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PubMed2001

Endothelial cell activation by myeloblasts: molecular mechanisms of leukostasis and leukemic cell dissemination.

Leukostasis and tissue infiltration by leukemic cells are poorly understood life-threatening complications of acute leukemia. This study has tested the hypothesis that adhesion receptors and cytokines secreted by blast cells play central roles in these reactions. Immunophenotypic studies showed that acute myeloid leukemia (AML) cells (n = 78) of the M0 to M5 subtypes of the French-American-British Cooperative Group expressed various amounts of adhesion receptors, including CD11a, b, c/CD18, CD49d, e, f/CD29, CD54, sCD15, and L-selectin. The presence of functional adhesion receptors was evaluated using a nonstatic adhesion assay. The number of blast cells attached to unactivated endothelium increased by 7 to 31 times after a 6-hour exposure of endothelium to tumor necrosis factor (TNF)-alpha. Inhibition studies showed that multiple adhesion receptors--including L-selectin, E-selectin, VCAM-1, and CD11/CD18--were involved in blast cell adhesion to TNF-alpha-activated endothelium. Leukemic cells were then cocultured at 37 degrees C on unactivated endothelial cell monolayers for time periods up to 24 hours. A time-dependent increase in the number of blasts attached to the endothelium and a concomitant induction of ICAM-1, VCAM-1, and E-selectin were observed. Additional experiments revealed that endothelial cell activation by leukemic myeloblasts was caused by cytokine secretion by blast cells, in particular TNF-alpha and IL-1 beta, and direct contacts between adhesion receptors expressed by blast cells and endothelial cells. Thus, leukemic cells have the ability to generate conditions that promote their own adhesion to vascular endothelium, a property that may have important implications for the pathophysiology of leukostasis and tissue infiltration by leukemic blast cells. (Blood. 2001;97:2121-2129)

Antigens, CD↗

PubMed2002

The tripeptide FEG ameliorates systemic inflammatory responses to rat intestinal anaphylaxis.

BACKGROUND: Food allergies are generally associated with gastrointestinal upset, but in many patients systemic reactions occur. However, the systemic effects of food allergies are poorly understood in experimental animals, which also offer the opportunity to explore the actions of anti-allergic drugs. The tripeptide D-phenylalanine-D-glutamate-Glycine (feG), which potentially alleviates the symptoms of systemic anaphylactic reactions, was tested to determine if it also reduced systemic inflammatory responses provoked by a gastric allergic reaction. RESULTS: Optimal inhibition of intestinal anaphylaxis was obtained when 100 microg/kg of feG was given 20 min before the rats were challenged with antigen. The increase in total circulating neutrophils and accumulation of neutrophils in the heart, developing 3 h and 24 h, respectively, after antigen challenge were reduced by both feG and dexamethasone. Both anti-inflammatory agents reduced the increase in vascular permeability induced by antigen in the intestine and the peripheral skin (pinna), albeit with different time courses. Dexamethasone prevented increases in vascular permeability when given 12 h before antigen challenge, whereas feG was effective when given 20 min before ingestion of antigen. The tripeptide prevented the anaphylaxis induced up regulation of specific antibody binding of a cell adhesion molecule related to neutrophil activation, namely CD49d (alpha4 integrin). CONCLUSIONS: Aside from showing that intestinal anaphylaxis produces significant systemic inflammatory responses in non-intestinal tissues, our results indicate that the tripeptide feG is a potent inhibitor of extra-gastrointestinal allergic reactions preventing both acute (30 min) and chronic (3 h or greater) inflammatory responses.

Anaphylaxis↗

PubMed2002

VLA-4-dependent and -independent pathways in cell contact-induced proinflammatory cytokine production by synovial nurse-like cells from rheumatoid arthritis patients.

Nurse-like stromal cell lines from the synovial tissue of patients with rheumatoid arthritis (RA-SNC) produce, on coculture with lymphocytes, large amounts of proinflammatory cytokines. In the present paper, we analyze the molecular events necessary for the induction of cytokine release from RA-SNC cells, and particularly the roles played by cell adhesion and the transmigration (also known as pseudoemperipolesis) of lymphocytes. For this purpose, the effects of various mAbs on the binding and transmigration of a human B-cell line, MC/car, were examined using a cloned RA-SNC line, RA-SNC77. To analyze the role of lymphocyte binding and transmigration on upregulated cytokine production by the RA-SNC77 cells, we used C3 exoenzyme-treated MC/car cells, which could bind to RA-SNC77 cells but could not transmigrate. Treatment with anti-CD29 or anti-CD49d mAb significantly reduced binding and transmigration of the MC/car cells. In contrast, the neutralizing anti-CD106/vascular cell adhesion molecule 1 mAb did not show any inhibitory effect. Likewise, none of the neutralizing mAbs against CD11a, CD18, CD44, CD49e, or CD54 showed significant effects. Binding of C3-treated or untreated MC/car cells to RA-SNC77 cells induced comparable levels of IL-6 and IL-8 production. In addition, the enhanced cytokine production by RA-SNC77 cells required direct lymphocyte contact via a very late antigen-4 (VLA-4)-independent adhesion pathway. These results indicate that, although both the VLA-4-dependent/vascular cell adhesion molecule 1-independent and the VLA4-independent adhesion pathways are involved in MC/car binding and subsequent transmigration, only the VLA4-independent adhesion pathway is necessary and sufficient for the enhanced proinflammatory cytokine production by RA-SNC77 cells. The transmigration process, which is dependent on Rho-GTPase, is not a prerequisite for this phenomenon.

Antibodies, Monoclonal↗

PubMed2003

Activation of A2A adenosine receptors inhibits expression of alpha 4/beta 1 integrin (very late antigen-4) on stimulated human neutrophils.

The alpha 4/beta 1 integrin very late antigen-4 (CD49d/CD29) is up-regulated on circulating neutrophils of septic patients. Although no individual agent mimics this effect of sepsis, we now report that following priming of human neutrophils with lipopolysaccharide or tumor necrosis factor alpha (TNF-alpha), addition of formyl-Met-Leu-Phe (fMLP) results in a "stimulated", sepsis-like, four- to fivefold rise in CD49d expression. TNF/fMLP stimulation also produced a similar increase in CD49d-mediated adhesion of neutrophils to a vascular cell adhesion molecule-1 (VCAM-1)-coated surface. Adenosine is a naturally occurring, anti-inflammatory mediator released from injured or inflamed tissues. We observed that stimulated neutrophil CD49d expression was decreased by activation of A(2A) adenosine receptors (A(2A)AR) with the selective agonist 4-[3-[6-amino-9-(5-ethylcarbamoyl-3,4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl]-cyclohexanecarboxylicacid methyl ester (ATL146e; EC(50)=6.4 nM). ATL146e (100 nM) also reduced the fraction of stimulated neutrophils that adhered to VCAM-1 from 38 +/- 6% to 27 +/- 5%. Inhibition of CD49d expression was equally inhibited by ATL146e, added before or after TNF priming, and was reversed by incubation with the A(2A)AR-selective antagonist 4-[2-[7-amino-2-(2-furyl) (1, 2, 4)triazolo(2,3-a) (1, 3, 5)triazin-5-yl-amino]ethyl]-phenol (ZM241385; 100 nM). A suboptimal ATL146e concentration (1 nM) combined with the type IV phosphodiesterase inhibitor rolipram (100 nM) synergistically decreased stimulated CD49d expression by >50%. The cyclic adenosine monophosphate (cAMP)-dependent kinase [protein kinase A (PKA)] inhibitor H-89 (10 microM) reversed the effect of ATL146e on stimulated CD49d expression. Other means of increasing cAMP in neutrophils also decreased stimulated CD49d expression. We conclude that adenosine binding to A(2A)AR counteracts stimulation of neutrophil CD49d integrin expression and neutrophil binding to VCAM-1 via a cAMP/PKA-mediated pathway.

Adenosine↗

PubMed2005

Relapse rates and enhancing lesions in a phase II trial of natalizumab in multiple sclerosis.

BACKGROUND: Natalizumab, a humanized monoclonal IgG4 antibody, is an alpha4-integrin antagonist, which inhibits the migration of inflammatory cells into the central nervous system, a key pathogenic mechanism in multiple sclerosis (MS). In a six month, phase II clinical trial of patients with relapsing MS, natalizumab significantly reduced the formation of new gadolinium-enhanced (Gd+) lesions and the number of clinical relapses. OBJECTIVE: To investigate the relationship of historical relapse rate and new Gd + lesion number with subsequent MS disease activity and natalizumab treatment in the phase II study. METHODS: Patients who participated in the phase II study were stratified into subgroups according to: (i) the number of relapses in the two years prior to entry into the study: 2 relapses (n = 108), 3 relapses (n =57), and >3 relapses (n =48); (ii) the number of new Gd + lesions at baseline (Month 0): 0 (n = 129), 1-2 (n =50), and >2 (n =33). Relapses and new Gd + lesions during the treatment phase of the trial were determined and compared for each subgroup. RESULTS: Both the prestudy relapse rate and number of new Gd + lesions at baseline were related to the subsequent risk of a relapse; baseline number of Gd + lesions was related to the likelihood of subsequent new Gd + lesion formation. There was a lower proportion of subjects with an on-study relapse and fewer new Gd + lesions in all natalizumab-treated subgroups when compared with their placebo counterpart; the difference was most apparent in the subgroups of patients with >3 relapses in the two years prior to study entry and >2 new Gd -- lesions at Month 0. CONCLUSIONS: There was a lower proportion of subjects with an on-study relapse in natalizumab-treated patients, particularly in those with a more active disease at study entry. Larger ongoing phase III studies will allow more definitive investigation of these preliminary subgroup findings.

Adult↗

PubMed2003

LDL activates signaling pathways leading to an increase in cytosolic free calcium and stimulation of CD11b expression in monocytes.

In the present study, we investigated the mechanisms by which plasma lipoproteins modulate the integrin-dependent adhesion properties of monocytes. LDL induced the expression of the monocyte CD11b in vitro as well as in vivo via intracellular signaling mechanisms involving calcium transients. The effect on CD11b transcription was specific for native LDL and was blocked by a neutralizing anti-LDL receptor antibody. Neither oxidized LDL nor HDL had any effect on CD11b expression. Although LDL stimulated CD11b surface expression, the integrins were not activated. To initiate the CD11b-specific adhesion to the endothelium, the engagement of chemokine receptor CCR2 and intact chemokine-to-integrin signaling was necessary. However, the activation of CCR2 with monocyte chemoattractant protein-1 not only stimulated the integrins preexisting on the cell surface, but also increased the number of CD11b molecules on the cell surface. This was particularly pronounced in THP-1 cells after treatment with LDL. In a previous study, we showed that LDL induces the expression of CCR2 in monocytes. We conclude that this may be the underlying cause of the enhanced chemokine effect on CD11b expression and activation observed with these cells.

Animals↗

PubMed2005

Menstrual cycle hormones induce changes in functional interactions between lymphocytes and decidual vascular endothelial cells.

During the secretory phase of the menstrual cycle, a natural killer (NK) cell subset expressing cluster of differentiation (CD)56bright appears in the decidualizing uterus and remains until onset of menses. If pregnancy occurs, decidual NK cells increase to become the predominant uterine lymphocytes of early pregnancy. To elucidate mechanisms of CD56bright cell recruitment to the uterus, an in vitro adhesion assay was used to assess the effect of the menstrual cycle, as well as cycle-associated hormones on adhesive properties of human lymphocytes. Adhesion of human peripheral blood lymphocytes to pregnant mouse lymph nodes and Peyer's Patches tissue sections was constant throughout the cycle. When uterine tissue was used as the substrate, adhesive CD56+ cells were found only in decidua basalis. Adhesion increased at the LH surge. Adhesion was mediated through both L-selectin and alpha4-integrin-dependent mechanisms. Furthermore, we observed increased adhesive function in CD56+ cells from male donors which had been cultured with estradiol or LH compared with cell aliquots cultured without additives. Lymphocytes adherent to mouse uterine tissue were predominantly CD56bright, suggesting that peripheral NK cells may be actively recruited to the uterus in an important, brief endocrine-regulated fashion at the time of ovulation to establish the decidual NK population of early pregnancy.

Adult↗

PubMed1997

Embryonic regulation of integrins beta 3, alpha 4, and alpha 1 in human endometrial epithelial cells in vitro.

In the present study, we examined the embryonic regulation of beta 3 integrin in human endometrial epithelial cells (EEC) at the protein level and analyzed putative embryonic factors responsible for this regulation. The model employed is based on a clinical in vitro fertilization program in which single human embryos were cocultured with EEC until blastocyst stage and then transferred back to the uterus. After embryo transfer, EEC wells were divided according to the embryonic status reached: EEC with embryos that achieved the blastocyst stage, EEC with arrested embryos, and EEC without embryos. Immunostaining for beta 3 was positive in plasma membrane of EEC. Flow cytometry showed a mean percentage of beta 3-stained cells of 24.1 +/- 5.7 in EEC cocultured with embryos that achieved the blastocyst stage (n = 13) vs. 9.5 +/- 1.6 (P < 0.05) in those EEC cultured with arrested embryos (n = 12). Immunostaining for alpha 1 and alpha 4 integrins was negative in EEC monolayers studied, regardless of the presence or absence of embryos, and these findings were confirmed by flow cytometry. The possibility that the embryonic IL-1 system and leukemia inhibitory factor were involved in the endometrial beta 3 up-regulation was investigated by neutralizing experiments demonstrating a significant inhibition of beta 3-stained cells when EEC monolayers were cultured in the presence of EEC/blastocyst-conditioned media with (n = 4) vs. without (n = 8) antihuman interleukin (IL)-1 alpha + IL-1 beta (1.65% vs. 14.6%; P < 0.05). Dose-response experiments further demonstrated an up-regulation of beta 3 positive cells when IL-1 alpha + IL-1 beta were added to the medium at a concentration of 10 pg/mL compared with control medium without added cytokines (40% vs. 20%, n = 4). The functional relevance of the EEC beta 3 up-regulation was tested using a mouse blastocyst adhesion assay. More mouse blastocysts attached to EEC previously in contact with human blastocyst (72.7%) compared with those EEC previously in contact with arrested embryos (40%). Our results demonstrate the selective effect of a developing human embryo on EEC expression of beta 3, which is maximal when a human blastocyst instead of an arrested embryo is considered. Furthermore, the embryonic IL-1 system seems to be involved in the EEC beta 3 up-regulation, reinforcing the concept of precise paracrine cross-talk between blastocyst and endometrial epithelium during embryonic implantation.

Adult↗

PubMed2006

EMILIN1 represents a major stromal element determining human trophoblast invasion of the uterine wall.

The detection of EMILIN1, a connective tissue glycoprotein associated with elastic fibers, at the level of the ectoplacental cone and trophoblast giant cells of developing mouse embryos (Braghetta et al., 2002) favored the idea of a structural as well as a functional role for this protein in the process of placentation. During the establishment of human placenta, a highly migratory subpopulation of extravillous trophoblasts (EVT), originating from anchoring chorionic villi, penetrate and invade the uterine wall. In this study we show that EMILIN1, produced by decidual stromal and smooth muscle uterine cells, is expressed in the stroma and in some instances as a gradient of increasing concentration in the perivascular region of modified vessels. This distribution pattern is consistent with the haptotactic directional migration observed in in vitro functional studies of freshly isolated EVT and of the immortalized HTR-8/SVneo cell line of trophoblasts. Function-blocking monoclonal antibodies against alpha4-integrin chain and against EMILIN1 as well as the use of EMILIN1-specific short interfering RNA confirmed that trophoblasts interact with EMILIN1 and/or its functional gC1q1 domain via alpha4beta1 integrin. Finally, membrane type I-matrix metalloproteinase (MT1-MMP) and MMP-2 were upregulated in co-cultures of trophoblast cells and stromal cells, suggesting a contributing role in the haptotactic process towards EMILIN1.

Cell Adhesion↗

PubMed2006

Altered membrane dynamics of quantum dot-conjugated integrins during osteogenic differentiation of human bone marrow derived progenitor cells.

Functionalized quantum dots offer several advantages for tracking the motion of individual molecules on the cell surface, including selective binding, precise optical identification of cell surface molecules, and detailed examination of the molecular motion without photobleaching. We have used quantum dots conjugated with integrin antibodies and performed studies to quantitatively demonstrate changes in the integrin dynamics during osteogenic differentiation of human bone marrow derived progenitor cells (BMPCs). Consistent with the unusually strong BMPC adhesion previously observed, integrins on the surface of undifferentiated BMPC were found in clusters and the lateral diffusion was slow (e.g., approximately 10(-11) cm2/s). At times as early as those after a 3-day incubation in the osteogenic differentiation media, the integrin diffusion coefficients increased by an order of magnitude, and the integrin dynamics became indistinguishable from that measured on the surface of terminally differentiated human osteoblasts. Furthermore, microfilaments in BMPCs consisted of atypically thick bundles of stress fibers that were responsible for restricting the integrin lateral mobility. Studies using laser optical tweezers showed that, unlike fully differentiated osteoblasts, the BMPC cytoskeleton is weakly associated with its cell membrane. Based on these findings, it appears likely that the altered integrin dynamics is correlated with BMPC differentiation and that the integrin lateral mobility is restricted by direct links to microfilaments.

Actin Cytoskeleton↗

PubMed2005

Developing therapeutics for the treatment of multiple sclerosis.

Multiple sclerosis (MS) is both a complex and chronic neurological disease of the CNS. This poses unique challenges for drug discovery in terms of delineating specific targets related to disease mechanisms and developing safe and effective molecules for clinical application. Preclinical animal models of MS provide the necessary test bed for evaluating the effects of novel therapeutic strategies. Because the clinical manifestations and pathological consequences of disease vary dramatically from individual to individual, as well as treatment response to existing therapies, this creates a significant research endeavor in terms of translating preclinical methodologies to the clinical domain. Potentially exciting treatments have emerged in the form of natalizumab (Tysabri), an alpha4 integrin antagonist, and more recently FTY720, a sphinogosine-1 phosphate receptor modulator, providing a compelling proof-of-principle from bench to bedside. However, further research is required to discharge safety concerns associated with these therapeutic avenues. Future prospects in the guise of disease-modifying therapies that target the inflammatory and neurodegenerative components of disease have come to the forefront of preclinical research with the sole aim of reducing the underlying irreversible progressive disability of MS. Significant progress with novel therapies will be made by implementing biomarker strategies that extrapolate robustly from animal models to the divergent patient populations of MS. The future therapeutic options for MS will depend on improvements in understanding the precise factors involved in disease onset and progression and subsequently the development of oral therapeutics that translate sustained benefit from the preclinical context into clinical reality.

Animals↗

PubMed2005

The role of alpha-4 integrin in the aetiology of multiple sclerosis: current knowledge and therapeutic implications.

Multiple sclerosis (MS) has been recognised as a disease since the mid-19th century. The delineation of its CNS pathology, revealing the presence of inflammatory demyelination and relative sparing of axons, was originally interpreted as evidence of infection. Despite many studies, a primary infectious aetiology of MS has not been found. However, the occurrence of acute demyelinating disease following a variety of infections and vaccinations, leading to MS in about a third of cases, provides evidence for the existence of an auto-allergic pathogenesis for the disease. Improved understanding of the role of the blood-brain barrier in protecting the CNS, and the mechanisms by which cells gain entry into the brain and spinal cord has advanced the understanding of MS. Evidence of the central role of the adhesion molecule alpha4beta1-integrin (very late activation antigen-4 [VLA-4]) for lymphocytes in endothelial transmigration into the CNS specifically, has provided a major insight into the pathogenesis of human demyelinating disease and its experimental model, experimental autoimmune encephalomyelitis (EAE). This finding has led to a new window of therapeutic opportunity in MS. Monoclonal antibodies to VLA-4 abrogate the development of EAE in sensitised animals and may actually reverse its clinical and pathological findings in manifest disease. Natalizumab, one such monoclonal antibody, which is administered intravenously, has been found to be a promising agent in the treatment of MS. Although single doses produced no improvement in the speed or quality of recovery from acute exacerbations of MS in a phase II trial, long-term administration (in phase II and phase III trials) have produced significant benefits with results showing both a marked reduction in the risk of new magnetic resonance imaging lesions and a significant reduction in the risk of exacerbations within 2 months of the initiation of therapy. Phase III double-blinded controlled trials have provided additional evidence of safety and a favourable impact on exacerbation rates over the 1 year of administration. Unfortunately, the success of natalizumab has been curtailed by three cases of progressive multifocal leukoencephalopathy, which have prompted the manufacturer to voluntary withdraw the drug from the market. An independent review board is currently investigating the safety of the drug to determine whether it should return to the market. The demonstration that selective modulation (blocking) of the adhesion molecule VLA-4 by natalizumab in MS, resembling that observed in experimental disease, represents a major advance in rational therapy.

Animals↗

PubMed2003

The effects of allergen and anti-allergic drugs on murine hemopoietic cells: moving targets, unusual mechanisms, and changing paradigms.

We used a variety of techniques to evaluate the effects of airway allergen exposure in mice on the responses of hemopoietic cells to cytokines and drugs in vitro and in vivo. Initial studies have shown that allergen exposure of sensitized mice leads to release of circulating mediators, that induce rapid upregulation of bone-marrow responses to IL-5 and GM-CSF. This may be related to glucocorticoids, because exogenous dexamethasone has similar effects on cultured murine bone-marrow, and because stress-induced glucocorticoids, in naïve or sensitized mice, have effects indistinguishable from those of allergen challenge in sensitized animals. Upregulation of eosinophil production is associated with an increased expression of alpha4 integrins, which may contribute to retention of these cells in the bone-marrow. Glucocorticoids regulate the adhesiveness, maturation and survival of eosinophils in murine bone-marrow culture, partly by counteracting the actions of Prostaglandin E2 and possibly other prostanoids. Allergen exposure of sensitized mice leads to accumulation of hemopoietic progenitors in the lungs, which differ from those in bone-marrow in growth properties and sensitivity to glucocorticoids. Lung transplantation has been used to demonstrate that the lung acts as a source of endocrine factors that promote hemopoietic cell accumulation, independently of damage caused by local allergic inflammation.

Allergens↗

PubMed2005

Biological therapies of autoimmune diseases.

The advances in the understanding of the pathogenesis of the autoimmune diseases have led to new treatment targets. Biological agents enhance or replace conventional immunosuppressive therapies in the treatment of autoimmune diseases. TNF-alpha has been validated as a good treatment target but the potential modalities also include the inhibition of the interaction between LFA-3 (lymphocyte function-associated antigen 3) and CD2, the blockade of the IL-1 receptors, the antibodies against the alpha4 integrins, the antibodies against B-cell CD20 and the inhibition of the activation of T-cells. The new treatments have had a major impact on inflammatory symptoms, the radiological damage and the anemia of chronic disease in rheumatoid arthritis and have substantially controlled the signs and symptoms in the spondylarthropathies group and plaque-type psoriasis. The efficacy of the biologic agents in systemic lupus erythematosus warrants further investigation but there have been some promising results in proliferative lupus nephritis. Several small studies have explored their use in the treatment of Sjogren's syndrome, adult onset Still's disease and several vasculitides but the results are still preliminary and warrant confirmation. The efficacy of the biological agents has been impressive. Susceptibility to infections has always been a major concern; a high level of suspicion is necessary and strategies should be implemented for the prevention, the rapid identification and pre-emptive therapy of such infections.

Animals↗

PubMed1994

Differential expression of integrins alpha 6 and alpha 4 determines pathways in human peripheral CD4+ T cell differentiation.

Integrins mediate leukocyte adhesion to vascular endothelium and thereby influence leukocyte recirculation. We have explored expression by peripheral blood T cells of beta 1 and beta 7 integrins, particularly alpha 4 beta 1 (VLA-4, CD49d), alpha 4 beta 7 (LPAM-1) and alpha 6 beta 1 (VLA-6, CD49f). Integrin expression differs between CD4+ cells and CD8+ cells in that CD4+ cells 1) are more heterogeneous, particularly for alpha 4; 2) express on the average less alpha 4 and beta 7; and 3) express on the average more alpha 6 and beta 1. 2D gel electrophoretic analysis was combined with flow cytometric analysis to determine which integrin chain pairs are expressed by the CD45RO- (naive) and CD45RO+ (memory) subsets of CD4+ cells. CD45RO- (naive) cells express homogeneously at intermediate levels the three integrin pairs alpha 6 beta 1, alpha 4 beta 1 and alpha 4 beta 7. Although 2D gel analysis suggests similar average integrin chain composition for CD45RO+CD4+ (memory) cells, flow cytometric analysis demonstrates multiple subsets of CD45RO+ cells differing markedly from each other and from naive cells in levels of expression of alpha 6 and alpha 4 integrins. There are a minimum of three CD45RO+ subsets: 1) alpha 4 beta 1hi alpha 6 beta 1hi alpha 4 beta 7neg, which comprises the majority of memory cells; 2) alpha 4 beta 7hi alpha 6 beta 1low presumptive gut-homing memory cells; and 3) alpha 6 beta 1hi alpha 4 beta 7neg alpha 4 beta 1neg, a previously unidentified subset expected to have unique migrational-functional properties. Of particular importance in these results are: the expression by CD4+ naive cells of alpha 6 beta 1, alpha 4 beta 1 and alpha 4 beta 7, the overall prominence and regulation of alpha 6 beta 1 on CD4+ cells, and the selective decreases as well as increases in alpha 4 beta 7 and alpha 4 beta 1 during CD4+ memory specialization. Taken together, these results suggest that differential regulation of expression of alpha 4 and alpha 6 integrin chains that accompany naive-to-memory transition in CD4+ cells are instrumental in generating functional subsets of CD4+ memory cells with specialized recirculation abilities.

CD4-Positive T-Lymphocytes↗

PubMed1994

Expanding roles for alpha 4 integrin and its ligands in development.

Interaction of alpha 4 integrins with vascular cell adhesion molecule-1 (VCAM-1) is classically important for immune function. However, we found recently that these receptors have a second role, in embryogenesis, where they mediate cell-cell interactions that are important for skeletal muscle differentiation. Here, we present evidence of an expanding role for these receptors in murine development. alpha 4 and VCAM-1 were found at embryonic sites of hematopoiesis, suggesting a role for these receptors during embryogenesis that parallels their hematopoietic function in adult bone marrow. During angiogenesis in the lung, alpha 4 and VCAM-1 were found on mesenchyme that gives rise to vascular endothelium and smooth muscle. alpha 4 persisted on the smooth muscle and the endothelium of newly forming vessels where it colocalized with its extracellular matrix ligand, fibronectin (FN). These patterns suggest several roles for alpha 4 integrins and their ligands in angiogenesis. alpha 4 was also found on neural crest derivatives where it colocalized with FN. alpha 4 was expressed selectively on cells in the dorsal root ganglia: it was apparent along ventral projections, but absent from dorsal projections, suggesting that alpha 4 integrins could be involved in defining neuronal fates. Although VCAM-1 was not expressed on most neural crest derivatives, it was found in the neural crest-derived outflow tract of the embryonic heart, where it colocalized with alpha 4. These results imply that alpha 4 integrins and their ligands could be important for migration or differentiation of neural crest. alpha 4 was also expressed on embryonic retina and FN was found on inductive mesenchyme surrounding the eye, suggesting a role for these proteins in eye development. Finally, based on their patterns of expression, we conclude that VCAM-1 only participates in a subset of interactions involving alpha 4 integrins, whereas FN appears to be the more general ligand.

Aging↗

PubMed1994

Expression of alpha 4 integrin mRNA and protein and fibronectin in the early chicken embryo.

alpha 4 integrins (alpha 4 beta 1 and alpha 4 beta 7) have been shown to mediate both cell-matrix adhesion to fibronectin and cell-cell adhesion to VCAM-1. These interactions have been suggested to contribute to hematopoiesis, lymphocyte homing, recruitment of inflammatory cells, neural crest cell migration and myogenesis. We report here the cloning of chicken alpha 4 cDNA and its use to define the patterns of expression of alpha 4 mRNA and protein in early chicken embryos (19-22 somite pairs), a stage at which neural crest cells can be examined at various points in their migration and somitic development and differentiation can also be observed at various stages. We observe widespread expression of both alpha 4 mRNA and protein, although the patterns of steady state expression do not conform precisely. Many neural crest cells contain significant levels of alpha 4 mRNA. Some neural crest cells express alpha 4 protein but its expression is transient and/or limited to a subset of these cells. alpha 4 is strongly expressed at both mRNA and protein levels by somitic cells and their derivatives in the sclerotome, dermatome and myotome and is also expressed in neural tube, otic placode, heart, gut endoderm and some other tissues. Comparison with the distributions of fibronectin shows that, although some alpha 4 expression occurs in locations consistent with a role in cell-matrix adhesion to fibronectin, alpha 4 is also expressed in other places where fibronectin is low or absent and a role for alpha 4 in cell-cell interactions appears more likely.

Amino Acid Sequence↗

PubMed1995

A direct binding assay for the vascular cell adhesion molecule-1 (VCAM1) interaction with alpha 4 integrins.

Vascular cell adhesion molecule-1 (VCAM1) is a member of the immunoglobulin (Ig) superfamily which interacts with the alpha 4 integrins alpha 4 beta 1 (very late antigen 4: VLA4) and alpha 4 beta 7, which are constitutively expressed on many leukocyte subsets and play a key role in cell trafficking and activation. Using a recombinant VCAM-IgG fusion protein (VCAM-Ig) as a soluble ligand for alpha 4 beta 1 we directly demonstrated by fluorescence analysis that the alpha 4 beta 1 receptor can exist in different affinity states on the cell surface, and that a high affinity state is induced by manganese ions or certain activating anti-beta 1 monoclonal antibodies (Jakubowski et al., 1995b). Here we have extended these observations by developing a rapid and reproducible assay using alkaline phosphatase (AP)-coupled VCAM-Ig (VCAM-Ig-AP) which measures the interaction between VCAM1 and alpha 4 integrins in a microtiter plate format. This assay has allowed us to evaluate directly the effects of metal ions, anti-beta 1 mAbs, and different cell types and species on the VCAM1/alpha 4 integrin interaction. Most importantly, the assay system provides a means to rapidly evaluate alpha 4 integrin-directed inhibitors without the complication of post-ligand binding events inherent in adhesion assays.

Animals↗