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PubMed1999

Expression of beta-integrin adhesion molecules in non-Hodgkin's lymphoma: correlation with clinical and evolutive features.

PURPOSE: To analyze beta-integrin expression in non-Hodgkin's lymphomas (NHLs) in order to assess its distribution among histologic subtypes and correlate with clinical features and outcome. PATIENTS AND METHODS: The expression of alpha2 through alpha6 and beta1 common chains of very late activation antigen (VLA ) molecules and alphaL (CD11a) and beta2 common (CD18) chains of leukocyte function-associated antigen 1 molecule were studied in 137 patients with NHL. Immunostaining was performed by a streptavidin-biotin alkaline phosphatase method, and integrin expression was semiquantitatively assessed. Correlation with clinical features was analyzed in 80 patients consecutively diagnosed as having immunocytoma (five cases), follicular lymphoma (19 cases), mantle-cell lymphoma (MCL; four cases), diffuse large-cell lymphoma (DLCL; 40 cases), lymphoblastic lymphoma (LL; six cases), anaplastic Ki-1-positive lymphoma (one case), and other peripheral T-cell lymphoma (five cases). RESULTS: MCL cells did not show alpha2 and alpha6 expression, whereas most expressed weak to moderate levels of alpha3, alpha4, and alpha5. LL mostly showed alpha2 to alpha5 expression, whereas alpha6 was observed in seven of 11 cases (higher proportion than that shown in other subgroups). Alpha chains of VLA molecules were present more frequently in T-cell than in B-cell lymphomas. Patients with moderate/strong alpha4, CD11a, and beta2 common chain expression presented more frequently with advanced stage and bone marrow infiltration. Moderate/strong alpha4, alpha5, and beta1 common chain expression correlated with extranodal involvement. In the subset of B-cell DLCL patients, negative/weak expression of alpha3 and alpha4 chains was related to a higher complete response rate. Moreover, negative or weak expression of alpha2, alpha3, alpha4, and beta1( )common chain had favorable significance for overall and failure-free survivals. CONCLUSION: In NHL, beta-integrin expression is related to histologic subtype. The expression pattern of these molecules probably influences disease dissemination and patients' prognoses.

CD18 Antigens↗
PubMed1999

Matrix adhesion characteristics of corneal myofibroblasts.

PURPOSE: To investigate the adhesion characteristics of corneal myofibroblasts in a cell culture model. METHODS: Immunocytochemistry, immunoprecipitation, western blot analysis, and attachment assays were used to evaluate matrix adhesion characteristics of myofibroblasts. RESULTS: Myofibroblasts, defined by their expression of the smooth muscle isoform of alpha-actin, were evaluated and compared with fibroblasts. Myofibroblasts had larger vinculin-containing focal adhesions and expressed more of the classic fibronectin receptor (FNR) alpha5beta1 per cell. However, myofibroblasts had less surface expression of the higher molecular weight alpha4 subunit of another FNR, alpha4beta1, than did fibroblasts. Myofibroblasts adhered more avidly in an integrin-dependent manner to fibronectin than did fibroblasts. The attachment to fibronectin was actin-dependent for both phenotypes, but the myofibroblasts' adhesion was more resistant to disruption by cytochalasin than were fibroblasts'. In addition to the previously described expression of a 135-kDa classic cadherin, myofibroblasts also expressed a 115-kDa mesenchymal cadherin, cadherin-11. CONCLUSIONS: Differentiation of corneal fibroblasts into myofibroblasts is associated with characteristics that would indicate that the latter have a special role in wound closure. The increase in focal and cell adhesion molecules that accompanies smooth muscle-specific actin expression provides the basis for the myofibroblasts' enhanced cell-fibronectin and cell-cell adhesion.

Animals↗
PubMed1998

The I domain of integrin leukocyte function-associated antigen-1 is involved in a conformational change leading to high affinity binding to ligand intercellular adhesion molecule 1 (ICAM-1).

On T cells the leukocyte integrin leukocyte function-associated antigen-1 (LFA-1) (CD11a/CD18) can be induced to bind its ligand intercellular adhesion molecule 1 (ICAM-1) (CD54) either by increasing the affinity of the receptor with Mg2+ and EGTA or by receptor clustering following activation with phorbol ester. The existence of these two adhesion-inducing pathways implies that alternative mechanisms might exist by which LFA-1 engages ICAM-1. The LFA-1 alpha subunit I domain contains a major binding site for ICAM-1. In this study we show that soluble LFA-1 I domain blocks ICAM-1 binding of the high affinity Mg2+-induced form of LFA-1 but not the phorbol ester-induced form. Under conditions of Mg2+-activation, the soluble I domain also prevents expression of an activation dependent epitope on LFA-1, implying that it inhibits a conformational change necessary for conversion to the high affinity form of this integrin. In addition, the binding of Mg2+-activated LFA-1 to ICAM-1 is blocked by peptides covering the alpha4-beta3 loop, the beta3-alpha5 loop, and the alpha5 helix of the I domain, whereas none of the peptides tested blocks phorbol ester-mediated adhesion. The blocking peptides localize to the same face of the crystal structure of the LFA-1 I domain and define an area that, during activation, may be involved in association of the I domain with another region of LFA-1, potentially the beta-propeller domain. This is the first evidence linking a structural domain of an integrin, in this case the I domain, with a particular activation mechanism.

Amino Acid Sequence↗
PubMed2005

Cardiomyopathy associated with microcirculation dysfunction in laminin alpha4 chain-deficient mice.

Laminin alpha4 chain is a component of extracellular matrix (ECM) laminin-8 and -9 and serves dual roles as a structure protein and as a signaling molecule. The abundance of laminin alpha4 chain transcripts in the heart suggests an important role of this protein in cardiovascular development and function. In this study, we demonstrate that laminin alpha4 deficient mice gradually develop cardiac hypertrophy with impaired function. We show that depletion of laminin alpha4 chain did not alter the levels of dystrophin-glycoprotein complex (DGC) components or affect cell membrane integrity. No alteration in integrin beta 1D protein was observed in terms of expression level or distribution pattern, indicating that the postnatal development of cardiac hypertrophy and cardiomyopathy in these mice is unlikely associated with the stability of sarcolemmal DGC and integrin complexes. Moreover, cardiomyocytes isolated from Lama4-/- mutant hearts maintained their contractility in vitro. In contrast, elevated levels of hypoxia-inducible factor 1alpha (Hif1alpha) and vascular endothelial growth factor A (Vegfa) transcripts, along with multiple foci of cardiomyocyte degeneration and fibrosis suggested sustained cardiac ischemia. Electron microscopy confirmed malformed blood vessels and wide pericapillary ECM spaces, suggesting the presence of microcirculation abnormalities in Lama4-/- mutant hearts. We thus conclude that mutation in the laminin alpha4 chain leads to abnormal cardiovascular ECM structure that cause insufficient oxygen supply to the heart and the subsequent ischemic cardiac phenotype observed. Our study links the genetic deficiency of an ECM protein to cardiomyopathy and implies a novel pathway of idiopathic cardiomyopathy in human.

Animals↗
PubMed1997

Integrin cross talk: activation of lymphocyte function-associated antigen-1 on human T cells alters alpha4beta1- and alpha5beta1-mediated function.

A regulated order of adhesion events directs leukocytes from the vascular compartment into injured tissues in response to inflammatory stimuli. We show that on human T cells, the interaction of the beta2 integrin leucocyte function-associated antigen-1 (LFA-1) with its ligand intercellular adhesion molecule-1 (ICAM-1) will decrease adhesion mediated by alpha4beta1 and, to a lesser extent, alpha5beta1. Similar inhibition is also seen when T cells are exposed to mAb 24, which stabilizes LFA-1 in an active state after triggering integrin function through divalent cation Mg2+, PdBu, or T cell receptor/ CD3 complex (TCR/CD3) cross-linking. Such cross talk decreases alpha4beta1 integrin-mediated binding of T cells to fibronectin and vascular cell adhesion molecule-1 (VCAM-1). In contrast, ligand occupancy or prolonged activation of beta1 integrin has no effect on LFA-1 adhesion to ICAM-1. We also show that T cell migration across fibronectin, unlike adhesion, is mediated solely by alpha5beta1, and is increased when the alpha4beta1-mediated component of fibronectin adhesion is decreased either by cross talk or the use of alpha4-blocking mAb. The ability of mAb 24 Fab' fragments to induce cross talk without cross-linking LFA-1 suggests signal transduction through the active integrin. These data provide the first direct evidence for cross talk between LFA-1 and beta1 integrins on T cells. Together, these findings imply that activation of LFA-1 on the extravasating T cell will decrease the binding to VCAM-1 while enhancing the subsequent migration on fibronectin. This sequence of events provides a further level of complexity to the coordination of T cell integrins, whose sequential but overlapping roles are essential for transmigration.

Anti-Allergic Agents↗
PubMed2004

Cyclic peptides from the loop region of the laminin alpha 4 chain LG4 module show enhanced biological activity over linear peptides.

Laminins, heterotrimeric glycoproteins in the basement membrane, are involved in diverse biological activities. So far, five alpha, three beta, and three gamma chains have been identified, and at least 15 laminin isoforms exist composed of various combinations of the different three chains. The major cell-surface receptors for laminins are integrins and proteoglycans, such as dystroglycans and syndecans. Previously, we reported that synthetic peptide A4G82 (TLFLAHGRLVFM, mouse laminin alpha4 chain residues 1514-1525) showed strong cell attachment and syndecan binding activities. On the basis of the crystal structure of the LG module and sequence alignment, A4G82 is located in the connecting loop region between beta-strands E and F in the laminin alpha4 chain LG4 module. Here, we have focused on the structural importance of this E-F loop region for the biological activity of the alpha4 chain LG4 module. To determine the importance of the loop structure, we synthesized peptide A4G82X (cyclo-A4G82X, Cys-TLFLAHGRLVFX-Cys, X= norleucine), which was cyclized via disulfide bridges at both the N- and C-termini. The cyclic peptides derived from A4G82X inhibited the heparin binding activity of the alpha4 chain G domain and promoted HT-1080 cell attachment better than the corresponding linear peptides. We determined FLAHGRLVFX as a minimal sequence of cyclo-A4G82X important for cell adhesion and heparin binding using a series of truncated peptides. Moreover, HT-1080 cell attachment to the cyclic peptides was more efficiently blocked by heparin than cell attachment to the linear peptides. Furthermore, the cyclic peptides showed significantly enhanced syndecan-2-mediated cell attachment activity. These results indicate that the activity of A4G82 is highly conformation-dependent, suggesting that the E-F loop structure is crucial for its biological activity.

Amino Acid Sequence↗
PubMed2003

Integrin-specific signaling pathways controlling focal adhesion formation and cell migration.

The fibronectin (FN)-binding integrins alpha4beta1 and alpha5beta1 confer different cell adhesive properties, particularly with respect to focal adhesion formation and migration. After analyses of alpha4+/alpha5+ A375-SM melanoma cell adhesion to fragments of FN that interact selectively with alpha4beta1 and alpha5beta1, we now report two differences in the signals transduced by each receptor that underpin their specific adhesive properties. First, alpha5beta1 and alpha4beta1 have a differential requirement for cell surface proteoglycan engagement for focal adhesion formation and migration; alpha5beta1 requires a proteoglycan coreceptor (syndecan-4), and alpha4beta1 does not. Second, adhesion via alpha5beta1 caused an eightfold increase in protein kinase Calpha (PKCalpha) activation, but only basal PKCalpha activity was observed after adhesion via alpha4beta1. Pharmacological inhibition of PKCalpha and transient expression of dominant-negative PKCalpha, but not dominant-negative PKCdelta or PKCzeta constructs, suppressed focal adhesion formation and cell migration mediated by alpha5beta1, but had no effect on alpha4beta1. These findings demonstrate that different integrins can signal to induce focal adhesion formation and migration by different mechanisms, and they identify PKCalpha signaling as central to the functional differences between alpha4beta1 and alpha5beta1.

Cell Adhesion↗
PubMed2000

Integrin expression by primary and immortalized human chondrocytes: evidence of a differential role for alpha1beta1 and alpha2beta1 integrins in mediating chondrocyte adhesion to types II and VI collagen.

OBJECTIVE: Chondrocytes have been shown to express beta1-containing integrins both in vitro and in situ, but their role in regulating chondrocyte function is poorly understood. The objective of this study was to determine how the relative expression of different integrins may be modulated in relation to the differentiated state and proliferative capacity of the chondrocyte. DESIGN: Integrin expression by four different cell lines of human chondrocytes immortalized with Simian virus 40 large T-antigen (SV40-TAg) was studied and compared to primary chondrocytes. Differences in alpha1 and alpha2 integrin subunit expression were utilized to further study the role of these integrins in mediating adhesion to types II and VI collagen. RESULTS: The overall cell-surface levels of beta1-containing integrins were higher on all four immortalized cell lines which expressed over 10-fold higher levels of alpha2 and alpha3 integrin subunits compared to primary cells. However, primary cells expressed higher levels of the alpha1 integrin subunit which was not expressed by T/C28a4 cells and expressed at variable and lower levels in the other lines. Levels of the alpha3 integrin subunit were significantly greater on the highly proliferative juvenile costal chondrocyte lines (T/C-28a4, C-2812, and C-20a4) compared to primary articular chondrocytes and tsT/AC-62 cells which were derived from adult articular chondrocytes. Expression of alpha5 was similar among primary cells and cell lines except on C-20/A4 cells which had an average of over 4-fold higher levels. None of the primary or immortalized chondrocytes tested expressed significant levels of alpha4. Cell adhesion assays revealed that both alpha1beta1 and alpha2beta1 could serve as chondrocyte adhesion receptors for types II and VI collagen. In cell lines expressing both integrins, alpha1beta1 was the preferential receptor for type VI collagen while alpha2beta1 was the preferential receptor for type II collagen. Rather than inhibiting adhesion, incubation with the alpha3 blocking antibody P1B5 increased adhesion of C-28/12 cells to both fibronectin and type II collagen by 67% and 100% respectively. CONCLUSIONS: Immortalization with SV40-TAg results in altered integrin expression by chondrocytes. Changes in the relative levels of alpha1, alpha2, and alpha3 subunits may significantly alter the manner in which chondrocytes interact with types II and VI collagen in the extracellular matrix.

Adult↗
PubMed2004

Different embryo-fetal toxicity effects for three VLA-4 antagonists.

BACKGROUND: VLA-4 (Very late antigen 4, integrin alpha4beta1) plays an important role in cell-cell interactions that are critical for development. Homozygous null knockouts of the alpha4 subunit of VLA-4 or VCAM-1 (cell surface ligand to VLA-4) in mice result in abnormal placental and cardiac development and embryo lethality. Objectives of the current study were to assess and compare the teratogenic potential of three VLA-4 antagonists. METHODS: IVL745, HMR1031, and IVL984 were each evaluated by the subcutaneous route in standard embryo-fetal developmental toxicity studies in rats and rabbits. IVL984 was also evaluated in mice. Fetuses were examined externally, viscerally, and skeletally. RESULTS: IVL745 did not cause significant maternal or fetal effects at doses up to 100 or 250 mg/kg/day in rats or rabbits, respectively. HMR1031 treatment resulted in marked maternal toxicity and slight fetal toxicity at the highest tested doses of 200 and 75 mg/kg/day in rats and rabbits, respectively. HMR1031 embryo-fetal effects consisted of slightly lower body weight and crown-rump length in rats and minor sternebral defects in rabbits. IVL984 treatment resulted in minimal maternal effects at doses up to 40, 15, and 100 mg/kg/day in rats, rabbits, and mice, respectively (excluding abortions in rabbits). However, marked developmental effects were observed at the lowest tested IVL984 doses, 1, 0.2, and 3 mg/kg/day in rats, rabbits, and mice, respectively. IVL984 embryo-fetal effects consisted of increased total post-implantation loss due to early resorptions and high incidences of cardiac malformations and skeletal malformations and/or variations. Notably, spiral septal defects were observed in up to 76% of rat fetuses and up to 58% of rabbit fetuses. CONCLUSIONS: Dramatic differences in teratogenic potential were observed: IVL745 was not teratogenic, HMR1031 caused slight embryo-fetal effects at maternally-toxic doses, and IVL984 was a potent teratogen at doses where direct maternal toxicity was limited to abortions in rabbits. Prominent effects of IVL984 included embryo lethality and cardiac malformations including spiral septal defects in three species.

Abnormalities, Drug-Induced↗
PubMed2000

New functions for non-collagenous domains of human collagen type IV. Novel integrin ligands inhibiting angiogenesis and tumor growth in vivo.

Collagen type IV is a major component of the basal lamina of blood vessels. Six genetically distinct collagen type IV chains have been identified and are distributed in a tissue-specific manner. Here we define a novel function for soluble non-collagenous (NC1) domains of the alpha2(IV), alpha3(IV), and alpha6(IV) chains of human collagen type IV in the regulation of angiogenesis and tumor growth. These NC1 domains were shown to regulate endothelial cell adhesion and migration by distinct alpha(v) and beta(1) integrin-dependent mechanisms. Systemic administration of recombinant alpha2(IV), alpha3(IV), and alpha6(IV) NC1 domains potently inhibit angiogenesis and tumor growth, whereas alpha1(IV), alpha4(IV), and alpha5(IV) showed little if any effect. These findings suggest that specific NC1 domains of collagen type IV may represent an important new class of angiogenesis inhibitors.

Amino Acid Sequence↗
PubMed2005

Evaluation of specificity and effects of monoclonal antibodies submitted to the Eighth Human Leucocyte Differentiation Antigen Workshop on rotavirus-cell attachment and entry.

Rotavirus infection of permissive cells is a multi-step process that requires interaction with several cell surface receptors. Integrins alpha2beta1, alpha4beta1, alphaXbeta2, and alphavbeta3 are involved in the attachment and entry into permissive cells for many rotavirus strains. However, possible roles of known partners of these integrins in this process have not been studied. Here, the specificities of new monoclonal antibodies directed to beta1 and beta2 integrins were determined using integrin-transfected cells. The ability of monoclonal antibodies to integrin partners CD82, CD151, CD321, and CD322 to bind rotavirus-permissive cell lines (MA104, Caco-2, and RD) and K562 cells expressing or lacking alpha4beta1 also was investigated. CD82 and CD151 were expressed on K562, alpha4-K562, and RD cells. CD321-specific antibodies bound K562, alpha4-K562, MA104, and Caco-2 cells. CD322 expression was detected on MA104 but not Caco-2 cells. Antibodies to CD82, CD151, CD321, and CD322 that bound these cells were investigated for their ability to inhibit cellular attachment and entry by rotaviruses. Antibody blockade of these integrin-associated proteins did not affect cell attachment or entry of the integrin-using rhesus rotavirus RRV or porcine rotavirus CRW-8, which uses alpha4beta1 integrin for infection. Antibody blockade of CD322 did not alter cell attachment or infectivity by human rotavirus strain RV-3, so RV-3 infection was independent of CD322. Overall, these studies indicate that CD82, CD151, CD321, and CD322 are unlikely to play a role in rotavirus-cell binding or entry.

Antibodies, Monoclonal↗
PubMed1998

Identification of ligand binding sites on integrin alpha4beta1 through chemical cross-linking.

We have used chemical cross-linking to identify sequences in integrin alpha4beta1 that are involved in its interactions with ligands. A recently described leucine-aspartic acid-valine (LDV)-based small molecule inhibitor of alpha4beta1 (BIO-1494), that contained a single reactive amino group for targeting the cross-linking, was used for these studies. The specificity of the interaction was defined by (i) the ability to block the interaction with a competitive inhibitor lacking the reactive group, (ii) the absolute requirement of divalent cations for cross-linking, and (iii) the lack of cross-linking to the functionally related integrin alpha4beta7. With ANB-NOS as the cross-linker, only the beta1 chain was labeled with BIO-1494, while with the more flexible cross-linker DSS both the alpha4 and beta1 chains were modified. Similar results were obtained when cross-linking was performed on K562 cells expressing alpha4beta1 but not on K562 cells expressing alpha2beta1. The site of cross-linking on the beta1 chain was localized by CNBr peptide mapping within residues 130-146, a region that contains the putative metal binding site DXSXS and for which analogous data had been generated with RGD binding to integrin alphaIIbbeta3. The striking similarity between the data we generated for an LDV ligand and published data for the RGD family supports the notion of a common ligand binding pocket formed by both integrin chains. The cross-linking strategy developed here should serve as a useful tool for studying alpha4beta1 function.

Amino Acid Sequence↗
PubMed2000

Production of recombinant soluble human integrin alpha4beta1.

Integrin alpha4beta1 is a major leukocyte adhesion receptor that is a key target for the development of anti-inflammatory therapeutics. With the dual long-term goals of developing a reagent for use in high-throughput inhibitor screening assays and for crystallisation trials and subsequent structure determination, we have generated a recombinant soluble alpha4beta1 receptor. Both subunits were truncated prior to the transmembrane domains by site-directed mutagenesis and expressed using baculovirus infection of insect cells. The molecular weights of the recombinant subunits were as expected for post-translationally unmodified protein. In addition, as observed for the native subunit, a proportion of the alpha4 subunit was proteolytically processed into two fragments. ELISA and solid phase ligand-binding assays were performed to investigate the folding and functionality of the soluble integrin. The data suggest that the receptor was correctly folded and that it bound recombinant ligands with similar kinetics to the native molecule.

Animals↗
PubMed2004

Critical period for a teratogenic VLA-4 antagonist: Developmental effects and comparison of embryo drug concentrations of teratogenic and non-teratogenic VLA-4 antagonists.

BACKGROUND: Integrins such as VLA-4 (Very late antigen 4, integrin alpha4beta1) play key roles in cell-cell interactions that are critical for development. Homozygous null knockouts of the VLA-4 alpha4-subunit or VCAM-1 (VLA-4 cell surface ligand) in mice result in failure of the allantois and chorion to fuse leading to interrupted placentation and cardiac development and embryo lethality. Embryo-fetal studies of three VLA-4 antagonists, IVL745, IVL984, and HMR1031 [Crofts et al., Birth Defects Res B 71:55-68 (this issue), 2004] with exposure on gestation days (GD) 6-17 (rat), 6-18 (rabbit) or 6-15 (mouse) showed that only IVL984 treatment resulted in embryo lethality and cardiac defects. Objectives of the current study were to determine the critical period for inducing IVL984-related embryo-fetal effects, and to test the hypothesis that these effects were due to higher embryo drug concentrations. METHODS: IVL984 was administered at 40 mg/kg/day to pregnant rats on GD 4 and 5, GD 6 and 7, GD 8 and 9, GD 10 and 11, or GD 12 and 13. Animals were euthanized on GD 21 and uteri and fetuses were examined. A treatment period of GD 10-12 was selected for subsequent toxicokinetic (TK) studies in which IVL984, HMR1031, or IVL745 was administered to pregnant rats and rabbits. On GD 12, maternal plasma, extra-embryonic tissue (placenta and amniotic fluid), and embryonic tissue were collected and analyzed for drug concentrations. RESULTS: In the IVL984 critical period study in pregnant rats, treatment on GD 10 and 11 resulted in increased post-implantation loss, skeletal variations, and spiral septal defects similar to those observed in standard embryo-fetal development studies with treatment throughout organogenesis. There were no embryo-fetal effects after treatment on GD 4 and 5, GD 6 and 7, or GD 8 and 9. There was a single aorta malformation after treatment on GD 12 and 13. In the TK studies, IVL745, HMR1031, and IVL984 were all detectable in embryonic tissue and there was no evidence for accumulation. Rat and rabbit embryo exposures (AUC or dose-adjusted AUC) on GD 12 could not explain the observed teratology (IVL984<HMR1031<IVL745). Further analyses incorporating pharmacological activity, clearance, and protein binding data provided a positive correlation between embryonic exposure and teratogenic potency. CONCLUSIONS: The critical period for IVL984 in the rat, GD 10 to 11, corresponds to the expression of alpha-4 integrin on the chorion and VCAM-1 on the allantois and myocardium as well as chorioallantoic fusion and formation of the spiral septum. Embryo drug levels adjusted for pharmacological activity, clearance, and protein binding provide a possible explanation for the differing teratogenic potency of IVL984, HMR1031, and IVL745.

Abnormalities, Drug-Induced↗
PubMed2004

An endothelial laminin isoform, laminin 8 (alpha4beta1gamma1), is secreted by blood neutrophils, promotes neutrophil migration and extravasation, and protects neutrophils from apoptosis.

During extravasation, neutrophils migrate through the perivascular basement membrane (BM), a specialized extracellular matrix rich in laminins. Laminins 8 (LN-8) (alpha4beta1gamma1) and 10 (LN-10) (alpha5beta1gamma1) are major components of the endothelial BM, but expression, recognition, and use of these laminin isoforms by neutrophils are poorly understood. In the present study, we provide evidence, using a panel of novel monoclonal antibodies against human laminin alpha4 (LNalpha4) chain, that neutrophils contain and secrete LN-8, and that this endogenous laminin contributes to chemoattractant-induced, alphaMbeta2-integrin-dependent neutrophil migration through albumin-coated filters. Phorbol ester-stimulated neutrophils adhered to recombinant human (rh) LN-8, rhLN-10, and mouse LN-1 (mLN-1) (alpha1beta1gamma1) via alphaMbeta2-integrin, and these laminin isoforms strongly promoted chemoattractant-induced neutrophil migration via the same integrin. However, only rhLN-8 enhanced the spontaneous migration. In addition, recruitment of neutrophils into the peritoneum following an inflammatory stimulus was impaired in LNalpha4-deficient mice. rhLN-8 also protected isolated neutrophils from spontaneous apoptosis. This study is the first to identify a specific laminin isoform in neutrophils and provides evidence for the role of LN-8 in the adhesion, migration, extravasation, and survival of these cells.

Aflatoxin B1↗
PubMed1999

Integrin subunits responsible for adhesion of human osteoblast-like cells to biomimetic peptide surfaces.

We have identified the integrin subunits responsible for the initial adhesion of human osteoblast-like cells to peptide-modified surfaces. Biomimetic peptide surfaces containing homogenous RGD (Arg-Gly-Asp), homogenous FHRRIKA (Phe-His-Arg-Arg-Ile-Lys-Ala), and a mixed ratio of FHRRIKA:RGD (25:75) were used to assess integrin-mediated adhesion. The RGD and FHRRIKA peptides were selected from the cell-binding and putative heparin-binding domains of bone sialoprotein. A panel of monoclonal antibodies against human alpha1, alpha2, alpha3, alpha4, alpha5, beta1, alpha(v), and alpha(v)beta3 was used to identify the subunits most dominant in mediating short-term (10 or 30 minutes) and long-term (4 hours) cell adhesion to the peptide surfaces. Anti-alpha2, anti-beta1, and anti-alpha(v) significantly (p < 0.05) diminished cell attachment to homogenous RGD surfaces following 30 minutes of incubation. After 4 hours of incubation on RGD-grafted surfaces, immunostaining of these integrin subunits revealed discrete localization of the alpha(v) subunit at the periphery of the cell (similar to focal contact points), whereas the alpha2 and beta1 subunits stained very diffusely throughout the cell. A radial-flow apparatus was used to determine the effect of anti-integrin antibodies on strength of cell detachment following 10 minutes of incubation on peptide-grafted surfaces. The strength of detachment from surfaces containing RGD was significantly reduced (p < 0.05) in the presence of anti-alpha2, anti-alpha(v), or anti-beta1 compared with controls (presence of preimmune mouse IgG). None of the antibodies significantly influenced cell attachment to homogenous FHRRIKA-grafted surfaces. These results demonstrate that initial (30 minutes) attachment of human osteoblast-like cells to homogenous RGD surfaces was mediated by the collagen receptor alpha2beta1 and the vitronectin receptor alpha(v)beta3, whereas only the vitronectin receptor governed longer term (longer than 30 minutes) adhesion (localization to focal contacts). The importance of distinct integrins in mediating the attachment of bone cells to RGD-immobilized surfaces indicates a strategy for engineering orthopaedic implants with a built-in surface specificity for cell adhesion.

Amino Acid Sequence↗
PubMed2004

The identification and optimization of orally efficacious, small molecule VLA-4 antagonists.

The identification of orally active, small molecule antagonists of the alpha4beta1 integrin, VLA-4, could lead to therapeutic agents with utility in a number of clinical settings, including asthma, multiple sclerosis and IBD. Starting from CDR3 sequences conserved among neutralizing alpha4 antibodies, peptides were identified that antagonized VLA-4 mediated adhesion in vitro. Through a series of structural modifications, these peptides evolved into small molecules that exhibited high potency and selectivity for VLA-4 in cell adhesion assays. Finally, through the optimization of physical and pharmacokinetic properties, compounds were identified that exhibited oral activity in animal models of asthma and multiple sclerosis.

Animals↗
PubMed2002

Modulation of integrin expression on rat bone marrow cells by substrates with different surface characteristics.

Biomaterials have been shown to be able to influence the growth and differentiation of osteogenic cells cultured on the surface. Although the precise mechanisms by which the materials influence osteogenic cells are unclear, it is possible that the materials manipulate the expression of integrins by the cells. We therefore studied the expression of a number of integrins by rat bone marrow (RBM) cells, after culture on culture polystyrene, on machined and grit-blasted titanium, and on calcium phosphate-coated titanium. Integrin expression was studied by FACS analysis. We found a large variation in the expression of integrins by cells in replicate experiments. After culture on polystyrene for 7 days, cells expressed alpha1, alpha2, alpha3, alpha5, alpha6, beta1, and beta3, although some of the subunits were expressed only occasionally. The cells did not express the alpha4 subunit. After culture of RBM cells for 8 days on coated and noncoated titanium substrates, cells always expressed alpha3, alpha5, alpha6, and beta1. The alpha1 and beta3 subunits were only expressed in some of the experiments. Frequently, the expression of alpha5, alpha6, and beta1 was higher on the coated than on the noncoated titanium substrates. Based on our results, we conclude that the studied materials are capable of influencing the expression of integrins by RBM cells cultured on relevant implant materials.

Animals↗