alpha subunit partners to beta1 and beta2 integrins during IL-4-induced foreign body giant cell formation

As beta1 and beta2 integrins were previously found to mediate adhesion during IL-4-induced foreign body giant cell (FBGC) formation, we pursued the identities of the alpha integrin partners of these adhesion receptors using our in vitro system of human monocyte-derived macrophage fusion. Immunoprecipitation with beta1 and immunoblotting reveal the presence of alpha5 and alphaV, as well as alpha2 and alpha3. alphaM and alphaX immunoprecipitate with beta2 but not with beta1. Immunocytochemistry coupled with confocal microscopy indicates that alpha5 and alphaX are poorly expressed on day 0. However, following the induction of fusion by IL-4 on day 3, they are each readily detectable in fusing macrophages/FBGC on day 7. In contrast, alphaM and alphaV are present throughout the culture period, with very strong alphaM expression on day 7. We also demonstrate expression and colocalization of alpha3, alpha5, or alphaV with beta1 on fusing macrophages/FBGC at this time point as well as strong colocalization of alphaM and alphaX with beta2 in FBGC and at fusion interfaces. Therefore, IL-4-induced FBGC are characterized by the expression of alphaMbeta2, alphaXbeta2, alpha5beta1, alphaVbeta1, alpha2beta1, and alpha3beta1, which indicates potential interactions with fragments of complement C3, fibrin(ogen), fibronectin, Factor X, and vitronectin, and possibly with certain collagens, laminin, and thrombospondin at sites of biomaterial implantation.     

Luteogenic hormones act through a vascular endothelial growth factor-dependent mechanism to up-regulate alpha 5 beta 1 and alpha v beta 3 integrins, promoting the migration and survival of human luteinized granulosa cells

The formation of the corpus luteum (CL) is critical for the establishment of a successful pregnancy. After ovulation, the CL develops from the remnants of the ovulated ovarian follicle. This process, which involves varying cell-matrix interactions, is poorly characterized. To understand the role and potential regulation of cell-matrix interactions in the formation of the CL, we investigated the expression and activity of the matrix protein fibronectin (FN) and several of its integrin receptors on luteinized granulosa cells (GCs). In situ, FN and several FN-binding integrins were detected around luteinizing GCs during the early luteal phase, although expression declined in the late luteal phase. In vitro, GCs released FN, and stimulation of these cells with human chorionic gonadotropin increased the surface expression of FN, alpha(5)beta(1), and alpha(v)beta(3). Up-regulation of these proteins on GCs was reproduced by stimulation with vascular endothelial growth factor (VEGF) and was inhibited by anti-VEGF antibody. Lastly, expression of alpha(5)beta(1) and alpha(v)beta(3) mediated adhesion to FN, facilitated migration, and prevented apoptosis. These data suggest that in vivo luteogenic hormones, in part through a VEGF-dependent mechanism, stimulate selected integrin-matrix adhesive interactions that promote the motility and survival of GCs and thus contribute to the formation and preservation of the CL.     

Role of the alpha3beta1 and alpha6beta4 integrins in tumor invasion

Integrin receptors are well-known mediators of cell adhesion that also have a fundamental role in controlling the migration of cells through tissues. Among the numerous members of a still growing family, two particular molecular complexes have turned out to be of key importance in tumor cell invasion of basement membranes, the α3β1 and α6β4 integrins. In this Review, we will focus on the role of these two receptors and the mechanisms by which they influence the invasion process. This revised version was published online in July 2006 with corrections to the Cover Date.     

Ameloblastoma and adenomatoid odontogenic tumor: the role of alpha2beta1, alpha3beta1, and alpha5beta1 integrins in local invasiveness and architectural characteristics

Ameloblastoma is an odontogenic neoplasm characterized by local invasiveness and a tendency toward recurrence, whereas adenomatoid odontogenic tumor (AOT) is an indolent neoplasm. The objective of the present study was to immunohistochemically analyze the role of alpha2beta1, alpha3beta1, and alpha5beta1 integrins in the cellular events and cell-matrix interactions that occur in these tumors and their consequent repercussions on the architectural arrangement and biologic behavior of these lesions. Paraffin-embedded specimens from 30 ameloblastomas (20 solid and 10 unicystic tumors) and 12 AOTs were submitted to immunohistochemistry using the catalyzed signal amplification system. A difference in the pattern of integrin expression was observed between the various histologic types of ameloblastoma. No significant difference in labeling intensity was observed between unicystic and solid ameloblastomas, but comparison between ameloblastomas and AOT showed a significantly stronger expression of alpha5beta1 integrin in the former (P < .05). Our findings suggest an important role of the integrins studied in the architectural characteristics of ameloblastomas and AOTs and a possible participation of alpha5beta1 integrin in the mechanism of local invasion of ameloblastomas.     

alpha(v)beta(3) Integrin in central nervous system tumors

alpha(v)beta(3) Is an integrin specifically expressed in endothelial cells of newly forming blood vessels. Integrin-mediated angiogenesis is hypothesized to play a central role in the development and the progression of central nervous system neoplasms. Accordingly, it is considered a potential target for antiangiogenic therapy. In the current study, we compare the expression of alpha(v)beta(3) in ependymomas, oligodendrogliomas, pilocytic astrocytomas, medulloblastomas, and vestibular schwannomas (acoustic neuromas). Samples of 5 tumors of each of the 5 tumor types were harvested surgically and frozen. After the pathological diagnosis was confirmed, immunohistochemistry was performed using an anti- alpha(v)beta(3) monoclonal antibody (LM609). The expression of alpha(v)beta(3) was assessed using a 4-tiered (0-3) grading scheme reflecting the percentage of positively staining vessels. All vestibular schwannomas demonstrated strong (grade 3) alpha(v)beta(3) expression. The expression was uniformly prominent in Antoni B regions of the tumors. Of 5 ependymomas, 4 demonstrated uniformly strong alpha(v)beta(3). Oligodendrogliomas, medulloblastomas, and pilocytic astrocytomas demonstrated more variable alpha(v)beta(3). alpha(v)beta(3) may contribute significantly to angiogenesis in vestibular schwannomas and ependymomas. Despite the high vascular density of oligodendrogliomas, pilocytic astrocytomas, and medulloblastomas, these tumors had variable moderate alpha(v)beta(3) expression. This discrepancy suggests temporal and/or regional variability in the angiogenesis in these types of tumor. This study provides the first demonstration of alpha(v)beta(3) expression in vestibular schwannomas, medulloblastomas, and pilocytic astrocytomas.     

CD9 is expressed on human endometrial epithelial cells in association with integrins alpha(6), alpha(3) and beta(1)

Recently we reported that CD9 is involved in the invasion of a trophoblast-like choriocarcinoma cell line, BeWo, probably through the regulation of integrin functions. Integrins have also been reported to be expressed in the human endometrium and it has been suggested that they play important roles in blastocyst implantation. This study used immunohistochemistry to investigate the expression of CD9 in the endometrium during the menstrual cycle. CD9 was found to be intensely expressed on the cell surface of the glandular epithelium throughout the menstrual cycle without any apparent differences in staining intensity. In addition, Western blotting analysis of the affinity-purified proteins confirmed that CD9 was associated with integrins beta(1), alpha(3) and alpha(6) in the human endometrium. Therefore it can be concluded that CD9, in association with integrins alpha(6), alpha(3) and beta(1), is a constitutive molecule of the endometrial glandular epithelium. These results also suggest that CD9 may be an important regulator of these integrins in the human endometrium.     

Adenoviral-encoded antigens are presented efficiently by a subset of dendritic cells expressing high levels of alpha(v)beta3 integrins

Dendritic cells (DC) play a central role in antigen presentation and are often targeted by adenoviral (Ad)-based gene therapy. However, DC lack the coxsackie-Ad receptor, and little is known about the process by which they acquire and present Ad-encoded antigens. We examined the expression of alpha(v)beta3 integrins (CD51/CD61) on mouse bone marrow-derived DC (BM-DC) and their susceptibility to transduction by Ad vectors. Less than 10% of BM-DC precursors expressed CD51, but expression increased over time in culture with granulocyte macrophage-colony stimulating factor (GM-CSF)/interleukin (IL)-4. After 7 days, 28 +/- 1.7% of CD11c+ DC expressed high levels of CD51 (CD51(hi)), and the remaining DC expressed low levels of CD51 (CD51(lo)). CD51(hi) CD express higher major histocompatibility complex type 1 (MHC I); however, both of the DC subsets expressed similar levels of MHC II and costimulatory molecules. When exposed to a first-generation Ad vector, transgene expression was restricted to the CD51(hi) DC subset and blocked by soluble peptides expressing an arginine, glycine, aspartic acid (RGD) sequence, confirming the role of integrins in viral entry. Consistent with this, a modified Ad expressing an RGD-binding sequence in its fiber knob (Ad-RGD) transduced the CD51(hi) DC subset with significantly higher efficiency. When BM-DC were transduced with an Ad-expressing ovalbumin (Ad-OVA), the CD51(hi) subset proved superior in activating OT-I (T cell receptor-OVA) T cells. Similar to in vitro effects, systemic administration of GM-CSF/IL-4 increased the expression of CD51 on splenic DC and rendered these cells susceptible to Ad transduction. These results suggest that a limited subset of DC expressing high levels of alpha(v)beta3 integrins is preferentially transduced by Ad vectors and activates CD8+ T cell responses against Ad-encoded antigens.     

Immunolocalization of alphaV,alpha3 and beta1 integrins in the human placenta with pre-eclampsia

Signs of pre-eclampsia are considered to be caused by maternal endothelial dysfunction due to circulating factors of placental origin. Integrins are a large family of cell surface, proteins that serve as receptors involved in cell-cell and cell-matrix interactions during placentation. Therefore, low expression of integrins or the lack of it may be encountered during pre-eclampsia. In the present study, we investigated the immunolocalisation of integrins alphaV, alpha3 and beta1 in placentas of normal and pre-eclamptic women. Thirty-two placentas from pre-eclamptic (n = 14) and normotensive (n = 18) women were used. Immunohistochemical staining was performed on formalin-fixed, paraffin-embedded tissue specimens, using anti-alphaV, anti-alpha3 and anti-beta1 antibodies and the indirect immunoperoxidase technique. A semi-quantitative grading system (HSCORE) was used to compare immunohistochemical staining intensities. Distribution patterns of alphaV, alpha3 and beta1 integrins were detected in cytotrophoblasts and Hofbauer cells in normal and pre-eclamptic placentas. Immunostaining of alphaV and beta1 integrins was slightly decreased in pre-eclamptic samples but alpha3 integrin immunostaining was similar in pre-eclamptic and normal placentas. Decreased immunostaining of integrins in the cytotrophoblasts may considered to be a structural basis for decreased placental perfusion in pre-eclampsia.     

Beta1 and beta2 integrins mediate adhesion during macrophage fusion and multinucleated foreign body giant cell formation

An in vitro system of interleukin (IL)-4-induced human monocyte-derived macrophage fusion was used to investigate the cell/substrate adhesive mechanisms that support multinucleated foreign body giant cell (FBGC) formation. Monocytes were cultured for 3 days and IL-4 was added to induce macrophage fusion and FBGC formation by day 7. Functionally defined anti-integrin antibodies demonstrated that initial monocyte adhesion is mediated by beta2 integrins, whereas during the induction of macrophage fusion by IL-4, an additional dependence on beta1 integrins is acquired. The combination of anti-beta1 plus anti-beta2 was most effective, reducing macrophage/FBGC adhesion to 10% of controls. Consistent with integrin-mediated signaling, the tyrosine kinase inhibitor genistein and the phosphatidylinositol-3-kinase inhibitors wortmannin and LY294002 also attenuated macrophage/FBGC adhesion. Confocal microscopic analysis revealed that beta2 integrins are present on monocytes after initial adhesion and are strongly expressed on fusing macrophages, particularly in peripheral cell areas, and on FBGCs. In contrast, beta1 integrins are not detected on monocytes but begin to appear during macrophage development and are strongly expressed on fusing macrophages and FBGCs. For the first time, these results demonstrate the IL-4-induced acquisition of cooperation between beta1 and beta2 integrins in the cell/substrate adhesive interactions that are required for multinucleated FBGC formation.     

Expression and function of beta(1) and beta(3) integrins of human mesothelial cells in vitro.

Mesothelial cells (MC) and extracellular matrix (ECM) components are thought to play a pivotal regulatory role during the inflammatory-reparative response of serosal membranes. Integrins are known to serve as cellular ECM receptors, but mesothelial integrin expression and its function, particularly its role for attachment to different ECM components, remain to be elucidated. The aim of the present study was to characterize the integrin expression of human omentum majus derived MC (HOMC) in vitro by immunohistochemistry and to investigate their functional significance with regard to HOMC adhesion to fibronectin (fn), vitronectin (vn), collagen IV (coll IV), and laminin (ln). Mesothelial cells in vitro strongly expressed beta(1), beta(3), alpha(2), alpha(3), alpha(5), and alpha(v) chains. A weak reactivity was found for alpha(1) and alpha(6), but no alpha(4) reactivity was detectable. Compared to the control, fn, vn, coll IV, and ln caused a significant 2.6-, 2.2-, 2-, and 1.6-fold increase of HOMC adhesion, respectively. Inhibition studies revealed that HOMC attachment to fn is mediated by alpha(5)beta(1), alpha(v)beta(1), and alpha(v)beta(3), with a synergistic effect of alpha(5)beta(1) and alpha(v)beta(3). Adhesion to vn is mediated by alpha(v)beta(1) and alpha(v)beta(3). Integrins alpha(1)beta(1), alpha(2)beta(1), and alpha(3)beta(1) mediate adhesion to coll IV and ln. We suggest that the integrin expression and function of mesothelial cells described here play an important role in the interaction of MC with the ECM, particularly during the acute and chronic inflammatory-reparative response of serosal membranes.     

Integrin alpha(v)beta(3) expression in colon carcinoma correlates with survival.

Integrin alpha(v)beta(3) is expressed by newly formed blood vessels in diseased and neoplastic tissue and can therefore be used as a marker for angiogenesis. We investigated its expression on the vasculature of 40 colon carcinomas using the anti-alpha(v)beta(3)-specific monoclonal antibody LM609. The average relapse-free interval and overall survival in patients suffering from colon carcinomas with high vascular expression of alpha(v)beta(3) integrin was significantly reduced compared with that in patients with low alpha(v)beta(3) integrin expressing tumor vasculature. Moreover, the expression level of alpha(v)beta(3) integrin correlated with the presence of liver metastases. In conclusion, we propose vascular expression of alpha(v)beta(3) integrin as a prognostic indicator for colon carcinoma.     

Involvement of alpha(v)beta3 integrin-like receptor and glycosaminoglycans in Candida albicans germ tube adhesion to vitronectin and to a human endothelial cell line

The present study was undertaken to investigate the expression of alpha(v)beta3 and alpha(v)beta5 integrin-like vitronectin receptors (VNRs) on Candida albicans germ tube and their involvement in its adhesion to vitronectin (VN) and human endothelial cells. By immunofluorescence and FACS analysis, several monoclonal antibodies directed against human alpha(v) or beta3 integrin subunit or alpha(v)beta3 and alpha(v)beta5 heterodimers, positively stained C. albicans germ tubes. C. albicans germ tubes specifically adhered (45-50%) to VN and this adhesion was markedly inhibited by RGD-, but not RGE-containing peptides. Adhesion of C. albicans germ tubes to VN was strongly inhibited by anti-alphav, anti-beta3 or anti-alpha(v)beta3, but not by alpha(v)beta5 monoclonal antibody. C. albicans germ tube adhesion to VN was also inhibited by glycosaminoglycans (GAGs) such as heparin or chondroitin sulphate. Finally, we show that C. albicans germ tubes adhere to the human EA.hy 926 endothelial cell line. This adhesion is markedly blocked by anti-beta3 monoclonal antibody, GRGDSP peptide or heparin, and is completely abolished by their combination. Overall these results indicate that C. albicans germ tube adherence to VN and to a human endothelial cell line is mediated by alpha(v)beta3, but not by alpha(v)beta5-like integrin, and depends on GAGs which may act by regulating alpha(v)beta3 integrin-like/VN adhesive interaction.     

Distribution of alpha3, alpha5 and alpha(v) integrin subunits in mature and immature human oocytes

The distribution of three integrin subunits, alpha3, alpha5 and alpha(v), in immature and mature human oocytes has been examined using immunofluorescence and confocal microscopy. The results demonstrate that both alpha5 and alpha(v) are present at the germinal vesicle stage, while alpha3 was only detected in oocytes after germinal vesicle breakdown, in metaphase I and II stage oocytes. The cortical concentration of integrin subunits alpha3 and alpha5 is consistent with their localization in the oolemma. In contrast, the homogeneous distribution of alpha(v) throughout the oocyte suggests the existence of cytoplasmic reservoirs of this protein in the oocyte.      

gp49B1-alpha(v)beta3 interaction inhibits antigen-induced mast cell activation

We have identified the integrin alpha(v)beta3 as a ligand for mouse gp49B1, thus identifying a new class of ligand for a member of the family of inhibitory immunoreceptors that bear C2-type immunoglobulin-like domains. The specific interaction was shown by both cell-protein and cell-cell binding assays. In addition, we found that the interaction of alpha(v)beta3 with gp49B1 on bone marrow-derived mouse mast cells inhibited antigen-induced immunoglobulin E-mediated cell activation. Because neither gp49B1 nor alpha(v)beta3 exhibit substantive allelic variation, their newly appreciated interaction may reflect an innate pathway for down-regulating the activity of mast cells.     

Functional role of alpha 2/beta 1 and alpha 4/beta 1 integrins in leukocyte intercellular adhesion induced through the common beta 1 subunit.

Whereas all of the integrins in the VLA protein subfamily are involved in cell-extracellular matrix interactions, only VLA-4 (through the alpha 4 subunit) has been implicated in the triggering of intercellular adhesion. Here we describe that the VLA protein beta 1 subunit (CD29) is also involved in the induction of homotypic cell aggregation. We have obtained three novel anti-beta 1 monoclonal antibodies (mAb) with the ability to induce cell aggregation on different leukocyte cell types. These mAb recognize an antigenic site on the common beta 1 chain of VLA proteins which is topographically and/or functionally distinct from other epitopes previously defined by several prototype anti-beta 1 mAb. Induction of cell aggregation by anti-beta 1 mAb is epitope specific, isotype and Fc independent, and displays kinetics similar to alpha 4-mediated aggregation. This cell aggregation requires an intact cellular metabolism, the presence of divalent cations in the extracellular medium, and the integrity of the cytoskeleton. We also have found that the Na+/H+ antiporter may be essential for this process. For Ramos cells, which bear only the VLA alpha 4/beta 1 heterodimer, intercellular adhesion induced through the VLA-beta 1 chain could be selectively inhibited by other anti-beta 1 mAb as well as by anti-alpha 4 mAb. Interestingly, anti-beta 1 mAb which induced strong aggregation of VLA-alpha 2- or VLA-alpha 4-transfected K562 cells, had minimal effect on the alpha 2- alpha 4- alpha 5+ K562 cell line. Furthermore, the beta 1-mediated induction of cell aggregation on alpha 2-K562- and alpha 4-K562-transfected cells was blocked by preincubation with either anti-alpha 2 or anti-alpha 4 mAb, respectively, as well as by other anti-beta 1 mAb. Interestingly, parental K562 cells were able to interact with both alpha 2- and alpha 4-transfected K562 cells, thus suggesting that counter-receptors for both integrins (VLA-2 and VLA-4) might exist on these cells. Together these results provide strong evidence supporting the involvement of alpha 2/beta 1 and alpha 4/beta 1 heterodimers in intercellular interactions and underline the pivotal role of the common beta 1 chain of VLA proteins in the integrin-mediated induction of cell aggregation.     

Low expression of beta 1, alpha 2 and alpha 3 subunits of VLA integrins in malignant mammary tumours.

The Very Late Antigens (VLAs) are alpha beta heterodimeric transmembrane proteins mediating cell-substratum as well as cell-cell interactions. Changes in their expression and/or function seem to occur in a number of invasive carcinomas and may at least in part explain their abnormal patterns of growth and differentiation. Using monoclonal antibodies to the beta 1 (DH12, A1A-5), alpha 2 (B1.515) and alpha 3 (E1.56) chains, VLA-2 (alpha 2 beta 1) and VLA-3 (alpha 3 beta 1) were studied on cryostat sections of three fibroadenomas and 43 invasive breast carcinomas (29 ductal, 14 lobular) by the avidin-biotin complex immunoperoxidase technique. In non-neoplastic breast tissue and in fibroadenomas VLA-2 and VLA-3 were expressed by myoepithelial cells and on the basolateral surface of the luminal cells. There was weak or absent expression of alpha 2, alpha 3 and the common beta 1 chain in the majority of invasive carcinomas compared to the adjacent normal breast epithelium and preinvasive (in-situ) carcinomas. In addition, the expression of the alpha 2 chain of VLA-2 was reduced significantly (P less than 0.005) in the poorly differentiated ductal breast carcinomas (Grade III) compared to the well (Grade I) and moderately (Grade II) differentiated ductal tumours. These data give further evidence that loss or down-regulation of VLA-2 and VLA-3 occur relatively frequently in invasive cancers, and, at least in the invasive ductal breast carcinomas. Loss of an extracellular matrix receptor controlling growth and differentiation seems to be one of the abnormalities underlying the progression towards an undifferentiated morphology.     

Distribution of beta 1, alpha 1, alpha 2 and alpha 3 integrin chains in basal cell carcinomas

The integrins are alpha beta heterodimeric transmembrane proteins mediating cell-substratum as well as cell-cell interactions. Changes in their expression and/or function seem to occur in a number of malignant epithelial neoplasms and may in part explain their abnormal patterns of growth and differentiation. Using monoclonal antibodies to the beta 1 (DH12), alpha 1 (TS2/7), alpha 2 (B1.515), and alpha 3 (E1.56) integrin chains, the alpha 1 beta 1 (VLA-1), alpha 2 beta 1 (VLA-2), and alpha 3 beta 1 (VLA-3) integrin receptors were studied on cryostat sections of 22 basal cell carcinomas (BCCs) and adjacent normal tissues by a standard peroxidase-antiperoxidase technique. In non-neoplastic skin, VLA-2 and VLA-3 were found in the basal layer, eccrine glands, and cells of the outer root sheath in which VLA-1 was detected. In BCCs, there was a considerably higher expression of VLA-2 and VLA-3 compared with epidermal basal cells but similar to that seen in hair bulb and outer root sheath. In two cases of nodular BCC showing evidence of regression, both VLA-2 and VLA-3 were completely negative, in contrast to non-regressing foci which were strongly positive. The high level of expression of two adhesion molecules (VLA-2 and VLA-3) involved in cell-substratum as well as cell-cell interactions may account for the more indolent pattern of growth characteristic of BCC and perhaps reflect its high degree of differentiation towards the hair follicle.     

Endometrial vascular and glandular expression of integrin alpha(v)beta3 in women with and without endometriosis

The integrin alpha(v)beta3 functions in both cell-cell and cell- extracellular matrix adhesion, and has reported roles in platelet aggregation, immune function, tissue repair, tumour invasion, angiogenesis and uterine receptivity. The aim of this study was to use immunohistochemistry to describe the vascular and glandular expression of integrin alpha(v)beta3 in formalin fixed, paraffin embedded endometrium obtained from women with (n = 29) and without (n = 24) endometriosis. The results showed a significant increase in the percentage of vessels expressing alpha(v)beta3 in the endometrium of women with endometriosis compared with controls (P = 0.0001). This difference was more pronounced in the secretory phase (P = 0.001) than the proliferative phase (P = 0.016). There was no correlation between vascular alpha(v)beta3 expression and the endothelial cell proliferation index (P > 0.05). Vascular sprouts were not observed in any of the 53 endometrial tissues obtained from women with or without endometriosis throughout the menstrual cycle. Results from semi- quantitative scoring of gland immunostaining showed that neither controls (P = 0.3329) nor the endometriosis group (P = 0.2260) had any significant changes in terms of alpha(v)beta3 expression between the different stages of the menstrual cycle. There was also no difference in glandular alpha(v)beta3 expression between women with and without endometriosis (P = 0.4302). These results provide evidence for increased endometrial angiogenesis in women with endometriosis compared with controls, and suggest that glandular expression of alpha(v)beta3 is not related to uterine receptivity per se.      

Regulation of MCP-3 and BRCA2 mRNA expression levels by beta(1) integrins.

The integrin cytoplasmic domain has been shown to modulate several cellular functions, including cell proliferation, adhesion, migration, and intracellular signaling. The beta(1) integrin subunits beta(1C) and beta(1A), which contain variant cytoplasmic domains, differentially affect cancer and normal cell functions. To identify target genes selectively regulated by these beta(1) cytoplasmic variants, stable cell transfectants expressing either beta(1A) or beta(1C) under the control of a doxycycline-inducible promoter were obtained using murine beta(1)-deficient GD25 cells. Screening of 1176 murine cDNAs using first-strand cDNA of mRNA isolated from either beta(1C)- or beta(1A)-expressing cells showed a striking differential expression of few genes. The differential expression of two genes, MCP-3 and BRCA2 (monocyte chemoattractant protein-3 and breast cancer susceptibility gene 2, respectively), whose products are involved, respectively, in chemotaxis and embryonic proliferation, was confirmed by Northern blot analysis. Increased MCP-3 and decreased BRCA2 mRNA levels in cells expressing beta(1C) compared to those in cells expressing beta(1A) were observed. Since beta(1C) and beta(1A) stable cell transfectants showed comparable adhesion to fibronectin, upregulation of MCP-3 and downregulation of BRCA2 mRNA levels did not appear to be due to a differential ability of the beta(1C) cells to adhere to the beta(1) ligand fibronectin. Overall, our data show that beta(1) integrin cytoplasmic domain variants control expression of downstream target genes in a differential manner without affecting cell adhesion.     

The beta2, alpha4, alpha5 integrins and selectins mediate chemotactic factor and endotoxin-enhanced neutrophil sequestration in the lung

Intravascular chemotactic factor activation of neutrophils (polymorphonuclear leukocytes; PMNLs), associated with actin polymerization resulting in PMNL stiffening, induces rapid and transient sequestration in the pulmonary vasculature and lung dysfunction. Recent studies have proposed that this sequestration is mediated by physical lodging of PMNLs because of loss of deformability. To examine the contribution of cell adhesion molecules in this process, we used blocking monoclonal antibodies (mAbs) to rat selectins and integrins in a model of PMNL margination (reflected by acute blood neutropenia) induced by N-formyl-met-leu-phe (FMLP) chemotactic factor infusion in normal or lipopolysaccharide (LPS)-primed rats. Blood PMNL levels dropped by 70% within 1 minute and for the duration of FMLP infusion (20 minutes) in normal or by 90% in LPS-primed rats. Pretreatment with mAbs to beta2(WT.3), VLA-4(TA-2 F(ab)(2)), and VLA-5 (HMalpha5 F(ab)(2)) in combination inhibited the decrease by 50% and to a greater degree than beta2 blockade alone (35% inhibition). F(ab)(2) mAbs to L-(HRL-3), P-(RMP-1), plus E-(RME-1) selectins had no effect but they potentiated inhibition by anti-beta2 + anti-VLA-4 + anti-VLA5 mAb treatment (69% inhibition, P < 0.05). Similar results were observed in the first 6 minutes in LPS-primed rats with complete inhibition of sequestration thereafter by combined selectin and integrin blockade. These results indicate that besides PMNL stiffening because of actin polymerization, both selectins and integrins substantially contribute to activated PMNL sequestration in the lung.