Functional characterisation of human pulmonary monocyte-like cells in lipopolysaccharide-mediated acute lung inflammation

Abstract

Background: We have previously reported the presence of novel subpopulations of pulmonary monocyte-like cells (PMLC) in the human lung; resident PMLC (rPMLC, HLA-DR(+)CD14(++)CD16(+)cells) and inducible PMLC (iPMLC, HLA-DR(+)CD14(++)CD16(-) cells). iPMLC are significantly increased in bronchoalveolar lavage (BAL) fluid following inhalation of lipopolysaccharide (LPS). We have carried out the first functional evaluation of PMLC subpopulations in the inflamed lung, following the isolation of these cells, and other lineages, from BAL fluid using novel and complex protocols.

Methods: iPMLC, rPMLC, alveolar macrophages (AM), neutrophils, and regulatory T cells were quantified in BAL fluid of healthy subjects at 9 hours post-LPS inhalation (n = 15). Cell surface antigen expression by iPMLC, rPMLC and AM and the ability of each lineage to proliferate and to undergo phagocytosis were investigated using flow cytometry. Basal cytokine production by iPMLC compared to AM following their isolation from BAL fluid and the responsiveness of both cell types following in vitro treatment with the synthetic corticosteroid dexamethasone were assessed.

Results: rPMLC have a significantly increased expression of mature macrophage markers and of the proliferation antigen Ki67, compared to iPMLC. Our cytokine data revealed a pro-inflammatory, corticosteroid-resistant phenotype of iPMLC in this model.

Conclusions: These data emphasise the presence of functionally distinct subpopulations of the monocyte/macrophage lineage in the human lung in experimental acute lung inflammation.

Keywords: Acute lung inflammation; Corticosteroid; Lipopolysaccharide; Macrophages; Monocytes; Multiparameter flow cytometry.

Figure 1

Cytocentrifuge preparations of cells in BAL fluid following LPS inhalation. iPMLC (A), rPMLC (B), neutrophils (C), lymphocytes (D) and alveolar macrophages (AM) (E) were isolated from BAL fluid with high purity using FACS. PMLC are similar in morphology to blood monocytes and are a similar size to neutrophils, larger than lymphocytes and smaller than AM (Scale bar = 20 μm). Typical morphological features of each lineage were observed i.e. multi-lobular neutrophils, lymphocytes with high nucleus to cytoplasm ratio, and “fried egg”-shaped AM.

Figure 2

Fluorescence activated cell sorting of cells in BAL fluid following LPS inhalation. Cells were identified in BAL fluid based upon their position on flow cytometry dot plots of size i.e. forward scatter (FSC-A) versus granularity i.e. side scatter (SSC-A) (A). PMLC were selected as HLA-DR + (B), and were subdivided into iPMLC and rPMLC subpopulations based upon their CD14 and CD16 expression (CD14++CD16- and CD14++CD16+, respectively; C). Representative samples of AM, iPMLC and rPMLC isolated from BAL fluid using FACS with >95% purity, and overlapped on SSC-A versus FSC-A dot plots are seen in (D). Like PMLC, it was possible to identify distinct populations of lymphocytes, neutrophils and alveolar macrophages in BAL fluid following LPS inhalation based upon their size and granularity (A). Lymphocytes were further selected for CD3 expression (E), neutrophils were classed as CD16+ HLA-DR- (F) and AM were selected as large CD16+ HLA- DR + cells (G).

Figure 3

Phagocytosis by cells in BAL fluid. Alveolar macrophages displayed a significantly increased capacity for phagocytosis compared to PMLC (P =0.0006 by Mann Whitney U test (n = 13)).

Figure 4

In vitro cytokine production by AM and iPMLC in response to treatment with dexamethasone. Log concentrations of IL-6 (Panels A-B), IL- 8 (Panels C-D) and TNFα (Panels E-F) in supernatants from flow-sorted, cultured AMs and iPMLCs. Data are shown paired (with/without incubation with dexamethasone) per 10,000 cells cultured; n = 8; statistical analysis was by Wilcoxon signed rank test; *P < 0.05.