Gerald S. Horan

Interleukin-1β Causes Acute Lung Injury via αvβ5 and αvβ6 Integrin–Dependent Mechanisms

Interleukin (IL)-1&bgr; has previously been shown to be among the most biologically active cytokines in the lungs of patients with acute lung injury (ALI). Furthermore, there is experimental evidence that lung vascular permeability increases after short-term exposure to IL-1 protein, although the exact mechanism is unknown. Therefore, the objective of this study was to determine the mechanisms of IL-1&bgr;–mediated increase in lung vascular permeability and pulmonary edema following transient overexpression of this cytokine in the lungs by adenoviral gene transfer. Lung vascular permeability increased with intrapulmonary IL-1&bgr; production with a maximal effect 7 days after instillation of the adenovirus. Furthermore, inhibition of the &agr;v&bgr;6 integrin and/or transforming growth factor-&bgr; attenuated the IL-1&bgr;–induced ALI. The results of in vitro studies indicated that IL-1&bgr; caused the activation of transforming growth factor-&bgr; via RhoA/&agr;v&bgr;6 integrin–dependent mechanisms and the inhibition of the &agr;v&bgr;6 integrin and/or transforming growth factor-&bgr; signaling completely blocked the IL-1&bgr;–mediated protein permeability across alveolar epithelial cell monolayers. In addition, IL-1&bgr; increased protein permeability across lung endothelial cell monolayers via RhoA- and &agr;v&bgr;5 integrin–dependent mechanisms. The final series of in vivo experiments demonstrated that pretreatment with blocking antibodies to both the &agr;v&bgr;5 and &agr;v&bgr;6 integrins had an additive protective effect against IL-1&bgr;–induced ALI. In summary, these results demonstrate a critical role for the &agr;v&bgr;5/&bgr;6 integrins in mediating the IL-1&bgr;–induced ALI and indicate that these integrins could be a potentially attractive therapeutic target in ALI.

Mice that lack activity of αvβ6- and αvβ8-integrins reproduce the abnormalities of Tgfb1- and Tgfb3-null mice

The arginine-glycine-aspartate (RGD)-binding integrins αvβ6 and αvβ8 activate latent TGFβ1 and TGFβ3 in vivo, but it is uncertain whether other RGD-binding integrins such as integrins αvβ5 and αvβ3 activate these TGFβ isoforms. To define the combined role of αvβ6- and αvβ8-integrin in TGFβ activation, we analyzed mice lacking function of both integrins by means of gene deletion and/or pharmacologic inhibition. Most Itgb6–/–;Itgb8–/– embryos die at mid-gestation; those that survive develop cleft palate–as observed in Tgfb3–/– mice. Itgb8–/– mice treated with an anti-αvβ6-integrin antibody develop severe autoimmunity and lack Langerhans cells–similar to Tgfb1-null mice. These results support a model in which TGFβ3-mediated palate fusion and TGFβ1-mediated suppression of autoimmunity and generation of Langerhans cells require integrins αvβ6 and αvβ8 but not other RGD-binding integrins as TGFβ activators.

Antibody-mediated blockade of integrin ?v?6 inhibits tumor progression in vivo by a transforming growth factor-?–regulated mechanism

The alpha(v)beta(6) integrin is up-regulated on epithelial malignancies and has been implicated in various aspects of cancer progression. Immunohistochemical analysis of alpha(v)beta(6) expression in 10 human tumor types showed increased expression relative to normal tissues. Squamous carcinomas of the cervix, skin, esophagus, and head and neck exhibited the highest frequency of expression, with positive immunostaining in 92% (n = 46), 84% (n = 49), 68% (n = 56), and 64% (n = 100) of cases, respectively. We studied the role of alpha(v)beta(6) in Detroit 562 human pharyngeal carcinoma cells in vitro and in vivo. Prominent alpha(v)beta(6) expression was detected on tumor xenografts at the tumor-stroma interface resembling the expression on human head and neck carcinomas. Nonetheless, coculturing cells in vitro with matrix proteins did not up-regulate alpha(v)beta(6) expression. Detroit 562 cells showed alpha(v)beta(6)-dependent adhesion and activation of transforming growth factor-beta (TGF-beta) that was inhibited >90% with an alpha(v)beta(6) blocking antibody, 6.3G9. Although both recombinant soluble TGF-beta receptor type-II (rsTGF-beta RII-Fc) and 6.3G9 inhibited TGF-beta-mediated Smad2/3 phosphorylation in vitro, there was no effect on proliferation. Conversely, in vivo, 6.3G9 and rsTGF-beta RII-Fc inhibited xenograft tumor growth by 50% (n = 10, P < 0.05) and >90% (n = 10, P < 0.001), respectively, suggesting a role for the microenvironment in this response. However, stromal collagen and smooth muscle actin content in xenograft sections were unchanged with treatments. Although further studies are required to consolidate in vitro and in vivo results and define the mechanisms of tumor inhibition by alpha(v)beta(6) antibodies, our findings support a role for alpha(v)beta(6) in human cancer and underscore the therapeutic potential of function blocking alpha(v)beta(6) antibodies.

Role of Integrin αvβ6 in Acute Lung Injury Induced by Pseudomonas aeruginosa

ABSTRACT Deletion of integrin αvβ6 has been associated with significant protection in experiments where lung injury was induced by bleomycin, lipophilic polysaccharides, and high tidal volume ventilation. This has led to the suggestion that antibody blockade of this integrin is a novel therapy for acute lung injury. We questioned whether β6 gene deletion would also protect against Pseudomonas aeruginosa-induced acute lung injury. Wild-type and littermate β6-null mice, as well as recombinant soluble TGF-β receptor type II-Fc (rsTGF-βRII-Fc) and anti-αvβ6 treated wild-type mice, were instilled with live P. aeruginosa. Four or 8 h after bacterial instillation, the mice were euthanized, and either bronchoalveolar lavage fluid or lung homogenates were obtained. Deletion of the β6 gene resulted in an overall increase in inflammatory cells in the lungs. Bacterial numbers from the lung homogenates of infected β6-null mice were significantly decreased compared to the numbers in the wild-type mice (1.6 × 106 CFU versus 4.2 × 106 CFU [P < 0.01]). There were no significant differences in P. aeruginosa-mediated increases in lung endothelial permeability between wild-type and β6-null mice. Similarly, pretreatment with the αvβ6 antibody or with rsTGF-βRII-Fc did not significantly affect the P. aeruginosa-induced lung injury or rate of survival compared to pretreatment with control immunoglobulin G. We conclude that deletion or inhibition of the integrin αvβ6 did not protect animals from P. aeruginosa-induced lung injury. However, these therapies also did not increase the lung injury, suggesting that host defense was not compromised by this promising new therapy.