CD11c+ alveolar macrophages are a source of IL-23 during lipopolysaccharide-induced acute lung injury

Abstract

Acute lung injury (ALI) is a severe pulmonary disease causing high numbers of fatalities worldwide. Innate immune responses are an integral part of the pathophysiologic events during ALI. Interleukin 23 (IL-23) is a proinflammatory mediator known to direct the inflammatory responses in various settings of infection, autoimmunity, and cancer. Interleukin 23 has been associated with proliferation and effector functions in T(H)17 cells. Surprisingly, little is known about production of IL-23 during ALI. In this study, we found expression of mRNA for IL-23p19 to be 10-fold elevated in lung homogenates of C57BL/6 mice after lipopolysaccharide (LPS)-induced ALI. Likewise, concentrations of IL-23 significantly increased in bronchoalveolar lavage fluids. Experiments with IL-23-deficient mice showed that endogenous IL-23 was required for production of IL-17A during LPS-ALI. CD11c-diphtheria toxin receptor transgenic mice were used to selectively deplete CD11c cells, the data suggesting that IL-23 production is dependent at least in part on CD11c cells during ALI. No alterations of IL-23 levels were observed in Rag-1-deficient mice as compared with wild-type C57BL/6 mice following ALI. The mouse alveolar macrophage cell line, MH-S, as well as primary alveolar macrophages displayed abundant surface expression of CD11c. Activation of these macrophages by LPS resulted in release of IL-23 in vitro. Our findings identify CD11c macrophages in the lung are likely an important source of IL-23 during ALI, which may be helpful for better understanding of this disease.

Figure 1

Expression and release of IL-23(p19) during acute lung injury (ALI). A, Microscopic images of the alveolar compartment from normal lungs 8 h after sham operation (Sham-OP, 40 μl PBS i.t.) or 8 h after LPS-induced ALI (40 μg in 40 μl i.t.), H&E stains, x40, scale bar: 50 μm, n=3/group. B, Real time PCR for IL-23(p19) mRNA in lung homogenates of C57BL/6 mice 8 h after LPS- ALI or Sham-OP, n=4/group. C, Time course for presence of IL-23(p19) in BAL fluids after LPS-ALI in C57BL/6 mice (n=5/group), ELISA. D, Detection of IL-23(p19) in BAL fluids 8 h after Sham-OP (n=6) or immune complex-induced ALI (IgGIC-ALI; n=7). * P < 0.05, ** P < 0.01.

Figure 2

Effects of genetic deficiency of IL-23 during LPS-ALI. A, LPS-ALI was performed with IL-23^−/− mice (n=6) or C57BL/6 mice (n=8) as controls with measurements of albumin leakage after 10 h (student’s t test, P=0.14). B, Detection of IL-17A concentrations in BALF from the same experiments as in frame A, ELISA. * P < 0.05.

Figure 3

CD11c⁺ cells are a source of IL-23(p19) during acute lung injury. A, CD11c-diphtheria toxin receptor transgenic mice (CD11c-DTR Tg) and C57BL/6 mice received diphtheria toxin (4 ng/g body weight i.t. together with 25 ng/g body weight i.p.) 24 h before LPS-ALI. Splenocytes were harvested 8 h after LPS-ALI and stained for CD45 and CD11c by flow cytometry. B, Cells in BALF from C57BL/6 and CD11c-DTR Tg mice were stained for CD45 and CD11c after LPS-ALI in mice from the same experiment as described above. C, Detection of IL-23(p19) by ELISA in BALF 8 h after LPS-ALI with depletion of CD11c⁺ cells in CD11c-DTR Tg (n=4) mice as compared to C57BL/6 mice (n=8) as controls. Both groups had received treatments with diphtheria toxin as described above. D, Comparison of IL-23(p19) concentrations in BAL fluids 8 h after LPS-ALI in Rag-1^−/− mice (n=5, deficient in T and B cells) and C57BL/6 mice (n=7).* P < 0.05, n.s. indicates not significant.

Figure 4

Phenotyping of mouse alveolar macrophages. A, MH-S macrophages were stained with anti-CD11c, anti-F4/80 and anti-CD11b antibodies while control cells were exposed to fluorochrome labeled isotype antibodies. Cells were then analyzed by flow cytometry. B, Primary alveolar macrophages were obtained from normal C57BL/6 mice by broncho-alveolar lavage. The abundancy of CD11c, F4/80 and CD11b surface markers was detected by flow cytometry as described above. Data are representative of 2 independent experiments.

Figure 5

Production of IL-23(p19) by alveolar macrophages, but not PMNs or alveolar epithelial cells, in vitro. A, MH-S macrophages were untreated (Ctrl) or incubated with LPS for 12 h followed by detection of IL-23 by ELISA in cell culture supernatants. B, Primary alveolar macrophages were obtained by broncho-alveolar lavage from healthy C57BL/6 mice and activated with LPS for 24 h in vitro with IL-23 detection. C, PMNs (C57BL/6) were incubated with LPS for 6 h followed by analysis of IL-23. D, Detection of IL-6 from PMNs, 6h. E, MLE-12 mouse type II like alveolar epithelial cells as unstimulated controls or in the presence of LPS with undetectable IL-23 after 20 h. F, Detection of IL-6 from MLE-12 cells, 20 h. LPS was used as 1 μg/ml in all experiments and all results were obtained by ELISA. * P < 0.05, ** P < 0.01, *** P < 0.001, n.s. indicates not significant. Data represent 3 independent experiments.