Acute lung injury (ALI), which is associated with a mortality of 30-40%, is attributable to inflammation that develops rapidly across the lung's vast vascular surface, involving an entire lung or even both lungs. No specific mechanism explains this extensive inflammatory spread, probably because of the lack of approaches for detecting signal conduction in lung capillaries. Here, we addressed this question by applying the photolytic uncaging approach to induce focal increases in Ca 2+ levels in targeted endothelial cells of alveolar capillaries. Uncaging caused Ca 2+ levels to increase not only in the targeted cell, but also in vascular locations up to 150 mm from the target site, indicating that Ca 2+ was conducted from the capillary to adjacent vessels. No such conduction was evident in mouse lungs lacking endothelial connexin 43 (Cx43), or in rat lungs in which we pretreated vessels with peptide inhibitors of Cx43. These findings provide the first direct evidence to our knowledge that interendothelial Ca 2+ conduction occurs in the lung capillary bed and that Cx43-containing gap junctions mediate the conduction. A proinflammatory effect was evident in that induction of increases in Ca 2+ levels in the capillary activated expression of the leukocyte adherence receptor P-selectin in venules. Further, peptide inhibitors of Cx43 completely blocked thrombin-induced microvascular permeability increases. Together, our findings reveal a novel role for Cx43-mediated gap junctions, namely as conduits for the spread of proinflammatory signals in the lung capillary bed. Gap junctional mechanisms require further consideration in the understanding of ALI.

Shedding of the extracellular domain of cytokine receptors allows the diffusion of soluble receptors into the extracellular space; these then bind and neutralize their cytokine ligands, thus dampening inflammatory responses. The molecular mechanisms that control this process, and the extent to which shedding regulates cytokine-induced microvascular inflammation, are not well defined. Here, we used real-time confocal microscopy of mouse lung microvascular endothelium to demonstrate that mitochondria are key regulators of this process. The proinflammatory cytokine soluble TNF-α (sTNF-α) increased mitochondrial Ca 2+ , and the purinergic receptor P 2 Y 2 prolonged the response. Concomitantly, the proinflammatory receptor TNF-α receptor-1 (TNFR1) was shed from the endothelial surface. Inhibiting the mitochondrial Ca 2+ increase blocked the shedding and augmented inflammation, as denoted by increases in endothelial expression of the leukocyte adhesion receptor E-selectin and in microvascular leukocyte recruitment. The shedding was also blocked in microvessels after knockdown of a complex III component and after mitochondria-targeted catalase overexpression. Endothelial deletion of the TNF-α converting enzyme (TACE) prevented the TNF-α receptor shedding response, which suggests that exposure of microvascular endothelium to sTNF-α induced a Ca 2+ -dependent increase of mitochondrial H 2 O 2 that caused TNFR1 shedding through TACE activation. These findings provide what we believe to be the first evidence that endothelial mitochondria regulate TNFR1 shedding and thereby determine the severity of sTNF-α-induced microvascular inflammation.

The liquid layer lining the pulmonary alveolar wall critically determines the lung's immune defense against inhaled pathogens, because it provides a liquid milieu in the air-filled alveolus for dispersal of immune cells and defensive surfactant proteins. However, mechanisms underlying formation of the liquid are unknown. We achieved visualization of the alveolar wall liquid (AWL) in situ in mouse lungs by means of optical-sectioning microscopy. Continuous liquid secretion was present in alveoli of wild-type (WT) mice under baseline conditions. This secretion was blocked by inhibitors of the cystic fibrosis transmembrane regulator (CFTR). The secretion was absent in Cftr ؊/؊ mice, and it was blocked when chloride was depleted from the perfusate of WT mice, providing the first evidence that CFTR-dependent chloride secretion causes AWL formation. Injected microparticles demonstrated flow of the AWL. The flow was blocked by CFTR inhibition and was absent in Cftr ؊/؊ mice. We conclude that CFTR-dependent liquid secretion is present in alveoli of the adult mouse. Defective alveolar secretion might impair alveolar immune defense and promote alveolar disease.Keywords: lung; mouse; CFTR; alveolar secretion; optical-sectioning microscopyThe thin liquid layer lining the alveolar wall of the lung constitutes an important component of the systemic defense against inhaled pathogens. The epithelial liquid establishes a liquid phase adjacent to the alveolar epithelium, enabling secreted phospholipids and proteins to maintain alveolar patency (1) and to establish alveolar immune defense (2). However, mechanisms underlying formation of the alveolar wall liquid (AWL) remain unidentified. In fact, the bulk of the evidence favors the view that in the adult lung, active mechanisms in the alveolar wall remove, rather than secrete, alveolar liquid (3).The difficulty is that studies of AWL have employed conventional morphologic approaches that are likely to alter physiologic liquid conditions in the alveolus (4, 5). To some extent, the liquid morphology was preserved by the application of lowtemperature electron microscopy that revealed continuity of the AWL (6). However, understanding of liquid formation and flow in the alveolar wall continues to be hampered by the lack of direct, real-time methods.Here, we address these issues through the first direct visualization of AWL by means of optical-sectioning microscopy of the mouse lung. Liquid secreted in proximal airways might drain (Received in original form September 13, 2006 and in final form January 25, 2007 ) The studies reported here were supported by NIH grants HL36024, HL64896, and HL78645 (J.B.); HL-75503 (K.P.); and HL080878 (C.E.P).Correspondence and requests for reprints should be addressed to Jahar

CD45 ؉ and collagen I-positive (Col ؉ ) fibrocytes are implicated in fibrogenesis in skin, lungs, and kidneys. Fibrocyte migration in response to liver injury was investigated using bone marrow (BM) from chimeric mice expressing luciferase (Col-Luc¡wt) or green fluorescent protein (Col-GFP¡wt) under control of the ␣1(I) collagen promoter and enhancer, respectively. Monitored by luciferase expression, recruitment of fibrocytes was detected in CCl 4 -damaged liver and in spleen. Migration of CD45 ؉ Col ؉ fibrocytes was regulated by chemokine receptors CCR2 and CCR1, as demonstrated, respectively, by 50% and 25% inhibition of fibrocyte migration in Col-Luc CCR2؊/؊ ¡wt and Col-Luc CCR1؊/؊ ¡wt mice. In addition to CCR2 and CCR1, egress of BM CD45 ؉ Col ؉ cells was regulated by transforming growth factor-␤1 (TGF-␤1) and liposaccharide in vitro and in vivo, which suggests that release of TGF-␤1 and increased intestinal permeability have important roles in fibrocyte trafficking. In the injured liver, fibrocytes gave rise to (myo)fibroblasts. In addition, a BM population of CD45 ؉ Col ؉ cells capable of differentiation into fibrocytes in culture was identified. Egress of CD45 ؉ Col ؉ cells from BM was detected in the absence of injury or stress in aged mice but not in young mice. Development of liver fibrosis was also increased in aged mice and correlated with high numbers of liver fibrocytes. In conclusion, in response to liver injury, fibrocytes migrate from BM to the liver. Their migration is regulated by CCR2 and CCR1 but is compromised with age.

One of the grant numbers in the Acknowledgments section was incorrect.The correct Acknowledgments section follows.For initial studies, gap peptides were provided by Scott Boitano, University of Arizona. This project was supported by NIH grants HL75503 to K. Parthasarathi and HL57556 and HL36024 to J. Bhattacharya.The authors regret this error.