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The airway is a primary portal of entry for noxious environmental stimuli that can trigger airway remodeling, which contributes significantly to airway obstruction in chronic obstructive pulmonary disease (COPD) and chronic asthma. Important pathologic components of airway remodeling include fibrosis and abnormal innate and adaptive immune responses. The positioning of fibroblasts in interstitial spaces suggests that they could participate in both fibrosis and chemokine regulation of the trafficking of immune cells such as dendritic cells, which are crucial antigen-presenting cells. However, physiological evidence for this dual role for fibroblasts is lacking. Here, in two physiologically relevant models - conditional deletion in mouse fibroblasts of the TGF-β-activating integrin αvβ8 and neutralization of αvβ8 in human COPD fibroblasts - we have elucidated a mechanism whereby lung fibroblast chemokine secretion directs dendritic cell trafficking, in a manner that is critically dependent on αvβ8-mediated activation of TGF-β by fibroblasts. Our data therefore indicate that fibroblasts have a crucial role in regulating both fibrotic and immune responses in the lung.

Brain arteriovenous malformations (BAVMs) are a rare but potentially devastating hemorrhagic disease. Transforming growth factor-beta signaling is required for proper vessel development, and defective transforming growth factor-beta superfamily signaling has been implicated in BAVM pathogenesis. We hypothesized that expression of the transforming growth factor-beta activating integrin, alphavbeta8, is reduced in BAVMs and that decreased beta8 expression leads to defective neoangiogenesis. We determined that beta8 protein expression in perivascular astrocytes was reduced in human BAVM lesional tissue compared with controls and that the angiogenic response to focal vascular endothelial growth factor stimulation in adult mouse brains with local Cre-mediated deletion of itgb8 and smad4 led to vascular dysplasia in newly formed blood vessels. In addition, common genetic variants in ITGB8 were associated with BAVM susceptibility, and ITGB8 genotypes associated with increased risk of BAVMs correlated with decreased beta8 immunostaining in BAVM tissue. These three lines of evidence from human studies and a mouse model suggest that reduced expression of integrin beta8 may be involved in the pathogenesis of sporadic BAVMs.

Trophic interactions between pulmonary epithelial and mesenchymal cell types, known as the epithelial-mesenchymal trophic unit (EMTU), are crucial in lung development and lung disease. Transforming growth factor (TGF)-beta is a key factor in mediating these interactions, but it is expressed in a latent form that requires activation to be functional. Using intact fetal tracheal tissue and primary cultures of fetal tracheal epithelial cells and fibroblasts, we demonstrate that a subset of integrins, alpha(v)beta(6) and alpha(v)beta(8), are responsible for almost all of the TGF-beta activation in the EMTU. Both alpha(v)beta(8) and alpha(v)beta(6) contribute to fetal tracheal epithelial activation of TGF-beta, whereas only alpha(v)beta(8) contributes to fetal tracheal fibroblast activation of TGF-beta. Interestingly, fetal tracheal epithelial alpha(v)beta(8)-mediated TGF-beta activation can be enhanced by phorbol esters, likely because of the increased activity of MT1-MMP, an essential co-factor in alpha(v)beta(8)-mediated activation of TGF-beta. Autocrine alpha(v)beta(8)-mediated TGF-beta activation by fetal tracheal fibroblasts results in suppression of both transcription and secretion of hepatocyte growth factor, which is sufficient to affect phosphorylation of the airway epithelial hepatocyte growth factor receptor, c-Met, as well as airway epithelial proliferation in a co-culture model of the EMTU. These findings elucidate the function and complex regulation of integrin-mediated activation of TGF-beta within the EMTU.

Brain hemorrhage is a severe complication of both neoplastic and nonneoplastic brain disease. Mice deficient in the alpha(v)beta8 integrin display defective brain vessel formation resulting in hemorrhage and perinatal death, but the mechanism of brain hemorrhage is unknown. Because the alpha(v)beta8 integrin is expressed by astrocytes and not expressed by endothelium, paracrine interactions between astrocytes and endothelial cells could contribute to the maintenance of brain vessel integrity. We have investigated the mechanisms underlying astrocytic-endothelial paracrine signaling and have found that integrin-mediated activation of transforming growth factor (TGF)-beta by astrocytes influences endothelial cell function. Thus, we identified the integrin alpha(v)beta8 in human perivascular glial cell processes surrounding developing blood vessels. Human astrocytic alpha(v)beta8 was a major cell surface receptor for latent TGF-beta, and alpha(v)beta8-dependent activation of TGF-beta was the major mechanism of TGF-beta activation in primary cultures of astrocytes or freshly dissociated fetal brain cells. This activation of TGF-beta was sufficient to inhibit endothelial migration in fibrin gels and to alter expression of genes affecting proteolytic and angiogenic pathways. Taken together, our data suggest that astrocytic alpha(v)beta8 acts as a central regulator of brain vessel homeostasis through regulation of TGF-beta activation and expression of TGF-beta-responsive genes that promote vessel differentiation and stabilization, most notably plasminogen activator inhibitor-1 and thrombospondin-1.

Field isolates of foot-and-mouth disease virus (FMDV) have been shown to use three alphav integrins, alphavbeta1, alphavbeta3, and alphavbeta6, as cellular receptors. Binding to the integrin is mediated by a highly conserved RGD motif located on a surface-exposed loop of VP1. The RGD tripeptide is recognized by several other members of the integrin family, which therefore have the potential to act as receptors for FMDV. Here we show that SW480 cells are made susceptible to FMDV following transfection with human beta8 cDNA and expression of alphavbeta8 at the cell surface. The involvement of alphavbeta8 in infection was confirmed by showing that virus binding and infection of the transfected cells are inhibited by RGD-containing peptides and by function-blocking monoclonal antibodies specific for either the alphavbeta8 heterodimer or the alphav chain. Similar results were obtained with a chimeric alphavbeta8 including the beta6 cytodomain (alphavbeta8/6), showing that the beta6 cytodomain can substitute efficiently for the corresponding region of beta8. In contrast, virus binding to alphavbeta6 including the beta8 cytodomain (alphavbeta6/8) was lower than that of the wild-type integrin, and this binding did not lead to infection. Further, the alphavbeta6 chimera was recognized poorly by antibodies specific for the ectodomain of alphavbeta6 and displayed a relaxed sequence-binding specificity relative to that of wild-type integrin. These data suggest that the beta6 cytodomain is important for maintaining alphavbeta6 in a conformation required for productive infection by FMDV.

Transforming growth factor (TGF)-beta is a potent multifunctional cytokine that is an essential regulator of epithelial proliferation. Because TGF-beta is expressed almost entirely in a latent state in vivo, a major source of regulation of TGF-beta function is its activation. A subset of integrins, alphavbeta8 and alphavbeta6, which are expressed in the human airway, has recently been shown to activate latent TGF-beta in vitro, suggesting a regulatory role for integrins in TGF-beta function in vivo. Here we have developed a novel, biologically relevant experimental model of human airway epithelium using intact human bronchial tissue. We have used this model to determine the function of integrin-mediated activation of TGF-beta in the airway. In human bronchial fragments cultured in vitro, authentic epithelial-stromal interactions were maintained and integrin and TGF-beta expression profiles correlated with profiles found in normal lung. In addition, in this model, we found that either the integrin alphavbeta8 or TGF-beta could inhibit airway epithelial cell proliferation. Furthermore, we found that one mechanism of integrin-alphavbeta8-dependent inhibition of cell proliferation was through activation of TGF-beta because anti-beta8 antibody blocked the majority (76%) of active TGF-beta released from bronchial fragments. These data provide compelling evidence for a functional role for integrin-mediated activation of TGF-beta in control of human airway epithelial proliferation in vivo.

Integrins, matrix metalloproteases (MMPs), and the cytokine TGF-beta have each been implicated in homeostatic cell behaviors such as cell growth and matrix remodeling. TGF-beta exists mainly in a latent state, and a major point of homeostatic control is the activation of TGF-beta. Because the latent domain of TGF-beta1 possesses an integrin binding motif (RGD), integrins have the potential to sequester latent TGF-beta (SLC) to the cell surface where TGF-beta activation could be locally controlled. Here, we show that SLC binds to alpha(v)beta8, an integrin expressed by normal epithelial and neuronal cells in vivo. This binding results in the membrane type 1 (MT1)-MMP-dependent release of active TGF-beta, which leads to autocrine and paracrine effects on cell growth and matrix production. These data elucidate a novel mechanism of cellular homeostasis achieved through the coordination of the activities of members of three major gene families involved in cell-matrix interactions.

The control of cell growth is regulated through coordinated responses to growth factors and cell-extracellular matrix (ECM) interactions. Integrins, the major family of cell-ECM receptors, are vital to these coordinated responses. Although much is known of the role of integrins in growth promotion, specific examples of integrin-mediated cell growth inhibition are few. On the basis of our findings that the integrin beta8 subunit is expressed in airway epithelial cells and is absent in lung cancers, we investigated the role and mechanism of the integrin alphavbeta8 in mediating growth inhibition. When introduced into either a lung or colon carcinoma cell line, beta8 inhibited cell growth without inducing apoptosis. Ligation of alphavbeta8 also induced cell rounding, inhibited focal contact formation, and initiated an inhibitory signaling pathway as demonstrated by increased expression of the cyclin-dependent kinase inhibitor p21Cip1. The cytoplasmic domain of beta8 was capable of both growth inhibition and causing cell shape changes as shown by the use of a chimeric integrin construct consisting of the beta8-cytoplasmic domain coupled to the beta6-extracellular domain. Finally, when tested in vivo, beta8 potently inhibited tumor growth in nude mice. Together, these results implicate alphavbeta8 as a novel growth-regulatory molecule of epithelial cells.