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. 2013 Nov 20;8(11):e81382.
doi: 10.1371/journal.pone.0081382. eCollection 2013.

Lung collagens perpetuate pulmonary fibrosis via CD204 and M2 macrophage activation

Mirjam Stahl  1 , Jonas Schupp, Benedikt Jäger, Michael Schmid, Gernot Zissel, Joachim Müller-Quernheim, Antje Prasse
Affiliations

Lung collagens perpetuate pulmonary fibrosis via CD204 and M2 macrophage activation

Mirjam Stahl et al. PLoS One. .

Abstract

Idiopathic pulmonary fibrosis is characterized by abundant collagen production and accumulation of alternatively activated macrophages (M2) in the lower respiratory tract. Mechanisms as to how alveolar macrophages are activated by collagen breakdown products are unknown. Alveolar macrophages were obtained by bronchoalveolar lavage from 30 patients with idiopathic pulmonary fibrosis (IPF) and 37 healthy donors (HD). Alveolar macrophages were cultured in the presence of collagen type I, III, IV and V monomers w/wo a neutralizing antibody against scavenger receptor I class A (CD204). Culture supernatants were assayed for the M2 markers CCL18, CCL2, and interleukin-1 receptor antagonist (IL-1ra) by ELISA. Furthermore, expression of phospho-Akt was measured using ELISA and expression of CD204 by RT-PCR and flow cytometry. Stimulation with collagen type I and III monomers significantly up-regulated CCL18, IL-1ra production of alveolar macrophages. Furthermore, expression of CCL2 and CD204 were up-regulated by collagen type I exposure. In addition, collagen type I stimulation increased pospho-Akt expression. Collagen type I effects were abrogated by neutralizing antiCD204 and a non-selective Phosphatidylinositide 3-kinase inhibitor (LY294002). Spontaneous CD204 expression of alveolar macrophages was significantly increased in patients with IPF. In conclusion, our findings demonstrate that monomeric collagen type I via CD204 induces phospho-Akt expression shifting alveolar macrophages to the profibrotic M2 type. Innate immune responses induced by collagen monomers might perpetuate pulmonary fibrosis.

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Conflict of interest statement

Competing Interests: Gernot Zissel and Antje Prasse serve as editors for PLOS ONE. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Collagen type I monomers increase M2 marker production by AM.
AM (1x106 cells/well and ml) from healthy donors (HD, n=23; Panel A) and IPF patients (IPF, n=14; Panel B) were cultured in serum-free medium with (coloured bar) or without (white bar) the presence of monomeric collagen type I for 24 hours. Note the 10fold difference in spontaneous M2 cytokine production between AM from IPF patients and HD. Collagen type I exposure of AM significantly enhanced CCL2, CCL18 and IL-1ra production. The effect of collagen type I monomers was more pronounced in AM from IPF. (*, p<0.05; **, p<0.001).
Figure 2
Figure 2. Collagen monomers increase M2 marker production by AM.
M2 cytokine production of BAL cells stimulated with monomers of various collagen types. M2 cytokine production of BAL cells from 5 additional patients with IPF are depicted in black and 5 additional experiments with BAL cells from healthy donors are depicted in grey. Panel A shows CCL18 production following stimulation with rat collagen-I (Col 1R; 100µg/ml), human collagen-I (Col I, 100 µg/ml), human collagen-III (Col III, 100 µg/ml), human collagen-IV (Col IV, 100 µg/ml), human collagen-V (Col V, 100 µg/ml). Panel B shows IL-1ra production following the same stimulation protocol. Panel C shows CCL2 production. *p<0.05.
Figure 3
Figure 3. CD204 expression is up-regulated in IPF patients.
In Panel A, relative level (RL) of CD204 mRNA expression of AM from IPF patients (n=20, grey) and healthy donors (n=18, white) is given. Similar results were obtained for CD204 relative fluorescence intensity (RFI) by flow cytometry. In Panel B, mean level of CD204 fluorescence intensity is depicted for naïve AM from IPF patients (n=10, grey) and healthy donors (HD, n=10, white). In Panel C representative original measurements of CD204 and isotype control by flow cytometry are depicted for healthy donor (HD) and patient with IPF. Exposure to collagen type I significantly increased CD204 protein and CD204 mRNA expression in AM of 5 healthy donors (Panel D and F) and IPF patients (Panel E and G). Box plots: horizontal lines represent median, 25 and 75 percentiles, and small lines characterize 10 and 90 percentiles (*, p<0.05; **, p<0.001).
Figure 4
Figure 4. Blocking of CD204 abrogates effects mediated by collagen type I monomers.
Preincubation of AM from HD (n=10, Panel A) and IPF patients (n=8, Panel B) with neutralizing antiCD204 (2µg/ml) prior to cell culture abrogated collagen type I monomers induced increase in CCL18 production. Mouse IgG1 served as control. Data are expressed by mean ± SD (*, p<0.05).
Figure 5
Figure 5. Collagen type I monomers increase Phospho-Akt expression via PI3kinase activation.
Stimulation of AM with collagen type I monomers resulted in a moderate increase in phospho-Akt expression, which was more pronounced in IPF patients (Panel A). Basal, constitutive phospho-Akt expression was high in AM from each 5 healthy donors and IPF patients. A non-selective inhibitor of PI3kinase, LY294002 (50 μM, DMSO as control), abrogated CCL18 production following stimulation with collagen type I monomers and decreased significantly spontaneous CCL18 production of AM (Panel B). Data are expressed by mean ± SD (*, p<0.05; **, p<0.005).
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