Histologic and ultrastructural analysis of the injured lung has been integral to current concepts of pathogenesis of acute lung injury/acute respiratory distress syndrome (ALI/ARDS). (a) A low-power light micrograph of a lung biopsy specimen collected two days after the onset of ALI/ARDS secondary to gram-negative sepsis demonstrates key features of diffuse alveolar damage, including hyaline membranes, inflammation, intra-alveolar red cells and neutrophils, and thickening of the alveolar-capillary membrane. (b) A higher-power view of a different field illustrates a dense hyaline membrane and diffuse alveolar inflammation. Polymorphonuclear leukocytes are imbedded in the proteinaceous hyaline membrane structure. The blue arrow points to the edge of an adjacent alveolus, which contains myeloid leukocytes. (c) An electron micrograph from a classic analysis of ALI/ARDS showing injury to the capillary endothelium and the alveolar epithelium. Abbreviations: A, alveolar space; BM, exposed basement membrane, where the epithelium has been denuded; C, capillary; EC, erythrocyte; EN, blebbing of the capillary endothelium; LC, leukocyte (neutrophil) within the capillary lumen. The histologic sections in panels a and b are used courtesy of Dr. K. Jones, University of California, San Francisco. Reprinted with permission from the American Thoracic Society.

(a) The normal alveolus and (b) the injured alveolus in the acute phase of acute lung injury and the acute respiratory distress syndrome. In the acute phase of the syndrome (b), there is sloughing of both the bronchial and alveolar epithelial cells; protein-rich hyaline membranes form on the denuded basement membrane. Neutrophils adhere to the injured capillary endothelium and marginate through the interstitium into the air space, which is filled with protein-rich edema fluid. In the air space, alveolar macrophages secrete cytokines; interleukin (IL)-1, -6, -8, and -10; and tumor necrosis factor α(TNF-α), which act locally to stimulate chemotaxis and activate neutrophils. IL-1 can also stimulate the production of extracellular matrix by fibroblasts. Neutrophils can release oxidants, proteases, leukotrienes, and other proinflammatory molecules such as platelet-activating factor (PAF). A number of anti-inflammatory mediators also present in the alveolar milieu include IL-1 receptor antagonist, soluble TNF receptor, autoantibodies against IL-8, and cytokines such as IL-10 and -11 (not shown). The influx of protein-rich edema fluid into the alveolus leads to the inactivation of surfactant. Abbreviation: MIF, macrophage-inhibitory factor. Adapted with permission from the Massachusetts Medical Society.

Neutrophil migration across epithelia can be considered in three sequential stages: adhesion, migration, and postmigration. The initial stage of neutrophil transepithelial migration is characterized by adhesion of the neutrophils to the basolateral epithelial membrane. Adhesion is mediated by ligation of CD11b/CD18 on the neutrophil surface to several molecules on the epithelial surface, including fucosylated glycoproteins; junctional adhesion molecule C ( JAM-C); and probably other, as-yet-unidentified molecules. After initial adhesion, neutrophils crawl along the epithelial cell membrane via sequential binding to a number of epithelial cell–surface molecules. Both epithelial and neutrophil CD47 molecules are involved during this stage, and CD47 on both cell types may bind to and signal through signal regulatory protein α (SIRPα). In addition, SIRPα probably signals via pathways that are independent of CD47 during neutrophil transepithelial migration. Once neutrophils have completely traversed the epithelial monolayer, they adhere to the apical epithelial surface, where they resist fluid flow and mechanical forces and constitute a defense barrier against invading microorganisms. See Reference for more details. Adapted with permission from the American Thoracic Society.

Migration of large numbers of neutrophils results in the death of epithelial cells with the formation of circular areas of denudation at the sites where neutrophils impale the monolayer. Calu-3 lung epithelial cells were grown to confluence on the underside of inverted semipermeable membranes in Transwell inserts. (a) In the control state, the epithelial monolayer is intact. However, when freshly isolated human neutrophils are added to the upper chamber (corresponding to the basolateral surface of epithelial cells) and induced to migrate in a physiological basolateral-to-apical direction across the epithelial monolayer by the addition of a chemoattractant (formyl-methionyl-leucyl-phenylalanine) to the lower chamber (apical surface), individual “scout” neutrophils migrate across the epithelium at specific sites. This initial breach of the epithelium results in a localized high concentration of chemoattractant at these sites. Therefore, as transmigration proceeds, the trailing neutrophils migrate towards the chemotactic gradient and follow in the “tracks” of the leading cells, crossing the monolayer at these sites. (b) The epithelial monolayer after neutrophil migration. The migration of large numbers of neutrophils at specific sites in the epithelial monolayer with the formation of clusters of neutrophils on the apical surface results in disruption of the monolayer and the creation of circular areas of denudation (arrows). The images represent the top-down view of the surface of the epithelial monolayer (not the cross section); the neutrophils have migrated from above the picture plane to below the picture plane.

Localization of markers of apoptosis in lung tissue sections from patients. (Left) Lung tissue sections from a patient who died with acute lung injury (ALI) or acute respiratory distress syndrome (ARDS). (Right) Lung tissue sections from a patient who died without pulmonary disease. (a–f ) Tissue sections that are counterstained with hematoxylin. ( g–j) Tissue sections that were imaged using differential interference contrast optics, without counterstain, because the epithelial cell immunostain reaction product was subtle. The rows of pictures are matched for one marker of apoptosis: (a,b) TUNEL, (c,d ) caspase-3, (e, f ) Bax, ( g, h) Bcl-2, and (i, j) p53. Cells lining, and in, the alveolar walls demonstrate more TUNEL-labeled nuclei, caspase-3-labeled cytoplasm (arrow), Bax-labeled cytoplasm (arrow), and p53-labeled cytoplasm (arrow) in the tissue sections from the patient who died with ALI or ARDS compared with the patient who died without pulmonary disease. However, Bcl-2-labeled cells lining, and in, the alveolar walls are more prominent in the tissue sections from the patient who died without pulmonary disease compared with the patient who died with ALI or ARDS, as expected. All of the panels are of the same magnification. Reprinted with permission from the American Society of Pathology.

A histologic section of acute respiratory distress syndrome in a human lung sample. Note the fluid-filled alveolar spaces with significant red blood cell infiltration. (Inset) Apical epithelial sodium channel (ENaC) staining and basolateral Na/K-ATPase staining in lung epithelia. Reprinted with permission from the American Physiological Society.

Histology sections (two examples for each condition) in the ex vivo perfused human lung lobes exposed to endotoxin with and without treatment with either allogeneic human mesenchymal stem cells (MSCs) or their conditioned medium (CM). Human lungs exposed to endotoxin with and without MSCs or their CM were fixed in 10% formalin at 4 h. Sections were stained with hematoxylin and eosin (magnification 10×). The instillation of human allogeneic MSCs (5 × 10⁶ cells) or their CM 1 h after endotoxin injury reduced the degree of edema and cellularity in the endotoxin-injured lung lobe. Abbreviation: LPS, lipopolysaccharide. Reprinted from Reference with permission.