Flow cytometric analysis of macrophages and dendritic cell subsets in the mouse lung

Abstract

The lung hosts multiple populations of macrophages and dendritic cells, which play a crucial role in lung pathology. The accurate identification and enumeration of these subsets are essential for understanding their role in lung pathology. Flow cytometry is a mainstream tool for studying the immune system. However, a systematic flow cytometric approach to identify subsets of macrophages and dendritic cells (DCs) accurately and consistently in the normal mouse lung has not been described. Here we developed a panel of surface markers and an analysis strategy that accurately identify all known populations of macrophages and DCs, and their precursors in the lung during steady-state conditions and bleomycin-induced injury. Using this panel, we assessed the polarization of lung macrophages during the course of bleomycin-induced lung injury. Alveolar macrophages expressed markers of alternatively activated macrophages during both acute and fibrotic phases of bleomycin-induced lung injury, whereas markers of classically activated macrophages were expressed only during the acute phase. Taken together, these data suggest that this flow cytometric panel is very helpful in identifying macrophage and DC populations and their state of activation in normal, injured, and fibrotic lungs.

Figure 1.

Gating strategy used to identify myeloid-cell subsets in the normal mouse lung. Cells were isolated from enzymatically digested mouse lungs, and after the exclusion of doublets and debris, immune cells were identified by CD45 staining. (A) A sequential gating strategy was first used to identify populations expressing specific markers: alveolar macrophages (MΦ) (Siglec F⁺ CD11b⁻ CD11c⁺ CD64⁺), CD103⁺ dendritic cells (DCs) (CD11c⁺ CD103⁺ CD24⁺), neutrophils (CD11b⁺ Ly6G⁺), and eosinophils (Siglec F⁺ CD11b⁺ CD11c⁻), followed by the identification of populations with overlapping expression patterns: interstitial macrophages (CD11b⁺ MHC II⁺ CD11c⁺ CD64⁺ CD24⁻), CD11b⁺ DCs (CD11b⁺ MHC II⁺ CD11c⁺ CD24⁺ CD64⁻), and monocytes (Mo)/undifferentiated macrophages (CD11b⁺ MHC II⁻ CD64^+/− Ly6C^lo). Scale bar in microphotographs = 5 μm. (B) Identification of macrophages and DCs using the minimal panel of surface markers. Both alveolar macrophages and CD103⁺ DCs are identified as CD11b⁻CD11c⁺ cells, and are further separated using CD64 and CD24, correspondingly. If necessary, MHC II can be used to confirm gating in CD103⁺ DCs (not shown). Gating on CD11b^hi cells allows for the separation of myeloid cells from lymphoid cells that either do not express this marker (T and B cells), or express it at intermediate level (natural killer cells). Granulocytes (neutrophils and eosinophils) can be gated out as CD24⁺CD11c⁻, and the identification of CD11b⁺ DCs (CD11b⁺ MHC II⁺ CD11c⁺ CD24⁻ CD64⁻), interstitial macrophages (CD11b⁺ MHC II⁺ CD11c⁺ CD64⁺ CD24⁻), and monocytes/undifferentiated macrophages can be continued as in the full panel (CD11b⁺ MHC II⁻ CD64^+/− Ly6C^lo). FSC, forward scatter; MHC II, major histocompatibility complex class II; SSC, side scatter.

Figure 2.

The phenotype of myeloid cells in mouse lungs changes during the course of bleomycin-induced lung injury. Left to right: Normal lung, 5 and 21 days after instillation of bleomycin. Top images were gated on CD45⁺ cells, with neutrophils and eosinophils gated out.

Figure 3.

Changes of myeloid-cell subsets in mouse lungs during bleomycin-induced lung injury (Days 5 and 21). (A) Numerical changes of myeloid-cell subsets were identified as described in Figure 1. Values represent means ± SEMs. Differences between groups were compared using one-way ANOVA. ***P < 0.001. (B) Expression of markers associated with classically (CD40, CD80, and CD86) and alternatively (CD71, CD206, and RELMα) activated macrophages on alveolar and interstitial macrophages during bleomycin-induced lung injury. Values represent means ± SEMs for median fluorescence intensity (MFI) for the given marker. Differences between groups were compared using one-way ANOVA. **P < 0.01. ***P < 0.001.