Translocation of influenza virus by migrating neutrophils.

Influenza, a predominantly upper respiratory tract infection, replicates in the respiratory epithelia and spreads by an unknown mechanism to the regional lymph nodes. Neutrophils, which accumulate during the early stages of the infection, may be involved in this process. An in vitro model system was used to examine the effect of migrating neutrophils on the permeability of the infected epithelium and on the spread of virus. Epithelial cells (MDCK) infected with influenza virus (WSN H1N1) maintained a stable transepithelial electrical resistance (a measure of epithelial permeability) for 12 hrs. However, when neutrophils migrated across the epithelium toward the virus budding on the apical surface of the epithelium (6 hrs. after infection), the transepithelial electrical resistance fell 24% (P less than 0.001). Neutrophils adhered specifically to the virus and to hemagglutinin expressed exclusively on the apical surface of the cells and phagocytized the free virions. In response to a chemotactic gradient, the infected neutrophils were able to leave the lumenal surface of the infected epithelium, and were able to migrate across the epithelium in equal numbers and at the same rate as uninfected neutrophils. Migration across infected monolayers from the lumenal to the ablumenal surface also caused a fall in resistance (21%, P less than 0.01). Electron microscopic examination of emigrating neutrophils revealed that the leukocytes transported the influenza virions within phagocytic vacuoles and on their surface to the ablumenal side of the monolayer. The results of these studies suggest that the passage of leukocytes across influenza-infected epithelia increases the permeability of the epithelium and provides a route for viral spread.

Differences in the ability of neutrophils and monocytes to traverse epithelial occluding junctions.

This study examines the effect of epithelial permeability on 1) the passage of the chemoattractant tritiated formyl methionyl-leucyl-phenylalanine (3H-fMLP; m.w. 437) and 2) the migration of leukocytes. In addition, it also compares the kinetics of neutrophil and monocyte transepithelial migration. As we had demonstrated with neutrophils (Milks et al.: Journal of Cell Biology 96:1241, 1983), when the permeability of the epithelium decreased, the accumulation of 3H-fMLP and the emigration of monocytes also decreased. When neutrophils and monocytes traversed epithelia with similar permeability, neutrophil accumulation was at least ninefold greater than that of monocytes at 30, 60, and 90 min. During the same time intervals, the number of neutrophil invasion sites/mm epithelium exceeded the number of monocyte invasion sites by at least fivefold, with approximately twice as many neutrophils as monocytes traversing each invasion site. These studies demonstrate that epithelial permeability affects the passage of the chemoattractant and the emigration of leukocytes. In addition, the enhanced ability with which neutrophils traverse occluding junctions compared to monocytes helps to explain, at least in part, the more rapid accumulation of neutrophils at inflammatory lesions.

In vitro studies of human monocyte migration across endothelium in response to leukotriene B4 and f-Met-Leu-Phe.

Relatively little is known about monocyte emigration from the vasculature or about the factors that regulate this process. In this study, a human in vitro model of a blood vessel wall was used for examination of monocyte transendothelial migration. Umbilical vein endothelial cells were grown to confluency on amnion connective tissue, and human monocytes were stimulated to cross the monolayer in response to the chemoattractants leukotriene B4 or f-Met-Leu-Phe. The pattern and time course of monocyte migration were similar for the two chemotactic factors. In both cases, approximately 40-50% of the adherent monocytes extended single or multiple pseudopods into the apical endothelial surface. This indenting behavior was also observed in the absence of chemotactic factors. It was not affected by the medium (M199 or Gey's) or method of monocyte isolation. Neutrophils also displayed this behavior, but only about half as many neutrophils as monocytes indented the endothelial surface. The integrity of the endothelium remained intact as the monocytes traversed the monolayer. When the monocytes reached the basal surface of the endothelium, they frequently wedged themselves between the basal surface of the endothelium and its basal lamina. The monocytes then invaded the basal lamina and accumulated in the connective tissue. In response to both f-Met-Leu-Phe and leukotriene B4, monocyte migration across the endothelium began as early as 10 minutes. The average rate of accumulation in the connective tissue peaked at 30 minutes; and by 60 minutes, 25-35% of the monocytes had traversed the monolayer. Approximately two to three times as many monocytes traversed the endothelium under conditions of chemotaxis as under conditions of chemokinesis or random migration. These studies provide the basis for understanding the process of monocyte migration out of the bloodstream and lay the foundation for the study of their differentiation into macrophages in the connective tissue.