Morphological and Cytochemical Characterization of Cells Infiltrating Mouse Lungs After Influenza Infection

Abstract

To initiate evaluation of the cell-mediated immunological response to influenza virus in a major site of disease, lung cells were obtained by transpleural lavage from lungs of uninfected mice and from those infected 3 or 6 days previously with 5 50% mouse infectious doses (MID 50 ) of avirulent (P3) or virulent (P9) influenza A Hong Kong (H3N2) virus. The number of cells recovered by lavage was dependent on the dose, time after inoculation, and the type of virus used for inoculation. Although lavage pools were shown to contain peripheral blood leukocytes, this contamination was shown to be consistently less than 5% of the total leukocytes harvested. Among the ca. 0.75 × 10 6 lavage cells obtained from each uninfected mouse, about 90% were macrophages or lymphocytes in approximately equal proportion. T, B, and null (lyphocytes lacking theta or surface immunoglobulin markers) lymphocytes averaged 23, 9, and 7% of cells in these suspensions, respectively. After infection with either P3 or P9 virus, increased numbers of activated macrophages and lymphoblasts were observed. The major change during P3 infection was an increase in absolute numbers of null lymphocytes. In contrast, during P9 infection, T and B lymphocytes and macrophages progressively increased in absolute numbers while null cells decreased. These data suggest that cell-mediated immunological responses to influenza virus occur in the lung during infection, but that the responses to virulent and avirulent variants may differ both qualitatively and quantitatively.

Appearance of Rosette-Forming Macrophages in the Lungs of Influenza Virus-Infected Mice

“…Previous studies have shown that the number of macrophages obtainable by lung lavage from a C3H mouse increases from about 126,000 before inoculation to 381,000 3 days after inoculation with the same dose of virulent influenza A/ HK virus as was used in the present studies, and to 1,240,000 by 6 days after inoculation (13). Therefore, the approximately 7-fold increase in the percentage of RFM between days 0 to 1 and days 2 to 3 actually represents about a 20-fold increase in the absolute number of RFM obtainable by lung lavage.…”

Cellular Changes in Lungs of Mice Infected with Influenza Virus: Characterization of the Cytotoxic Responses

“…More direct information about the nature of the local response to influenza virus infection was provided in a previous report from this laboratory which described the types of leukocytes obtained by lavaging the lungs of immunocompetent BALB/c mice (33). Among the approximately 750,000 leukocytes from the lungs of uninfected animals, about 90% were macrophages or lymphocytes in roughly equal proportions.…”

“…Leukocyte collection and characterization. Methods used in this laboratory for harvesting peripheral blood leukocytes and cells from the lung, lymph node, and spleen have been previously described (33). Briefly, heparinized peripheral blood was obtained from the orbital sinus and fractionated on Ficoll-Isopaque gradients.…”

“…Fluorescent antibody procedures. Cells bearing surface immunoglobulin were quantitatively assessed by direct immunofluorescence (31) using fluorescein isothiocynate-conjugated goat anti-mouse gamma globulin (Cappel Laboratories, Downingtown, Pa.) as described previously (33). All slides were examined at 400x with a Leitz Othoplan fluorescent microscope equipped with phase optics and Ploem illumination.…”

“…Cells bearing the theta antigen were identified by indirect immunofluorescence (19) by using identical conditions but labeling with rabbit anti-thymocyte serum and staining with fluoroscein isothiocyanateconjugated goat anti-rabbit gamma globulin (Cappel Laboratories). The production and adsorption of the rabbit anti-thymocyte serum have been described (33).…”