Journal
EXPERIMENTAL LUNG RESEARCH
Volume 31, Issue 3, Pages 323-339Publisher
TAYLOR & FRANCIS INC
DOI: 10.1080/01902140590918524
Keywords
flow cytometry; influenza A; macrophages; recruited macrophages; resident macrophages
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Funding
- NHLBI NIH HHS [HL-69763, HL-56176, HL-48889, HL-03910] Funding Source: Medline
- NIAID NIH HHS [AI-46534] Funding Source: Medline
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Laser flow cytometric analysis was used in conjunction with in vivo labeling with the lipophilic fluorescent dye DiIC(18)(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 DiIC, 8(5)-DS. At 4 hours, dye-positive leukocytes were present in both BAL and blood of normal mice, indicating that DiIC(18)(5)-DS rapidly crossed the pulmonary endothelial-epithelial barrier. At 4 days after dye injection, 98% of BAL cells were DiIC(18)(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 (similar to 0.16% of total peripheral blood leukocytes) were DiIC(18)(5)-DS positive at, 6 days after injection, whereas >95% of BAL leukocytes were strongly ye-positive on days 6 to 28. When DiIC(18)(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., DiIC(18)(5)-DS negative, Mac-3 positive) at 4 awl 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.
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