Neutrophils and monocytes initially accumulate in response to influenza infection. However, very little is known about how these leukocytes are attracted to sites of viral infection, how they recognize virus-infected cells, the effect they have on the course of the viral infection or in maintaining the integrity of the epithelium. To address these issues an in vitro model of influenza infection will be used. How leukocytes are attracted to virus infected epithelium (MDCK) will be determined. The adherence of human neutrophils and monocytes to infected epithelial cells (or LLC-PK1 cells transfected with the hemagglutinin gene of A/Japan/305/57(H2N2 and its inhibition with monoclonal antibodies (mab) will be used to determine: 1) the ability of these leukocytes to recognize virus-infected epithelial 2) the stage in the replication cycle when this occurs and 3) if this binding is to the viral hemagglutinin molecule expressed on the surface of infected cells. To examine if there is a relationship between leukocyte viral binding molecules and other leukocyte adhesion- promoting receptors (complement receptors CRl and CR3, and the receptor for the Fc region of IgG (FcR)), mab to these receptors will be used to inhibit neutrophil agglutination by influenza virus and to form ligand-coated surfaces which will trap these receptors on the adherent cell membrane of the leukocytes. The ability of virus to bind to the CRl, CR3 or FcR receptor-free surface will be determined with (35S) methionine-labelled virus and by electron microscopy. The leukocyte molecules which bind influenza strain WSN(HlNl) can also be redistributed to the adherent surface of the leukocyte. Using this methodology one can then examine if these binding molecules are involved in the binding of other influenza serotypes or other viruses. The effect of leukocyte accumulation on the progression of the viral infection and on the permeability of the infected epithelium will be determined by continuous transepithelial electrical resistance studies. Finally, the effect of leukocyte passage on the polarized distribution of virions and endogenous and viral antigens in the epithelial cell membrane will be determined by immunofluorescence and ultrastructural immunocytochemistry. The results of these studies should provide insight into how leukocytes are attracted to and recognize influenza-infected epithelia and the role they play in limiting viral spread and initiating recovery.
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