title: Discrimination of resident and infiltrated alveolar macrophages by flow cytometry in influenza A virus-infected mice.
abstract: Laser flow cytometric analysis was used in conjunction with in vivo labeling with the lipophilic fluorescent dye DiIC18(5)-DS to discriminate resident alveolar macrophages from newly infiltrating monocytes/macrophages in mice with and without pulmonary influenza A virus infection. Leukocytes in bronchoalveolar lavage (BAL) and peripheral blood were analyzed by 2-color flow cytometry as a function of time following intravenous injection of DiIC18(5)-DS. At 4 hours, dye-positive leukocytes were present in both BAL and blood of normal mice, indicating that DiIC18(5)-DS rapidly crossed the pulmonary endothelial-epithelial barrier. At 4 days after dye injection, 98% of BAL cells were DiIC18(5)-DS positive, and almost all of these were monocytes/macrophages based on labeling with fluorescein isothiocyanate (FITC)-conjugated antibody to the Mac-3 marker. Only 3.2% +/- 0.3% of peripheral blood monocytes (approximately 0.16% of total peripheral blood leukocytes) were DiIC18(5)-DS positive at 6 days after injection, whereas > 95% of BAL leukocytes were strongly dye-positive on days 6 to 28. When DiIC18(5)-DS was injected in mice 6 days prior to intranasal challenge with influenza A, flow cytometry indicated that 57.8% 5.6% and 60.7% +/- 8.5% of macrophages/monocytes in BAL were newly infiltrated (i.e., DiIC18(5)-DS negative, Mac-3 positive) at 4 and 7 days, respectively, post viral infection. The discrimination of subpopulations of resident and newly recruited macrophages in BAL should facilitate future mechanistic studies on pulmonary infection and inflammatory lung injury.
authors.affiliation: Department of Anesthesiology, University at Buffalo-SUNY School of Medicine, Biomedical Research Building, Room 247, 3435 Main Street, Buffalo, NY 14214, USA. bdavidso@buffalo.edu
authors.forename: Bruce A
authors.initials: BA
authors.lastname: Davidson
authors.forename: Carleton C
authors.initials: CC
authors.lastname: Stewart
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Russo
authors.forename: Patricia R
authors.initials: PR
authors.lastname: Chess
authors.forename: Paul R
authors.initials: PR
authors.lastname: Knight
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D000943:Antigens, Differentiation; D001773:Blood Cells / Q000473:pathology; D001992:Bronchoalveolar Lavage Fluid / Q000166:cytology; D002232:Carbocyanines; D002465:Cell Movement; D002469:Cell Separation; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D009980:Influenza A virus; D016676:Macrophages, Alveolar / Q000276:immunology / Q000473:pathology*; D008297:Male; D051379:Mice; D009000:Monocytes / Q000276:immunology / Q000473:pathology; D009976:Orthomyxoviridae Infections / Q000097:blood / Q000276:immunology / Q000473:pathology*
chemical_list: D000911:Antibodies, Monoclonal; D000943:Antigens, Differentiation; D002232:Carbocyanines; D005456:Fluorescent Dyes; C036763:monocyte-macrophage differentiation antigen; C024286:3,3'-dioctadecylindocarbocyanine; D016650:Fluorescein-5-isothiocyanate