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PubMed2005

Physiological basis for novel drug therapies used to treat the inflammatory bowel diseases. I. Immunology and therapeutic potential of antiadhesion molecule therapy in inflammatory bowel disease.

Adhesion molecules regulate the influx of leukocytes in normal and inflamed gut. They are also involved in local lymphocyte stimulation and antigen presentation within the intestinal mucosa. In intestinal inflammation, many adhesion molecules are upregulated, but alpha4-integrins most likely hold a key position in directing leukocytes into the inflamed bowel wall. Therapeutic compounds directed against trafficking of leukocytes have been designed and are being developed as a novel class of drugs in the treatment of Crohn's disease and ulcerative colitis. This review deals with the immunological aspects of leukocyte trafficking focused on gut homing of T cells. Second, the changes in adhesion molecules and T cell trafficking during intestinal inflammation are discussed. Finally, we review the clinical data that have been gathered with respect to the therapeutic potential and the safety of antiadhesion molecule treatment. Antegren, or natalizumab, a humanized anti-alpha4 integrin IgG4 antibody, has been most extensively evaluated and may be close to registration. A more specific humanized alpha4beta7-integrin MLN-02 has shown preliminary clinical efficacy in ulcerative colitis, and both antergren and MLN-02 appear to be very safe. Trials with the anti-ICAM-1 antisense oligonucleotide ISIS-2302 in steroid refractory Crohn's disease have provided conflicting efficacy data. In the near future, some of these novel biological agents may prove valuable therapeutic tools in the management of refractory inflammatory bowel disease, although it is too early to define the patient population that will benefit most from these agents.

Antibodies, Monoclonal↗

PubMed2006

Natalizumab.

Medical therapeutics has entered a new and exciting era with the development of biological agents that target specific sites. In the studies performed to date, natalizumab, a humanized monoclonal antibody to alpha4 integrin, when administered intravenously at monthly intervals, appeared to be highly beneficial with respect to decreasing both relapse rate and contrast-enhancing lesions in multiple sclerosis. Salutary actions on other disease parameters were also observed. However, treatment with natalizumab was complicated by the development of a rare demyelinating disease of the brain--progressive multifocal leukoencephalopathy (PML)--that developed in three patients in clinical trials of the drug. Currently, the estimated incidence for the development of PML with natalizumab treatment is 1:1,000 after approximately 18 months of therapy; however, as the development of PML is a stochastic event, it is quite possible that higher rates will be observed after longer treatment courses. Curiously, other opportunistic infections were not observed at higher rates in the treated groups when compared to controls, suggesting a specific association between natalizumab, and perhaps other alpha4 integrin blockers, and PML. Postmarketing surveillance studies should answer this and other questions related to the use of natalizumab.

Antibodies, Monoclonal↗

PubMed2006

Fatalities in natalizumab treatment--a 'no go' for leukocyte recirculation approaches?

Natalizumab (Tysabri), Biogen Idec/Elan) is a humanised neutralising antibody directed against alpha4 integrin expressed by leukocytes. Although it is an effective therapy for multiple sclerosis (MS), the serious adverse effect of progressive multifocal leukoencephalopathy (PML) resulted in its voluntary withdrawal from the market by Biogen Idec/Elan in February 2005. This has raised debates on whether PML was caused by blocking leukocyte trafficking-mediated immune suppression or by other effects through targeting alpha4 integrin per se. The authors propose that natalizumab-associated PML is a target-specific side effect predominantly due to the combination of: i) blocking leukocyte trafficking to peripheral organs resulting in reduced immune surveillance; ii) mobilisation of PML-causative JC virus-carrying bone marrow precursor cells and splenic marginal zone B cells; and iii) migration of these cells to sites of inflammation such as the brain. Therefore, combination of these effects is, so far, specific for the target alpha4 integrin and should not occur in general when interfering with other targets involved in leukocyte trafficking.

Antibodies, Monoclonal↗

PubMed1997

Defining extracellular integrin alpha-chain sites that affect cell adhesion and adhesion strengthening without altering soluble ligand binding.

It was previously shown that mutations of integrin alpha4 chain sites, within putative EF-hand-type divalent cation-binding domains, each caused a marked reduction in alpha4beta1-dependent cell adhesion. Some reports have suggested that alpha-chain "EF-hand" sites may interact directly with ligands. However, we show here that mutations of three different alpha4 "EF-hand" sites each had no effect on binding of soluble monovalent or bivalent vascular cell adhesion molecule 1 whether measured indirectly or directly. Furthermore, these mutations had minimal effect on alpha4beta1-dependent cell tethering to vascular cell adhesion molecule 1 under shear. However, EF-hand mutants did show severe impairments in cellular resistance to detachment under shear flow. Thus, mutation of integrin alpha4 "EF-hand-like" sites may impair 1) static cell adhesion and 2) adhesion strengthening under shear flow by a mechanism that does not involve alterations of initial ligand binding.

Amino Acid Sequence↗

PubMed2005

Neutrophils, nitric oxide, and microvascular permeability in severe sepsis.

STUDY OBJECTIVES: Alterations in microvascular permeability are prevalent in patients with sepsis; a recent study reported that patients with septic shock had increased capillary filtration coefficient (Kf), a noninvasive index of microvascular permeability. We aimed to determine whether patients with severe sepsis had increased Kf, and whether the magnitude of Kf correlated with indexes of nitric oxide activity and neutrophil activation. DESIGN: Single-center, prospective study. SETTING: Twenty-five-bed ICU of a medical college-affiliated teaching hospital. PATIENTS: Fifteen ICU patients with severe sepsis based on the American College of Chest Physicians/Society of Critical Care Medicine consensus criteria of 1992, and 10 nonseptic ICU patients as control subjects. INTERVENTIONS: Kf was measured by venous congestion plethysmography, plasma nitrate/nitrite (NOx) by chemiluminescence, and neutrophil expression of alpha4-integrin (an index of neutrophil activation) by flow cytometry. MEASUREMENTS AND RESULTS: Septic patients had higher Kf than nonseptic control subjects. Kf of septic patients was 5.6 +/- 0.6 x 10(-3) mL.min(-1).100 mL tissue(-1).mm Hg(-1) (mean +/- SEM, mL.min(-1).100 mL tissue(-1).mm Hg(-1) = Kf units [KfU]) as compared to 3.9 +/- 0.5 x 10(-3) KfU in nonseptic ICU patients (p < 0.05). There was no correlation between plasma NOx and Kf, or between neutrophil alpha4-integrin expression and Kf in patients with sepsis. Septic patients with clinical evidence of edema had significantly higher Kf (p < 0.05) than nonedematous septic patients. CONCLUSIONS: ICU patients with severe sepsis have increased Kf, a noninvasive index of microvascular water permeability. The magnitude of hyperpermeability did not correlate with NOx levels or one index of neutrophil activation (alpha4-integrin expression). Presence of peripheral edema in these patients was associated with increased Kf, and may represent a simple, clinical indicator of altered microvascular permeability in sepsis.

Adult↗

PubMed2006

Altered CD4+/CD8+ T-cell ratios in cerebrospinal fluid of natalizumab-treated patients with multiple sclerosis.

BACKGROUND: Treatment with natalizumab, a monoclonal antibody against the adhesion molecule very late activation antigen 4, an alpha4beta(1) integrin, was recently associated with the development of progressive multifocal leukoencephalopathy, a demyelinating disorder of the central nervous system caused by JC virus infection. OBJECTIVE: To test the effect of natalizumab treatment on the CD4(+)/CD8(+) T-cell ratios in cerebrospinal fluid (CSF) and peripheral blood. DESIGN: Prospective longitudinal study. SETTING: Academic and private multiple sclerosis centers. PATIENTS: Patients with multiple sclerosis (MS) treated with natalizumab, untreated patients with MS, patients with other neurologic diseases, and human immunodeficiency virus-infected patients. MAIN OUTCOME MEASURES: CD4(+) and CD8(+) T cells were enumerated in CSF and peripheral blood. The mean fluorescence intensity of unbound alpha4 integrin on peripheral blood CD4(+) and CD8(+) T cells was analyzed before and after natalizumab therapy. RESULTS: Natalizumab therapy decreased the CSF CD4(+)/CD8(+) ratio of patients with MS to levels similar to those of human immunodeficiency virus-infected patients. CD4(+)/CD8(+) ratios in peripheral blood in patients with MS progressively decreased with the number of natalizumab doses, but they remained within normal limits. Six months after the cessation of natalizumab therapy, CSF CD4(+)/CD8(+) ratios normalized. The expression of unbound alpha4 integrin on peripheral blood T cells decreases with natalizumab therapy and was significantly lower on CD4(+) vs CD8(+) T cells. CONCLUSIONS: Natalizumab treatment alters the CSF CD4(+)/CD8(+) ratio. Lower expression of unbound alpha4 integrin on CD4(+) T cells is one possible mechanism. These results may have implications for the observation that some natalizumab-treated patients with MS developed progressive multifocal leukoencephalopathy.

Adolescent↗

PubMed1999

Direct demonstration of P-selectin- and VCAM-1-dependent mononuclear cell rolling in early atherosclerotic lesions of apolipoprotein E-deficient mice.

Apolipoprotein E-deficient (ApoE-/-) mice develop atherosclerotic lesions throughout the arterial tree, including the carotid bifurcation. Although the expression of adhesion molecules such as ICAM-1, vascular cell adhesion molecule-1 (VCAM-1), and P-selectin on endothelium that overlie atherosclerotic plaques has been implicated in monocyte recruitment to developing lesions, monocyte adhesion in atherosclerotic vessels has not been observed directly. To investigate which adhesion molecules may be important in monocyte adhesion to atherosclerotic lesions, an isolated mouse carotid artery preparation was developed and perfused with mononuclear cells. We show rolling and attachment of the human monocytic cell line U937 and the mouse monocyte-macrophage cell line P388D1 in carotid arteries from 10- to 12-week-old ApoE-/- and C57BL/6 wild-type mice fed a Western-type diet (21% fat wt/wt) for 4 to 5 weeks. No rolling was observed in carotid arteries from C57BL/6 or BALB/c wild-type mice fed a chow diet and little was observed in BALB/c mice fed a Western-type diet. This model represents early lesion development as shown by minimal macrophage infiltration in the intima of carotid arteries from ApoE-/- mice fed a Western-type diet. Rolling was observed at shear stresses that were characteristic of the low-shear recirculation zone near the carotid bifurcation. Mononuclear cell attachment and rolling were significantly inhibited by monoclonal antibody blockade of P-selectin or its leukocyte ligand P-selectin glycoprotein ligand-1. Rolling velocities increased after monoclonal antibody blockade of mononuclear cell alpha4-integrin or VCAM-1, which indicates that alpha4-integrin interacting with VCAM-1 stabilizes rolling interactions and prolongs monocyte transit times.

Animals↗

PubMed2000

Laminin alpha4 and integrin alpha6 are upregulated in regenerating dy/dy skeletal muscle: comparative expression of laminin and integrin isoforms in muscles regenerating after crush injury.

The expression of laminin isoforms and laminin-binding integrin receptors known to occur in muscle was investigated during myogenic regeneration after crush injury. Comparisons were made between dystrophic 129ReJ dy/dy mice, which have reduced laminin alpha2 expression, and their normal littermates. The overall histological pattern of regeneration after crush injury was similar in dy/dy and control muscle, but proceeded faster in dy/dy mice. In vitro studies revealed a greater yield of mononuclear cells extracted from dy/dy muscle and a reduced proportion of desmin-positive cells upon in vitro cultivation, reflecting the presence of inflammatory cells and "preactivated" myoblasts due to ongoing regenerative processes within the endogenous dystrophic lesions. Laminin alpha1 was not detectable in skeletal muscle. Laminin alpha2 was present in basement membranes of mature myofibers and newly formed myotubes in control and dy/dy muscles, albeit weaker in dy/dy. Laminin alpha2-negative myogenic cells were detected in dy/dy and control muscle, suggesting the involvement of other laminin alpha chains in early myogenic differentiation, such as laminin alpha4 and alpha5 which were both transiently expressed in basement membranes of newly formed myotubes of dy/dy and control mice. Integrin beta1 was expressed on endothelial cells, muscle fibers, and peripheral nerves in uninjured muscle and broadened after crush injury to the interstitium where it occurred on myogenic and nonmyogenic cells. Integrin alpha3 was not expressed in uninjured or regenerating muscle, while integrin alpha6 was expressed mainly on endothelial cells and peripheral nerves in uninjured muscle. Upon crush injury integrin alpha6 increased in the interstitium mainly on nonmyogenic cells, including infiltrating leukocytes, endothelial cells, and fibroblasts. In dy/dy muscle, integrin alpha6 occurred on some newly formed myotubes. Integrin alpha7 was expressed on muscle fibers at the myotendinous junction and showed weak and irregular expression on muscle fibers. After crush injury, integrin alpha7 expression extended to the newly formed myotubes and some myoblasts. However, many myoblasts and newly formed myotubes were integrin alpha7 negative. No marked difference was observed in integrin alpha7 expression between dy/dy and control muscle, either uninjured or after crush injury. Only laminin alpha4 and integrin alpha6 expression patterns were notably different between dy/dy and control muscle. Expression of both molecules was more extensive in dy/dy muscle, especially in the interstitium of regenerating areas and on newly formed myotubes. In view of the faster myogenic regeneration observed in dy/dy mice, the data suggest that laminin alpha4 and integrin alpha6 support myogenic regeneration. However, whether these accelerated myogenic effects are a direct consequence of the reduced laminin alpha2 expression in dy/dy mice, or an accentuation of the ongoing regenerative events in focal lesions in the muscle, requires further investigation.

Animals↗

PubMed1998

A role of chondroitin sulfate glycosaminoglycan binding site in alpha4beta1 integrin-mediated melanoma cell adhesion.

We have previously reported that alpha4beta1 (but not alpha5beta1) integrin-mediated melanoma cell adhesion is inhibited by removal of cell surface chondroitin sulfate glycosaminoglycan (CSGAG), suggesting that melanoma chondroitin sulfate proteoglycan plays a role in modulating the adhesive function of alpha4beta1 integrin. In the current study, we demonstrated that alpha4beta1 integrin binds to CSGAG. We have identified a peptide from within alpha4 integrin termed SG1 (KKEKDIMKKTI) that binds to cell surface melanoma chondroitin sulfate proteoglycan, indicating that SG1 represents a CSGAG binding site within the alpha4 integrin subunit. Soluble SG1 inhibits alpha4beta1 integrin-mediated human melanoma cell adhesion to CS1. Polyclonal antibody generated against the peptide inhibits melanoma cell adhesion to CS1, and the inhibition is reversed by Mn2+ and an activating monoclonal antibody anti-beta1 (8A2). Additionally, pretreatment of cells with anti-SG1 IgG inhibits the expression of the monoclonal antibody 15/7 epitope in the presence of soluble CS1 peptide, suggesting that anti-SG1 IgG prevents ligand binding by alpha4beta1 integrin. These results demonstrate that alpha4beta1 integrin interacts directly with CSGAG through SG1 site, and that this site can affect the ligand binding properties of the integrin.

Antibodies↗

PubMed1999

Lymphocyte migration in lymphocyte function-associated antigen (LFA)-1-deficient mice.

Using lymphocyte function-associated antigen (LFA)-1(-/-) mice, we have examined the role of LFA-1 and other integrins in the recirculation of lymphocytes. LFA-1 has a key role in migration to peripheral lymph nodes (pLNs), and influences migration into other LNs. Second, the alpha4 integrins, alpha4beta7 and alpha4beta1, have a hitherto unrecognized ability to compensate for the lack of LFA-1 in migration to pLNs. These findings are confirmed using normal mice and blocking LFA-1 and alpha4 monoclonal antibodies. Unexpectedly, vascular cell adhesion molecule (VCAM)-1, which is essential in inflammatory responses, serves as the ligand for the alpha4 integrins on pLN high endothelial venules. VCAM-1 also participates in trafficking into mesenteric LNs and Peyer's patch nodes where mucosal addressin cell adhesion molecule 1 (MAdCAM-1), the alpha4beta7-specific ligand, dominates. Both alpha4beta1, interacting with ligand VCAM-1, and also LFA-1 participate in substantial lymphocyte recirculation through bone marrow. These observations suggest that organ-specific adhesion receptor usage in mature lymphocyte recirculation is not as rigidly adhered to as previously considered, and that the same basic sets of adhesion receptors are used in both lymphocyte homing and inflammatory responses.

Animals↗

PubMed2001

A CD2-green fluorescence protein-transgenic mouse reveals very late antigen-4-dependent CD8+ lymphocyte rolling in inflamed venules.

Intravital microscopy allows detailed analysis of leukocyte trafficking in vivo, but fails to identify the nature of leukocytes investigated. Here, we describe the development of a CD2-enhanced green fluorescence protein (EGFP)-transgenic mouse to characterize lymphocyte trafficking during inflammation in vivo. A CD2-EGFP plasmid construct including the CD2 promoter, the EGFP transgene, and the CD2 locus control region was injected into B6CBA/F1 pronuclei. EGFP+ offspring were backcrossed into C57BL/6 mice for six generations. Flow cytometry demonstrated that all peripheral blood EGFP+ cells were positive for CD2 and negative for the granulocyte Ag Ly 6-G (GR-1). EGFP(high) cells stained positive for CD2, CD3, CD8, TCR beta-chain, and NK1.1 but did not express the B cell and monocyte markers CD45RA, CD19, and CD11b. In vitro stimulation assays revealed no difference in lymphocyte proliferation and IL-2 secretion between EGFP+ and EGFP- mice. Intravital microscopy of untreated or TNF-alpha-treated cremaster muscle venules showed EGFP+ cells in vivo, but these cells did not roll or adhere to the vessel wall. In cremaster muscle venules treated with both TNF-alpha and IFN-gamma, EGFP(high) cells rolled, adhered, and transmigrated at a rolling velocity slightly higher (11 microm/s) than that of neutrophils (10 microm/s). Blocking alpha4 integrin with a mAb increased rolling velocity to 24 microm/s. These findings show that CD8+ T cells roll in TNF-alpha/IFN-gamma-pretreated vessels in vivo via an alpha4 integrin-dependent pathway.

Animals↗

PubMed2005

The extracellular matrix protein mindin serves as an integrin ligand and is critical for inflammatory cell recruitment.

Leukocyte recruitment to inflammation sites depends on interactions between integrins and extracellular matrix (ECM). In this report we show that mice lacking the ECM protein mindin exhibit severely impaired recruitment of neutrophils and macrophages in 4 different inflammation models. Furthermore, neutrophils directly bind to immobilized mindin, and mindin matrix mediates neutrophil migration in vitro. The adhesion of neutrophils to mindin is blocked by anti-integrin alpha4, anti-integrin alpha(M), and anti-integrin beta2 antibodies. We also show that HEK-293 cells transfected with cDNA encoding these integrins exhibit enhanced binding to immobilized mindin matrix and the increased binding can be blocked by anti-integrin antibodies. Our results suggest that mindin serves as a novel ligand for integrins and mindin-integrin interactions are critical for inflammatory cell recruitment in vivo.

Animals↗

PubMed1998

Human eotaxin induces alpha 4 and beta 2 integrin-dependent eosinophil accumulation in rat skin in vivo: delayed generation of eotaxin in response to IL-4.

The CC chemokine eotaxin, originally purified from bronchoalveolar lavage fluid of sensitized guinea pigs following allergen challenge, is a potent eosinophil-selective chemoattractant. In the present study, we have used (111)In-eosinophils and human eotaxin to characterize the profile of chemokine-induced eosinophil accumulation in vivo in rat skin. Intradermally injected eotaxin caused a dose-dependent accumulation of (111)In-eosinophils. Time course studies indicated that the response was rapid, since all the accumulation occurred within the first 1 to 2 h of eotaxin injection. The i.v. administration of anti-intercellular adhesion molecule-1, anti-vascular cell adhesion molecule-1, or anti-alpha4 integrin mAbs significantly inhibited the eosinophil accumulation induced by 100 pmol of human eotaxin by 73, 43, and 67%, respectively. Further, when (111)In-eosinophils were pretreated in vitro with anti-alpha4 integrin or anti-beta2 integrin mAbs, or with a combination of both mAbs, eotaxin-induced responses in vivo were reduced by 52, 49, and 68%, respectively. Eosinophil accumulation induced by intradermal IL-4, but not that induced by TNF-alpha or leukotriene B4, appeared to be mediated in part by endogenously generated eotaxin. Anti-eotaxin Abs significantly inhibited (54%) the later phases (24-28 h) but not the early phase (0-4 h) of the response to IL-4. This was consistent with eotaxin mRNA expression peaking at 18 h after IL-4 injection. Our findings show that human eotaxin is a potent inducer of eosinophil accumulation in vivo, this response being dependent on alpha4 integrin/vascular cell adhesion molecule-1 and beta2 integrin/intercellular adhesion molecule-1 adhesion pathways. Further, the eosinophil accumulation in response to IL-4 is partly mediated by endogenously generated eotaxin.

Animals↗

PubMed2003

Lymphocyte homing to bronchus-associated lymphoid tissue (BALT) is mediated by L-selectin/PNAd, alpha4beta1 integrin/VCAM-1, and LFA-1 adhesion pathways.

Bronchus-associated lymphoid tissue (BALT) participates in airway immune responses. However, little is known about the lymphocyte-endothelial adhesion cascades that recruit lymphocytes from blood into BALT. We show that high endothelial venules (HEVs) in BALT express substantial levels of VCAM-1, in marked contrast to HEVs in other secondary lymphoid tissues. BALT HEVs also express the L-selectin ligand PNAd. Anti-L-selectin, anti-PNAd, and anti-LFA-1 mAbs almost completely block the homing of B and T lymphocytes into BALT, whereas anti-alpha4 integrin and anti-VCAM-1 mAbs inhibit homing by nearly 40%. alpha4beta7 integrin and MAdCAM-1 are not involved. Importantly, we found that mAbs against alpha4 integrin and VCAM-1 significantly block the migration of total T cells (80% memory phenotype) but not naive T and B cells to BALT. These results suggest that an adhesion cascade, which includes L-selectin/PNAd, alpha4beta1 integrin/VCAM-1, and LFA-1, targets specific lymphocyte subsets to BALT. This high level of involvement of alpha4beta1 integrin/VCAM-1 is unique among secondary lymphoid tissues, and may help unify lymphocyte migration pathways and immune responses in BALT and other bronchopulmonary tissues.

Animals↗

PubMed2003

Matrix metalloproteinase production by COOH-terminal heparin-binding fibronectin fragment in rheumatoid synovial cells.

Fibronectin with IIICS region is present in rheumatoid synovium, and fibronectin fragments are increased in rheumatoid joints. We investigated the ability of COOH-terminal heparin-binding fibronectin fragment (COOH-HBFN-f) containing IIICS to induce matrix metalloproteinase (MMP) production and the role of mitogen-activated protein kinase (MAPK) pathway and CS-1 sequence that can bind alpha4beta1 integrin in MMP induction by COOH-HBFN-f in rheumatoid synovial fibroblasts (RSF). When RSF in monolayer culture were incubated with COOH-HBFN-f, COOH-HBFN-f stimulated the production of MMP-1, MMP-3, and MMP-13 by RSF in association with activation of extracellular signal-regulated kinase, p38 MAPK, and c-Jun NH(2)-terminal kinase. Immunoprecipitation of cell lysates demonstrated the presence of alpha4 integrin in cultured RSF. Similar to COOH-HBFN-f, treatment with CS-1 synthetic peptide derived from IIICS resulted in increased MMP production and activation of the kinases, although the MMP levels were low. Preincubation of RSF with anti-alpha4 integrin antibody resulted in partial suppression of the COOH-HBFN-f-stimulated MMP production. Inhibition studies using protein kinase inhibitors (PD98059 and SB203580) showed that those MAPK pathways contributed to MMP up-regulation by COOH-HBFN-f and CS-1. Thus, the present results have clearly shown that COOH-HBFN-f and CS-1 stimulate MMP production in association with activation of MAPK pathways in RSF. Integrin alpha4beta1 may be partially involved in the MMP induction by COOH-HBFN-f.

Arthritis, Rheumatoid↗

PubMed1999

EC3, a novel heterodimeric disintegrin from Echis carinatus venom, inhibits alpha4 and alpha5 integrins in an RGD-independent manner.

EC3, a heterodimeric disintegrin (Mr = 14,762) isolated from Echis carinatus venom is a potent antagonist of alpha4 integrins. Two subunits called EC3A and EC3B were isolated from reduced and alkylated EC3 by reverse-phase high performance liquid chromatography. Each subunit contained 67 residues, including 10 cysteines, and displayed a high degree of homology to each other and to other disintegrins. EC3 inhibited adhesion of cells expressing alpha4beta1 and alpha4beta7 integrins to natural ligands vascular cell adhesion molecule 1 (VCAM-1) and mucosal addressin cell adhesion molecule 1 (MadCAM-1) with IC50 = 6-30 nM, adhesion of K562 cells (alpha5beta1) to fibronectin with IC50 = 150 nM, and adhesion of alphaIIbbeta3 Chinese hamster ovary cells to fibrinogen with IC50 = 500 nM; it did not inhibit adhesion of alphavbeta3 Chinese hamster ovary cells to vitronectin. Ethylpyridylethylated EC3B inhibited adhesion of Jurkat cells to immobilized VCAM-1 (IC50 = 6 microM), whereas EC3A was inactive in this system. The MLDG motif appeared to be essential for activity of EC3B. Linear MLDG peptide inhibited the adhesion of Jurkat to VCAM-1 in a dose-dependent manner (IC50 = 4 mM), whereas RGDS peptide was not active at the same concentration. MLDG partially inhibited adhesion of K562 cells to fibronectin (5-10 mM) in contrast to RGDS peptide (IC50 = 3 mM), inhibiting completely at 10 mM.

Amino Acid Sequence↗

PubMed1998

Rat neutrophils express alpha4 and beta1 integrins and bind to vascular cell adhesion molecule-1 (VCAM-1) and mucosal addressin cell adhesion molecule-1 (MAdCAM-1).

The alpha4 integrins, which are constitutively expressed on all human leukocyte subtypes except neutrophils, interact with vascular cell adhesion molecule-1 (VCAM-1) and mucosal addressin cell adhesion molecule (MAdCAM-1) on endothelium to mediate selective recruitment of leukocyte subpopulations, other than neutrophils, to sites of inflammation. However, here we report that a different paradigm of leukocyte recruitment may exist in the rat. Flow cytometric analysis of rat neutrophils using a panel of monoclonal antibodies which recognize rat alpha4 and beta1 integrins showed consistent, low levels of expression. Although alpha4 was expressed at lower levels on neutrophils than all other rat leukocytes, this level of expression was sufficient to mediate significant levels of alpha4- and beta1-dependent neutrophil adhesion to rat and human VCAM-1, and alpha4-dependent, but beta1-independent, adhesion to human MAdCAM-1. These data suggest that rat neutrophils, unlike other species, may use alpha4 integrins to traffic to sites of inflammation in vivo.

Animals↗

PubMed1997

Rat mast cell lines bind to the vascular cell adhesion molecule-1 (VCAM-1) and the mucosal addressin cell adhesion molecule-1 (MAdCAM-1).

Mast cells are key mediators of allergy and inflammation. Increased mast cell numbers are observed in the gut during helminth infestation and at many sites of inflammation. To determine whether mast cells express functional receptors for endothelial cell adhesion molecules, we studied the adhesion of two rat mucosal-type mast cell lines RBL-1 and RCMC-1 to transfected mucosal addressin cell adhesion molecule-1 (MAdCAM-1) and VCAM-1. Both mast cell lines expressed high levels of alpha4 integrins on their surface and bound to CHO cells transfected with VCAM-1. Anti-alpha4 mAbs, TA-2 and L25, inhibited the specific adhesion of the mast cells to VCAM-1 by about 92 and 63%, respectively. Both of the mast cell lines also demonstrated an increased adhesion to CHO cells transfected with MAdCAM-1. The adhesion of RBL-1 to MAdCAM-1 was also significantly inhibited by the anti-alpha4 mAbs TA-2, L25, and HP2/1 by 39, 76, and 42%, respectively. In addition, RBL-1 cells adhered to both VCAM-1 and MAdCAM-1 under both static and nonstatic (shear) conditions, and this was also inhibited by the anti-alpha4 mAb TA-2. Thus, mucosal-type mast cell lines express functional alpha4 integrins that can mediate adhesion to VCAM-1 and MAdCAM-1. These results suggest a mechanism for mast cell accumulation at sites of inflammation and in the gut.

Animals↗