88+/-0.17, mean+/-S.E., control=0.36+/-0.028, n=8-24, P<0.05). But IFN-gamma did not increase the expression of VCAM-1. Addition of dexamethasone (10 micrometer) and TGF-beta1 (1ng, 10ng/ml) blunted IL-1beta and TNF-alpha induced expression of VCAM-1. Therefore, VCAM-1 could be inducible in human glomerular endothelial cells, and it was regulated by dexamethasone and TGF-beta1. The negative findings in histopathology may reflect the transience of VCAM-1 expression and does not necessarily exclude an important role of this molecule in the early stages of renal disease.
Capillaries ; Dexamethasone* ; Endothelial Cells* ; Endothelium ; Enzyme-Linked Immunosorbent Assay ; Humans ; Integrin alpha4beta1 ; Monocytes ; Neutrophils ; Phagocytosis ; T-Lymphocytes ; Transforming Growth Factor beta ; Transforming Growth Factor beta1 ; Tumor Necrosis Factor-alpha ; Vascular Cell Adhesion Molecule-1*

Multiple sclerosis (MS) is the most common demyelinating disease affecting the central nervous system of young adults living in the western world. MS should be strongly suspected when a young adult develops one or more neurological episodes consistent with damage to white matter within the central nervous system (CNS), especially when these affect the optic nerves, brainstem, or spinal cord. The patient with relapses, each of which can be attributed to demyelination in the CNS, requires no investigation prior to establishing the diagnosis of clinically definite MS. For a diagnosis of MS, separate anatomical sites within the CNS must have been affected on different occasions, typically three. MS in Asian populations is characterized by the selective and dominant involvement of the optic nerve and spinal cord with some incidence of brainstem lesions. 35-40% of MS cases in Korea are of this optico-spinal type with or without brainstem lesions. Reported cases of neuromyelitis optica spectrum disease (NMOSD), causing severe optic neuritis (ON) and/or longitudinally extensive transverse myelitis, either monophase or with a relapse-remitting pattern, some of which were diagnosed previously as the optico-spinal form of MS in Asia, have increased annually in Korea with the development of the NMO-IgG or aquaporin4-antibody detecting technique. NMO-IgG detection is very important in the diagnosis of early stage of NMOSD and the differentiation of MS and other demyelinating disease. Many new convenient oral drugs or very potent intravenous monoclonal antibodies for targeting VLA-4, CD20, and CD52 may decrease the annual relapse rate and burden of brain-spinal cord lesionsin MS.
Antibodies, Monoclonal ; Asia ; Asian Continental Ancestry Group ; Brain Stem ; Central Nervous System ; Demyelinating Diseases ; Humans ; Incidence ; Integrin alpha4beta1 ; Korea ; Multiple Sclerosis ; Myelitis, Transverse ; Neuromyelitis Optica ; Optic Nerve ; Optic Neuritis ; Recurrence ; Spinal Cord ; Western World ; Young Adult

OBJECTIVETo study the effects of very late antigen(VLA) antagonist BIO-1211 on eosinophil chemotaxis, recruitment and mediator release.

METHODSEosinophil chemotaxis was induced by platelet activating factor(PAF) in vitro and eosinophil recruitment and release were determined in vivo.

RESULTVLA antagonist BIO-1211 inhibited eosinophil chemotaxis induced by PAF. The inhibitory rates at 4x10(-11), 4x10(-10), 4x10(-9) mol x L(-1) were 24.9%, 29.9%, and 31.3%, respectively. Pretreatment by BIO-1211 1, 3 and 10 mg x kg(-1) intraperitoneally inhibited the recruitment of eosinophils in PAF in the rat induced by Sephadex in a dose dependent manner. Inhibitory rates were 60.3%, 68.9%, and 72.9%(P<0.05), respectively. BIO-1211 did not inhibit eosinophil peroxidase(EPO) release from eosinophils.

CONCLUSIONBIO-1211 inhibits eosinophil chemotaxis and recruitment, alleviates local inflammation, and may represent a new type of drug for allergic diseases.

Animals ; Cell Movement ; drug effects ; Chemotaxis, Leukocyte ; drug effects ; Dose-Response Relationship, Drug ; Eosinophil Peroxidase ; Eosinophils ; drug effects ; physiology ; Integrin alpha4beta1 ; antagonists & inhibitors ; physiology ; Male ; Oligopeptides ; pharmacology ; Peroxidases ; secretion ; Platelet Activating Factor ; pharmacology ; Rats ; Rats, Sprague-Dawley

To explore the effect of ligustrazine on the expression of adherent molecule VCAM-1/VLA-4 of bone marrow cells in syngenic bone marrow transplantation (BMT) mice, the mice were divided into 3 groups: normal group (which received no treatment), BMT control group and ligustrazine-treated groups. BMT mouse models were established. The BMT control group and the ligustrazine-treated group were orally administered 0.2 ml saline per mouse and 2 mg ligustrazine per mouse, respectively, twice a day. On the day 7, 14, 21, 28 after BMT, mice were respectively killed. Bone marrow nucleated cells were detected, and then the expression of VCAM-1/VLA-4 was assayed by immunohistochemistry, RT-PCR and flow cytometry analysis, respectively. The results showed that in ligustrazine-treated group, the accounts of bone marrow nucleated cells on the day 7, 14, 21, 28 after BMT were all higher than that in BMT control group. The expression level in the ligustrazine-treated group was significantly higher than that in the BMT control group (P < 0.05 or P < 0.01). It is concluded that ligustrazine can enhance VCAM-1/VLA-4 expression in bone marrow after syngenic bone marrow transplantation in mice, which may be related to the mechanisms underlying the ligustrazine accelerating hematopoietic reconstitution in allogenic bone marrow transplantation.
Animals ; Bone Marrow Cells ; drug effects ; metabolism ; Bone Marrow Transplantation ; methods ; Flow Cytometry ; Immunohistochemistry ; Integrin alpha4beta1 ; biosynthesis ; genetics ; Male ; Mice ; Mice, Inbred BALB C ; Pyrazines ; pharmacology ; Reverse Transcriptase Polymerase Chain Reaction ; Transplantation, Isogeneic ; Vascular Cell Adhesion Molecule-1 ; biosynthesis ; genetics

OBJECTIVETo study the protective effect of bone marrow stromal cells (BMSCs) upon childhood leukemia cells and the influence of VLA-4 antibody in vitro on leukemia cell apoptosis.

METHODSBMSCs from children with acute leukemia-were isolated by human lymphocyte separation medium. BMSCs (adherent) and leukemia cells (suspended) were cultured in vitro. This study included four groups: leukemia cells alone (control), leukemia cells+BMSCs, leukemia cells+BMSCs supernatant and leukemia cells+BMSCs+VLA-4 antibody. The apoptosis rate of leukemia cells in the four groups was determined by Annexin Ⅴ-FITC double-labeled flow cytometry. The expression of survivin and bcl-2 genes in leukemia cells was ascertained by RT-PCR.

RESULTSThe apoptosis rate of leukemia cells in the leukemia cells+BMSCs and the leukemia cells+BMSCs supernatant groups was lower than that in the control group (P<0.05). Compared with the leukemia cells+BMSCs and the leukemia cells+BMSCs supernatant groups, the apoptosis rate of leukemia cells in the VLA-4 antibody group increased significantly (P<0.05). In the VLA-4 antibody group, the apoptosis rate of leukemia cells increased with prolonged culture time. There were significant differences in the apoptosis rate between 12 hrs and 24 hrs after VLA-4 antibody treatment (P<0.01). The expression of survivin and bcl-2 genes in leukemia cells from the VLA-4 antibody groups was reduced compared with that from the leukemia cells+BMSCs and the leukemia cells+BMSCs supernatant groups (P<0.05).

CONCLUSIONSBMSCs play protective roles on leukemia cells. VLA-4 antibody can block the adhesion between BMSCs and leukemia cells and promote leukemia cell apoptosis.

Adolescent ; Antibodies ; therapeutic use ; Apoptosis ; Bone Marrow Cells ; physiology ; Child ; Child, Preschool ; Female ; Genes, bcl-2 ; Humans ; Infant ; Inhibitor of Apoptosis Proteins ; Integrin alpha4beta1 ; immunology ; Leukemia ; pathology ; therapy ; Male ; Microtubule-Associated Proteins ; genetics ; Stromal Cells ; physiology