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Prominent beta-5 gene expression in the cardiovascular system and in the cartilaginous primordiae of the skeleton during mouse development.

The alpha v beta (alpha(v)beta5) heterodimer has been implicated in many biological functions, including angiogenesis. We report the beta5 gene expression pattern in embryonic and foetal mouse tissues as determined by Northern blotting and in situ hybridization. During the earliest stages, beta5 mRNA is widespread in the mesoderm. During later developmental stages, it remains mostly confined to tissues of mesodermal origin, although probable inductive effects trigger shifts of beta5 gene expression from some mesenchymatous to epithelial structures. This was observed in the teeth, skin, kidneys, and gut. Of physiological importance is the beta5 labeling in the developing cardiovascular and respiratory systems and cartilages. Furthermore, early beta5 gene expression was observed within the intra- and extraembryonic sites of hematopoiesis. This suggests a major role for beta5 in the hematopoietic and angiogenic stem cells and thus in the development of the vascular system. Later, the beta5 gene was expressed in endothelial cells of the vessels developing both by angiogenesis and vasculogenesis in the lung, heart, and kidneys. Moreover, the beta5 hybridization signal was detected in developing cartilages but not in ossified or ossifying bones. beta5-Integrin is a key integrin involved in angiogenesis, vasculogenesis, hematopoiesis, and bone formation.

Animals↗

Bovine gamma delta T cell subsets express distinct patterns of chemokine responsiveness and adhesion molecules: a mechanism for tissue-specific gamma delta T cell subset accumulation.

Subsets of gammadelta T cells localize to distinct tissue sites in the absence of exogenous Ag stimulation or development of effector/memory cells. Selective lymphocyte homing from the blood into tissues is controlled by a multistep process involving vascular and lymphocyte adhesion molecules, and G protein-linked chemokine receptors. The role of these mechanisms in the tissue tropism of gammadelta T cells is still poorly understood. In this study, we demonstrate that a subset of gammadelta T cells, most of which express an antigenically distinct TCR and are characterized by coexpression of CD8, selectively accumulated in tissues that expressed high levels of the mucosal vascular addressin, mucosal addressin cell adhesion molecule 1. These cells expressed higher levels of alpha(4)beta(7) integrins than other gammadelta T cell subsets and selectively migrated to the CCR7 ligand secondary lymphoid-tissue chemokine (CCL21). Integrin activation by CCL21 selectively increased CD8(+)gammadelta T cell binding to recombinant mucosal addressin cell adhesion molecule 1. These results suggest that the tropism of circulating CD8(+)gammadelta T cells for mucosal tissues is due, at least in part, to selective developmental expression of adhesion molecules and chemokine receptors.

Animals↗

Alterations in granule matrix and cell surface of focal adhesion kinase-deficient mast cells.

Focal adhesion kinase (FAK) is a nonreceptor protein tyrosine kinase that plays an important role in many cellular processes and is tyrosine phosphorylated after FcepsilonRI aggregation in mast cells. In mice, null mutation of the fak gene results in a lethal phenotype in which the embryos fail to develop past day 8.5 of gestation. To study the role of FAK in these mast cells, 8.5-day embryos were isolated and placed in culture with IL-3 and stem cell factor (SCF). Although FAK was not required for the development of mast cells in culture, the FAK(-/-) embryo-derived mast cells had several distinct characteristics. Compared with the controls, the mast cells that lack FAK were less metachromatic and by electron microscopy had granules that appeared largely electron lucid, although their histamine content was unchanged. The FAK-deficient mast cells had a reduction in the content of chondroitin/dermatan sulfate, the major glycosaminoglycan component of the granular matrix. The FAK-deficient cells had fewer microvilli that were fused with each other, giving the cell surface a ruffled appearance. There was also a 3-fold increase in the number of cells highly expressing beta(7) integrin. However, signal transduction from the high affinity IgE receptor for the secretion of histamine was similar in the wild-type, heterozygote, and the FAK-deficient cells. The FcepsilonRI-induced tyrosine phosphorylation of paxillin, Crk-associated tyrosine kinase substrate (CAS), and mitogen-activated protein kinase proteins was independent of FAK. These results indicate that FAK plays a role in regulating the glycosaminoglycan content of the secretory granules and influences the cell surface morphology of mast cells.

Animals↗

Critical role of T cell-dependent serum antibody, but not the gut-associated lymphoid tissue, for surviving acute mucosal infection with Citrobacter rodentium, an attaching and effacing pathogen.

Citrobacter rodentium uses virulence factors similar to the enteropathogenic Escherichia coli to produce attaching and effacing lesions in the distal colon of mice. We used this infection model to determine components of adaptive immunity needed to survive infection. During acute infection, wild-type mice develop breaks across infected epithelial surfaces but resolve infection. Surprisingly, mice markedly deficient in mucosal lymphocyte populations from beta(7) integrin deficiency resolve infection, as do CD8alpha-/- or TCR-delta-/- mice. In contrast, CD4-/- or TCR-beta-/- mice develop polymicrobial sepsis and end-organ damage, and succumb during acute infection, despite epithelial damage similar to wild-type mice. B cell-deficient (MuMT-/-) or B and T cell-deficient (recombinase-activating gene 2-/-) mice develop severe pathology in colon and internal organs, and deteriorate rapidly during acute infection. Surviving mice develop robust Citrobacter-specific serum IgM responses during acute infection, whereas mice that succumb do not. However, CD4-/- mice receiving serum Igs from infected wild-type mice survive and clear the infection. Our data show that survival of apparently self-limited and luminal mucosal infections requires a systemic T cell-dependent Ab response against bacteria that enter through damaged mucosa. These findings have implications for understanding host defense against mucosal infections, including the pathogenesis of these diseases in immunocompromised populations.

Abscess↗

Cathepsin-L influences the expression of extracellular matrix in lymphoid organs and plays a role in the regulation of thymic output and of peripheral T cell number.

Nackt mice, which are deficient in cathepsin-L (CTSL), show an early impairment during positive selection in the context of class II MHC molecules and as a consequence, the percentage and absolute number of CD4(+) thymocytes are significantly decreased. In this study, we show that lymph nodes from nackt mice are hypertrophied, showing normal absolute numbers of CD4(+) T cells and marked increases in the number of CD8(+) T lymphocytes. Basal proliferative levels are increased in the CD4(+) but not in the CD8(+) population. Lymph node T cells show increases in the expression of alpha(5), alpha(6), and beta(1) integrin chains. These alterations correlate with increases in the expression of extracellular matrix (ECM) components in lymph nodes. Interestingly, laminin, fibronectin, and collagen I and IV are markedly decreased in nackt thymus which shows an augmented output of CD8(+) cells. These results demonstrate that a mutation in the Ctsl gene influences the levels of ECM components in lymphoid organs, the thymic output, and the number of T cells in the periphery. They further raise the possibility that, by regulating the level of expression of ECM components in lymphoid organs, CTSL is able to broadly affect the immune system.

Animals↗

Cutting edge: CD4+CD25+ regulatory T cells impaired for intestinal homing can prevent colitis.

Transfer of CD4(+)CD45RB(high) T cells into RAG(-/-) mice causes colitis, which can be prevented by CD4(+)CD25(+) regulatory T cells (Treg). Colitis induction by CD4(+)CD45RB(high) T cells requires beta(7) integrin-dependent intestinal localization, but the importance of beta(7) integrins for Treg function is unknown. In this study, we show that beta(7)(-/-) Treg were effective in preventing colitis. Treg expanded in vivo to the same extent as CD4(+)CD45RB(high) T cells after transfer and they did not inhibit CD4(+)CD45RB(high) T cell expansion in lymphoid tissues, although they prevented the accumulation of Th1 effector cells in the intestine. beta(7)(-/-) Treg were significantly reduced in the large intestine, however, compared with wild-type Treg, and regulatory activity could not be recovered from the intestine of recipients of beta(7)(-/-) Treg. These data demonstrate that Treg can prevent colitis by inhibiting the accumulation of tissue-seeking effector cells and that Treg accumulation in the intestine is dispensable for colitis suppression.

Adoptive Transfer↗

Enhanced FTY720-mediated lymphocyte homing requires G alpha i signaling and depends on beta 2 and beta 7 integrin.

The immunomodulatory drug FTY720 interferes with sphingosine-1-phosphate (S1P) receptor signaling leading to lymphocyte retention in secondary lymphoid organs and consequently to profound lymphopenia in the peripheral blood. The molecular mechanisms transduced by S1P receptors upon being triggered by its native ligand, S1P, or by FTY720, are largely unknown. In this study we analyze the role of beta2 and beta7 integrin and their ligands ICAM-1, VCAM-1, and MadCAM-1 on lymphocyte homing in the presence of FTY720. We demonstrate that this drug facilitates homing of lymphocytes single-deficient of either beta2 or beta7 integrin but not of beta2-deficient lymphocytes, which in addition were blocked by anti-beta7 integrin Abs. Enhanced lymphocyte homing is preceded by increased adherence of integrin-deficient as well as wild-type lymphocytes to high endothelial venules (HEV) in FTY720-treated animals. Elevated adherence to HEV requires intact lymphocyte Galphai signaling that cannot be stably imprinted on lymphocytes even after prolonged exposure to FTY720. Thus, FTY720 influences lymphocyte homeostasis not only by suppressing lymphocyte egress from lymph nodes but also by facilitating lymphocyte homing across HEV in an integrin-dependent fashion.

Adjuvants, Immunologic↗

Identification of a TLR-independent pathway for Borrelia burgdorferi-induced expression of matrix metalloproteinases and inflammatory mediators through binding to integrin alpha 3 beta 1.

Borrelia burgdorferi stimulates a robust inflammatory response at sites of localization. Binding of borrelial lipoproteins to TLR-2 is one pathway important in the host response to B. burgdorferi. However, while TLR-2 is clearly important in control of infection, inflammation is actually worsened in the absence of TLR-2 or the shared TLR adapter molecule, MyD88, suggesting that there are alternative pathways regulating inflammation. Integrins are cell surface receptors that play an important role in cell to cell communications and that can activate inflammatory signaling pathways. In this study, we report for the first time that B. burgdorferi binds to integrin alpha(3)beta(1) and that binding of B. burgdorferi to this integrin results in induction of proinflammatory cytokines, chemokines, and end-effector molecules such as matrix metalloproteinases in primary human chondrocyte cells. Expression of these same molecules is not affected by the absence of MyD88 in murine articular cartilage, suggesting that the two pathways act independently in activating host inflammatory responses to B. burgdorferi. B. burgdorferi-induced alpha(3) signaling is mediated by JNK, but not p38 MAPK. In summary, we have identified a new host receptor for B. burgdorferi, integrin alpha(3)beta(1); binding of B. burgdorferi to integrin alpha(3)beta(1) results in the release of inflammatory mediators and is proposed as a TLR-independent pathway for activation of the innate immune response by the organism.

Adaptor Proteins, Signal Transducing↗

Addressing the role of cell adhesion in tumor cell dormancy.

The dissemination of tumor cells prior to the surgical resection of early stage tumors poses a serious risk to the disease free survival of cancer patients. This risk arises from the latent capacity of these cells to form solid metastatic lesions after a prolonged period of dormancy, exacerbated by the fact that these cells are often refractory to adjuvant chemotherapeutic protocols. Ensuring the long term survival of cancer patients therefore necessitates an understanding of the mechanisms of tumor cell dormancy and the accompanying drug resistance. Experiments designed to compare the biological behavior of metastatic versus nonmetastatic variants of tumor cells provide evidence that there exists a phenomenon of single-cell dormancy which may depend on a reciprocal dialogue between the tumor cell and the tissue microenvironment. Through a combination of 3-dimensional cell culture technique and in vivo models investigators are now beginning to elucidate the molecular mechanisms underlying this phenomenon. Here we review the results of a series of experiments describing the role of cell adhesion events in dictating tumor cell behavior, including the balance between proliferation and dormancy, and the acquisition of drug resistance.

Animals↗

Expression of T-lineage early developmental markers by cells establishing atopic dermatitis skin infiltrates.

The association between the atopic dermatitis, eczema and T-cell immunodeficiency disorders are well known, thus suggesting that bone marrow T-precursors could use the micro-environment of the skin as an extrathymic site for compensatory ontogenesis. In keeping with this hypothesis, we analyzed the atopic dermatitis skin lymphocytic infiltrate phenotypes to establish their ontogenetic stage of development. Cryostatic sections (4 microns) obtained from acute lesional skin biopsies of six patients with extrinsic atopic dermatitis were processed with indirect immunoperoxidase, using a panel of first-step monoclonal antibodies (mAb) specific to CD104 (integrin beta 4 chain), CD90w (Thy 1 antigen), CD44 (phagocytic glycoprotein-1; Pgp-1), CD1a and the DNA polymerase terminal deoxynucleotidyl transferase (TdT). Within the lymphocytic dermal infiltrate different levels of immunoreactivity were observed with respect to CD104, CD90w and CD1a. A strong, spread staining was also detected for mAb specific to Pgp-1 and TdT. Together, the reported features indicate that the atopic dermatitis skin-homing lymphocytes express immunophenotypes which are distinctive of the early T-ontogeny.

Adult↗

[Correlation of cell apoptosis induction with expression of human beta5 integrin on hematopoietic cells].

OBJECTIVE: To investigate the function of alpha(v)beta5 integrin in hematopoietic cells. METHODS: Beta5 integrin cDNA was expressed in K562 cells through a retroviral vector system. The changes of the alpha(v)beta5 and alpha(v)beta3 integrins expression in apoptosis and differentiation induced by serum depletion were observed. RESULT: The beta5 integrin cDNA failed to express in K562 cells after the introduction of the beta5 integrin retrovirus vector pG beta5CHT. Imbalanced expression of alpha(v)beta3 and alpha(v)beta5 integrins occurred during apoptosis and differentiation induced by serum depletion. Treatment of hematopoietic cells with anti-alpha(v)beta5 monoclonal antibody inhibited the cells apoptosis. CONCLUSION: Overexpression of the alpha(v)beta5 integrin cDNA in hematopoietic cells was associated with the inhibition of cell proliferation and apoptosis.

Apoptosis↗

Digital image analysis system for the quantification of infiltrates and cell adhesion molecules in inflammatory cardiomyopathy.

BACKGROUND: We attempted to develop a digital image analysis (DIA) system for endomyocardial biopsies (EMBs) to reliably quantify a) biopsy quality, b) immunohistochemically-marked infiltrates, and c) cell adhesion molecules (CAMs) in relation to net heart area (HA) for the semi-automated diagnosis of inflammatory cardiomyopathy (InfCM). MATERIAL/METHODS: 140 EMBs from dilated cardiomyopathy (DCM) patients and 14 autopsy heart samples (controls) were immunostained for T-lymphocytes (CD2, CD3, CD4, CD8), beta(2)-integrin+ infiltrates (CD18, LFA-1, Mac-1) and CAMs (immunoglobulin superfamily: ICAM-1, HLA class I, HLA DR, VCAM-1, CD58; selectins: CD62E and CD62P; and the beta(1)-integrin chain CD29). EMB quality was assessed visually on a three-point scale. Infiltrates were quantified visually (per hpf) and by DIA (per mm2 HA). CAM expression was evaluated semiquantitatively and by DIA (area fraction [AF]: stained area relative to HA). RESULTS: DIA-evaluated HA correlated significantly with the visual assessment of EMB quality. The visual evaluation of both infiltrates and CAMs correlated significantly with the respective DIA-based quantification. DIA-quantified CAM-AF and infiltrates were discriminated by the CAM classification (CAMs+: n=87; 62%) compared to controls. DIA-quantified CAM immunoreactivity correlated significantly with the DIA-quantified counter-receptor+ infiltrates. DIA evaluation of biopsy quality, infiltrates, and CAMs was devoid of inter- and intraobserver variability. CONCLUSIONS: The DIA system presented here enables standardized and observer-independent assessment of EMB quality and intramyocardial inflammation (density of infiltrates and CAM expression) in DCM biopsies related to HA. Our data confirm that endothelial CAM count and counter-receptor+ immunocompetent infiltration are interdependent pathogenic and diagnostic hallmarks of InfCM.

Adult↗

[Molecular mechanism of the sensitivity of myocardial cell injury after severe hypoxia].

OBJECTIVE: To explore the molecular mechanism of the sensitivity of myocardial cell injury after severe hypoxia. METHODS: Differential expression of the genes in rat myocardium and skeletal muscles after severe hypoxia for 6 hours was determined by DNA chip containing 4096 mice genes. RESULTS: There were high expressions of 125 genes in skeletal muscles and 111 genes in myocardial cells after 6 hours of severe hypoxia. Among them, high expression genes in myocardial cells included those encoding apoptosis activator Mtd, complement C3, vascular cell adhesion molecule-1, integrin beta subunit and lipopolysaccharide binding proteins (LBP). CONCLUSION: Activation of apoptosis of myocardial cells and inflammatory mediators played important roles in myocardial cell injury after 6-hour severe hypoxia.

Animals↗

Interactions of a laminin-binding peptide from a 33-kDa protein related to the 67-kDa laminin receptor with laminin and melanoma cells are heparin-dependent.

A laminin-binding peptide (peptide G), predicted from the cDNA sequence for a 33-kDa protein related to the 67-kDa laminin receptor, specifically inhibits binding of laminin to heparin and sulfatide. Since the peptide binds directly to heparin and inhibits interaction of another heparin-binding protein with the same sulfated ligands, this inhibition is due to direct competition for binding to sulfated glycoconjugates rather than an indirect effect of interaction with the binding site on laminin for the 67-kDa receptor. Direct binding of laminin to the peptide is also inhibited by heparin. This interaction may result from contamination of the laminin with heparan sulfate, as binding is enhanced by the addition of substoichiometric amounts of heparin but inhibited by excess heparin and two heparin-binding proteins. Furthermore, laminin binds more avidly to a heparin-binding peptide derived from thrombospondin than to the putative receptor peptide. Adhesion of A2058 melanoma cells on immobilized peptide G is also heparin-dependent, whereas adhesion of the cells on laminin is not. Antibodies to the beta 1-integrin chain or laminin block adhesion of the melanoma cells to laminin but not to peptide G. Thus, the reported inhibition of melanoma cell adhesion to endothelial cells by peptide G may result from inhibition of binding of laminin or other proteins to sulfated glycoconjugate receptors rather than from specific inhibition of laminin binding to the 67-kDa receptor.

Amino Acid Sequence↗

[Selection and identification of human keratinocyte stem cells in vitro].

OBJECTIVE: To investigate the selection and identification of human keratinocyte stem cells(KSC) in vitro. METHODS: According to the characteristics of KSC which can adhere to extracellular matrix very fast, we selected 3 groups of different time(5 minutes, 20 minutes and 60 minutes) and unselected as control group. And the cells were identified by monoclone antibody of beta 1-integrin and cytokeratin 19 (Ck19), then the image analysis was done. Furthermore we analyzed the cultured cells with flow cytometer(FCM) and observed the ultrastructure of the cell by transmission electron microscope(TEM). RESULTS: The cell clones formed in all groups after 10 to 14 days, while the cells of 5 minute group grew more slowly than those of the other groups, however, the clones of this group were bigger. The expression of beta 1-integrin and Ck19 were found in all groups. The positive rate of beta 1-integrin was significant difference between 5 minute group and the other groups (P < 0.05). And the expression of Ck19 was no significant difference between 5 minute group and 20 minute group(P > 0.05), and between 60 minute group and control group. But significant difference was observed between the former and the later groups(P < 0.05). The result of FCM showed that most cells of the 5 minute group lied in G1 period of cell cycle, which was different from those of the other groups. At the same time, the cells of 5 minute group were smaller and contained fewer organelles than those of the other groups. CONCLUSION: The above results demonstrate that the cells of 5 minute group have a slow cell cycle, characteristics of immaturity, and behaving like clonogenic cells in vitro. The cells have the general anticipated properties for KSC. So the KSC can be selected by rapid attachment to extracellular matrix and identified by monoclone antibody of beta 1-integrin and Ck19.

Antibodies, Monoclonal↗

IGF-1 modulation of rat cardiac fibroblast behavior and gene expression is age-dependent.

The collagenous extracellular matrix (ECM) forms a stress-tolerant network that is essential for proper function of the vertebrate heart. Profound changes have been detected in the interstitial ECM concurrent with developmental and disease processes of the heart. These alterations in either the organization or accumulation of ECM components markedly affect myocardial function. Studies have shown that a number of biochemical factors, including angiotensin II, transforming growth factor-beta, and insulin-like growth factors, modulate collagen expression by heart fibroblasts, however, few studies have examined the differential effects of these factors on fibroblasts from animals of different physiological backgrounds. The present studies were carried out to determine whether cardiac fibroblasts isolated from different aged animals (fetal, neonatal, and adult) have diverse responses to insulin-like growth factor-1 (IGF-1). Fibroblasts isolated from fetal, neonatal, and adult rat hearts were treated with IGF-1, and several downstream responses were measured, including collagen gel contraction, adhesion to ECM, and expression of interstitial collagen and integrins. IGF-1 affected these parameters to different degrees, depending on the age of the animal from which the fibroblasts were isolated. These experiments indicate that IGF-1 is a potent modulator of fibroblast behavior in general; however, significant differences are apparent in the responsiveness of cells to this growth factor depending on the age of the animal of origin. Future experiments will be directed at determining how the in vivo chemical and biomechanical environment affects the response of heart fibroblasts to growth factors such as IGF-1.

Aging↗

[Immunohistochemical expression of integrin alphav, beta5 and beta3 in ovarian epithelial carcinoma: correlation with drug resistance of chemotherapy].

OBJECTIVE: To investigate the possible correlation between the expression of integrin alphav, beta5 and beta3 in tumor tissues and the response to chemotherapy and survival of patients with ovarian epithelial carcinoma. METHODS: Seventy-seven patients with ovarian epithelial carcinoma from January 1996 to December 2002 were entered into the study. All subjects were matched up with the inclusion criteria and followed up till December 2003. Each patient received cytoreductive surgery and systematic chemotherapy. Patients were divided into drug resistant group (n = 26) and drug sensitive group (n = 51). The antigens of integrin alphav, beta5 and beta3 in ovarian tumour tissues were tested by immunohistochemistry. Logistic regression was employed to analyze the correlation between drug resistance and factors including patients' age, clinical stage, pathological degree and type of tumors, chemotherapy methods, operative results and the expression of integrin alphav, beta5 and beta3 in tumors. The disease prognosis was analyzed by multivariate COX regression. RESULTS: In the drug resistance group, the expressions of integrin alphav and beta5 were significantly higher than that in the drug sensitive group (P = 0.004 and 0.001). The expression of integrin beta3 was not statistically different in the two groups (P = 0.668). According to multivariate analysis, the expression of integrin alphav and beta5 and clinical stage were three independent factors correlated with drug resistance and disease prognosis for the patients with ovarian epithelial carcinoma (P = 0.014, 0.030, 0.042 for drug resistance; and P = 0.045, 0.031, 0.001 for prognosis respectively). CONCLUSION: The high expressions of integrin alphav and beta5 in ovarian epithelial carcinoma are risk factors for drug resistance and poor disease prognosis.

Adenocarcinoma↗

Expression and signaling activity of Wnt-5a/discoidin domain receptor-1 and Syk plays distinct but decisive roles in breast cancer patient survival.

PURPOSE: The loss of Wnt-5a, a G-protein-coupled receptor ligand, or Syk, an intracellular kinase, has in separate studies been associated with poor prognosis of breast cancer patients. Both proteins are involved in cell adhesion, a key event in epithelial cancer metastasis. Here, we have investigated whether Syk is part of the Wnt-5a/discoidin domain receptor-1 (DDR1) signaling pathway and if a signaling interaction of these proteins is important for breast cancer-specific survival. EXPERIMENTAL DESIGN: The signaling interactions between Wnt-5a/DDR1 and Syk were addressed in mammary cell lines. Their mRNA and protein levels and the respective clinical correlates were investigated in 94 cases of primary breast cancer. RESULTS: The expression of Wnt-5a and Syk correlated in four of five tumor cell lines. However, despite a constitutive association between Syk and the Wnt-5a-dependent adhesion receptor DDR1, we found no evidence of a Wnt-5a/DDR1-mediated activation of Syk. Instead, beta(1) integrins initiate the adhesion-induced activation of Syk. In tumors from breast cancer patients, the protein expression of Wnt-5a and Syk were differently regulated at the translational and transcriptional level, respectively. Analysis of breast cancer-specific survival revealed that the presence of Wnt-5a and Syk in primary tumors has good predictive value for a favorable outcome. Intriguingly, a simultaneous loss of both proteins did not reduce survival more than loss of either. CONCLUSIONS: Despite the difference in regulation of Wnt-5a and Syk protein expression and their lack of signaling interaction, our clinical data indicate that a favorable prognosis in breast cancer requires the expression and signaling activity of both.

Breast Neoplasms↗