We previously reported that chronic alcohol ingestion in an experimental rat model increased TGFβ1 and that this is associated with alcohol-mediated epithelial dysfunction . Further, although there was no evidence of TGFβ1 release into the alveolar space during baseline or ‘unstressed’ conditions, there was a marked increase in the activation and release of TGFβ1 into the alveolar space in response to acute endotoxemia, further enhancing alveolar epithelial barrier disruption . These experimental findings suggested that this could be a contributing mechanism underlying the strong association between alcohol abuse and an increased risk of acute lung injury . In the present study, although we again determined that TGFβ1 is not activated and released into the extracellular fluid spontaneously, it is activated in situ in the non-septic alcoholic lung. This mechanism could explain why even in the absence of an acute stress such as sepsis there is nevertheless alveolar epithelial barrier dysfunction even in the otherwise healthy experimental animals during chronic alcohol ingestion as reflected by increased paracellular leak of radiolabeled albumin . We show here that (1) alveolar macrophages from alcohol-fed rats have increased expression of TGFβ1 and that the TGFβ1protein is membrane-bound, (2) co-culture of alcohol-primed macrophages and epithelial cells disrupted alveolar epithelial barrier function in a αvβ6- and TGFβ-dependent manner, (3) treating alveolar epithelial cells with the lysates of alveolar macrophages from alcohol-fed rats decreased their barrier function and this effect was antagonized by co-treating with an anti-TGFβ1 antibody, (4) alveolar epithelial cells from alcohol-fed rats had increased expression of the integrin chains αv and β6, and (5) treating alveolar epithelial cells with active TGFβ1 in vitro also increased the expression of these integrin chains, suggesting a forward feedback in which TGFβ1 induces the expression of the integrin that activates it.
The lung is comprised of multiple cell types including alveolar epithelial cells and macrophages in close vicinity, and cells communicate with each other, either in a paracrine manner through locally secreted cytokines/growth factors, or in a juxtacrine manner via cell-associated cytokines or growth factors. We identified membrane-bound TGFβ1 on alveolar macrophages suggesting a juxtacrine interaction with adjacent epithelial cells. Whether TGβ1 is stored in the extracellular matrix or on the surface of alveolar macrophages, it is present as a latent complex within a prodomain that shields it from binding to its receptors. The binding of the αv chain to an RGD sequence in the prodomain and exertion of force on this domain changes its conformation and activates TGFβ1, which can then bind to TGFβ receptors and initiate a wide range of intracellular signals.
Cell surface integrins regulate cell growth, migration, and survival. The αvβ6 integrin is a transmembrane glycoprotein that is mainly expressed by injured epithelium . Integrins participate in activation of growth factors and initiate intracellular signaling cascades in response to receptor binding . The integrin αvβ6 binds to the latency-associated peptide leading to activation of TGFβ1 . Studies in αvβ6 knockout mice showed a deficiency in TGFβ1 activation by the epithelium and increased inflammation in response to injury and infection. Further, transgenic mice with a targeted deletion of the β6-integrin developed exaggerated lung inflammation  that was prevented by restoring β6 expression. Interestingly, bleomycin treatment leads to lung fibrosis due to increased activation of TGFβ1 . In vivo, the αvβ6 integrin is an activator of TGFβ1, which stimulates fibroblast proliferation and collagen production and has been implicated in fibrosis .
TGFβ1 is known to regulate many biological processes. Cells produce TGFβ1 as a latent complex and the active peptide must be released from this complex in order to be activated and bind its receptors. Activation of TGFβ1 within the epithelium by the αvβ6 integrin plays a role in many diseases , including airway hyperresponsiveness in allergic asthma . Other integrins such as αvβ5 are implicated in TGFβ1 activation in myofibroblast differentiation in fibrotic lungs . In contrast to stress fibers used by fibroblasts and other contractile cells, epithelial cells exert force on latent TGFβ using actin/myosin . Nevertheless, in both cell types mechanotransducers are involved in TGFβ1 activation. In idiopathic pulmonary fibrosis, the lung epithelium plays a key role in the fibrotic response and integrin-mediated activation of TGFβ1 has been implicated as a primary driver of this pathophysiology . In fact, the activation of TGFβ1 by αvβ6 has been proposed as a potential therapeutic target for fibrotic lung diseases. In experimental model of airway fibrosis Mitchell et al. showed that chronic alcohol ingestion was associated with amplification of airway fibrosis through increase in IL-13 signalling . Interestingly, IL-13 modulates TGFβ1 signalling during airway fibrosis, and alcohol’s priming effect for increased IL-13 signalling may play a role in lung transplantation related injury. Integrin αvβ6 plays a role in acute lung injury induced by Pseudomonas aeruginosa and deletion of this integrin provides protection in experimental models of lung injury due to bleomycin or high tidal volume ventilation. Lung biopsies from patients with a diagnosis of IPF show staining for integrin αvβ6 within pneumocytes , and partial inhibition of TGFβ using integrin αvβ6 antibodies was effective in blocking murine pulmonary fibrosis without inducing an inflammatory response. Beta 6 integrin expression increased within the alveolar epithelium in radiation induced fibrosis model  and anti-αvβ6 therapy prevented fibrosis. Many αv integrins play a role in preventing inappropriate vascular growth and controlling vascular permeability, and studies in mice lacking the beta 6 subunit found a role for integrin-mediated TGFβ1 activation in pulmonary and renal fibrosis, acute lung injury, and pulmonary emphysema . These studies elucidate the important potential contributions of αvβ6-mediated activation of TGFβ1 in many diseases.
Cytokines such as TGFβ1 have multiple and often diverse functions on different cell types. Activated TGFβ1 in situ can disrupt epithelial cell function and barrier integrity causing edema. In addition, active TGFβ1 can favor proliferation of fibroblasts and this has potential to lead to fibrosis or can decrease immune function of macrophage . Thus, alcohol’s damaging effects on the lung involve multiple cell types and interactions between them. One of the mechanisms by which chronic alcohol abuse leads to oxidative stress includes activation of renin-angiotensin system in the lung. Alcohol-induced amplification of the renin-angiotensin system appears to be the major cause of the alveolar epithelial oxidant stress and TGFβ1-mediated barrier disruption . Examining cell-cell interactions in the lung such as done in the current study may discover, in future, interconnected pathophysiological targets for alcohol abuse.