Accurate and simple discrimination of mouse pulmonary dendritic cell and macrophage populations by flow cytometry: Methodology and new insights

Abstract

BackgroundThe need to accurately discriminate dendritic cells (DCs) and macrophages (Mphs) in mouse lungs is critical given important biological differences. However, a validated flow cytometry–based method is still lacking, resulting in much confusion between both cell types.MethodsSingle‐cell suspensions freshly obtained from collagenase‐digested lung tissue were stained with a CD11c‐specific monoclonal antibody, detected using a PE‐Cy5 or APC‐conjugated secondary reagent. Cellular immunophenotype was simultaneously explored using a panel of PE‐conjugated markers. The FL1 or FITC‐detection channel was reserved for the assessment of autofluorescence.ResultsCD11c‐bright cells were heterogeneous and displayed a bimodal distribution with regard to autofluorescence (AF). CD11c+/low‐AF cells were lineage‐negative and showed features compatible with myeloid DCs. This was confirmed by morphology, potent T‐cell stimulatory function in a mixed‐leukocyte reaction, surface expression of MHCII and costimulatory molecules, and further immunophenotypical criteria, including the expression of Mac‐1 and absence of CD8α. In contrast, CD11c+/high‐AF cells displayed the features of pulmonary Mphs, including typical Mph morphology, very weak induction of T‐cell proliferation, low to absent expression of MHCII and costimulatory molecules, and very low levels of Mac‐1 as well as F4/80. We also show that only CD11c+/high‐AF cells strongly expressed the macrophage marker MOMA‐2, while interestingly Mac‐3 was expressed at high levels by CD11c+/high‐AF and low‐AF alike.ConclusionsThis study shows that the combination of CD11c‐expression and autofluorescence is necessary and sufficient to accurately separate DCs from macrophage subpopulations in mouse lungs. © 2004 Wiley‐Liss, Inc.

Distinct Differentiation Potential of Blood Monocyte Subsets in the Lung

“…Staining for MHC-II revealed that CD11c ϩ CX 3 CR1/GFP ϩ cells also express high levels of MHC-II, whereas CD11c ϩ CX 3 CR1/GFP Ϫ cells are MHC-II low (Fig. 1E), supporting their definition as DC and M⌽, respectively (31). Interestingly, we recently reported the same CX 3 CR1 expression pattern for small intestinal lamina propria DC and M⌽ (33).…”

“…1A). In addition, lung and alveolar M⌽, but not DC, are autofluorescent (31,32). In this study, we show that lung M⌽ and DC also differ in their expression of the chemokine receptor CX 3 CR1.…”

“…Lung DC can be further characterized as CD11c ϩ MHC-II high cells, whereas lung M⌽ are CD11c ϩ MHC-II low (21,31). Staining for MHC-II revealed that CD11c ϩ CX 3 CR1/GFP ϩ cells also express high levels of MHC-II, whereas CD11c ϩ CX 3 CR1/GFP Ϫ cells are MHC-II low (Fig.…”

Dynamics of lung macrophage activation in response to helminth infection

“…Although F4/80 has been used traditionally as a macrophage-specific surface marker, its use in the lung is compromised by low levels of F4/80 on lung macrophages and expression levels that vary in the context of inflammation (Fig. 2) [48,49]. To increase the purity of lung macrophage preparations for procedures such as adoptive transfers, it may be necessary to add a step to specifically deplete the lung DCs [50].…”

Pulmonary Mycobacterial Granuloma

“…These two populations share similar cell surface makers and are broadly defined by their expression of complement receptors: CR3(CD11b/CD18) and CR4(CD11c/CD18). 31 Thus we first examined the tissue localization and accumulation of CD11bϩ and CD11cϩ cells in mycobacterium-infected lung by immunohistochemistry. By day 21, there was a large accumulation of both CD11bϩ and CD11cϩ cells in the lung, with the CD11bϩ cells being more abundant in the granuloma ( Figure 1A).…”