Discrimination of resident and infiltrated alveolar macrophages by flow cytometry in influenza A virus-infected mice

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 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 (∼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., DiIC ₁₈(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.