New concepts of IL-10-induced lung fibrosis: fibrocyte recruitment and M2 activation in a CCL2/CCR2 axis

Abstract

IL-10 is most commonly recognized as an anti-inflammatory cytokine possessing immunosuppressive effects necessary for regulated resolution of proinflammation. However, its role in the development of fibrosis during inflammatory resolution has not been clear. Few prior studies have linked IL-10 with the inhibition of fibrosis principally on the basis of regulating inflammation thought to be driving fibroproliferation. In contrast, in a model of long-term overexpression of IL-10, we observed marked induction of lung fibrosis in mice. The total cell number retrieved by bronchoalveolar lavage (BAL) increased 10-fold in the IL-10 overexpression (IL-10 OE) mice, with significant infiltration of T and B lymphocytes and collagen-producing cells. The presence of increased fibrocytes, isolated from collagenase-digested lungs, was identified by flow cytometry using dual staining of CD45 and collagen 1. Quantitative PCR analysis on an array of chemokine/chemokine receptor genes showed that receptor CCR2 and its ligand, CCL2, were highly upregulated in IL-10 OE mice, suggesting that IL-10-induced fibrocyte recruitment was CCL2/CCR2 specific. Given the prior association of alternatively activated (M(2)) macrophages with development of fibrosis in other disease states, we also examined the effect of IL-10 OE on the M(2) macrophage axis. We observed significantly increased numbers of M(2) macrophages in both BAL and whole lung tissue from the IL-10 OE mice. Administration of rabbit anti-CCL2 antiserum to IL-10 OE mice for three consecutive weeks significantly decreased fibrosis as evidenced by lung hydroxyproline content, compared with mice that received preimmune rabbit serum. These results indicate that overexpression of IL-10 induces fibrosis, in part, by fibrocyte recruitment and M(2) macrophage activation, and likely in a CCL2/CCR2 axis.

Figures

Fig. 1.

IL-10 overexpression in the lung induces fibrosis. Both control and IL-10 overexpression (OE) mice were fed with tetracycline chow for 1 and 2 mo to induce IL-10 overexpression. In a third group, mice were removed of tetracycline-containing chow after 1 mo of IL-10 overexpression and maintained on regular food for another month before experimental analysis. A: lung sections were stained with Masson's trichrome. Staining represents 6 individual mice per group. Magnification: ×40, ×100, and ×400. B: total lung collagen levels were measured with the hydroxyproline method. Results are ± SE of 5 animals and represent 2 independent experiments of a total number of 9–10 individual animals per group.

Fig. 2.

IL-10 overexpression induces lymphocytic infiltration into the alveolar space. BAL cells were harvested by repeated lavage as described in Materials and Methods. A: lymphocytic cell population in the IL-10 OE mice was identified on the FSC/SSC plot. B: frequency of macrophages, T cells, and B cells from the bronchoalveolar lavage (BAL) fluids were determined by surface markers F4/80, CD3 and CD19 by flow cytometry. C: total BAL cell numbers retrieved from control mice and IL-10 OE mice. Results are ± SE of 3 animals and represent 3 independent experiments of a total number of 9–10 individual animals per group.

Fig. 3.

mRNA/protein and immunocytochemistry analysis of collagen expression from BAL cells. BAL cells harvested from control and IL-10 OE mice were allowed to adhere for 2 h at 37°C. After removal of suspension cells, adherent cells were subjected to total RNA isolation (A) or whole cell lysate preparation (B; see Materials and Methods). A: total RNA was extracted by Trizol method and quantitative (Q)-PCR was performed for collagen 1, collagen 3, and fibronectin gene expression. Results are ± SE of 4 animals and represent 2 independent experiments of a total number of 9–10 individual animals per group. B: fibronectin and collagen 1 protein were analyzed by Western blot. Equal loading was examined by probing for GAPDH. C: BAL cells from IL-10 OE mice were cultured for 14 days in vitro, typsin digested, and plated on a chamber slide. Immunocytochemistry staining of collagen 1-positive cells was described in Materials and Methods. Parallel staining with normal rabbit IgG was used as negative control. Staining represents 3 independent experiments of 9 individual animals per group. Magnification: ×400.

Fig. 4.

IL-10 overexpression induces alternative activation of macrophages. Both control and IL-10 OE mice were fed with tetracycline-containing chow for 1 mo. BAL cells harvested from control and IL-10 OE mice were allowed to adhere for 2 h at 37°C and then, after removal of suspended cells, were subjected to total RNA preparation and Q-PCR analysis for FIZZ1, Arg1 and ST2 gene expression (A) or Western blot analysis for Arg1 and FIZZ1 protein expression (B), and immunocytochemistry analysis with anti-FIZZ1 antibody (C, top) and anti-Arg1 antibody (C, bottom) for intracellular staining of FIZZ1 and Arg1. Magnification: ×400. D: lung immunohistochemical staining of F4/80 and FIZZ1. Paraffin-embedded lung sections were subjected to immunohistochemistry staining with anti-F4/80 and anti-FIZZ1 antibodies to distinguish lung macrophages and to identify alternative activated macrophages. Stronger signals and accumulation of F4/80-positive cells were found around the bronchial epithelial areas in IL-10 OE mice. While control mice showed negative staining of FIZZ1, FIZZ1 was highly expressed on bronchial epithelial cells and in the infiltrated macrophages from the IL-10 OE mice. Magnification: ×200 and ×100.

Fig. 4.

IL-10 overexpression induces alternative activation of macrophages. Both control and IL-10 OE mice were fed with tetracycline-containing chow for 1 mo. BAL cells harvested from control and IL-10 OE mice were allowed to adhere for 2 h at 37°C and then, after removal of suspended cells, were subjected to total RNA preparation and Q-PCR analysis for FIZZ1, Arg1 and ST2 gene expression (A) or Western blot analysis for Arg1 and FIZZ1 protein expression (B), and immunocytochemistry analysis with anti-FIZZ1 antibody (C, top) and anti-Arg1 antibody (C, bottom) for intracellular staining of FIZZ1 and Arg1. Magnification: ×400. D: lung immunohistochemical staining of F4/80 and FIZZ1. Paraffin-embedded lung sections were subjected to immunohistochemistry staining with anti-F4/80 and anti-FIZZ1 antibodies to distinguish lung macrophages and to identify alternative activated macrophages. Stronger signals and accumulation of F4/80-positive cells were found around the bronchial epithelial areas in IL-10 OE mice. While control mice showed negative staining of FIZZ1, FIZZ1 was highly expressed on bronchial epithelial cells and in the infiltrated macrophages from the IL-10 OE mice. Magnification: ×200 and ×100.

Fig. 5.

IL-10 overexpression induces recruitment of fibrocytes into the lung. A, B: identification of lung fibrocytes. Whole lung leukocytes were isolated by collagenase digestion method as described in Materials and Methods. After surface staining with CD45-PerCP Cy5.5, cells were fixed and permeabilzed with paraformaldehyde and 0.1% saponin, followed by intracellular staining with either control rabbit IgG (A) or anti-collagen 1 rabbit IgG (B). Finally, cells were stained with donkey anti-rabbit PE-coupled secondary and subjected to flow cytometry analysis. Percentage of fibrocytes was calculated by subtracting the percentage of cells stained with the irrelevant control IgG from the specific collagen 1 staining percentage. C: total lung fibrocyte numbers. Lung fibroctye numbers = total lung leukocyte numbers × fibrocyte percentage. Data represent n = 4 per group and were repeated 3 times.

Fig. 6.

Upregulation of CCL2/CCR2 is responsible for the IL-10-induced lung fibrocyte recruitment. A: BAL cells harvested from control and IL-10 OE mice were allowed to adhere for 2 h at 37°C and then, after removal of suspended cells, were subjected to total RNA preparation and Q-PCR analysis for CCR2, CCR4, and CXCR4 gene expression. B: whole lung total RNA was extracted by Trizol method and was subjected to Q-PCR analysis for CCL2, CCL19, CCL21b, and CXCL12 gene expression. C: CCL2 levels in the BAL fluids were measured by ELISA analysis. Results are ± SE of 4 animals and represent 2 independent experiments of a total number of 9–10 individual animals per group.

Fig. 7.

IL-10 directly induces M2 activation in cultured BAL macrophages. A and B: IL-13 upregulation in IL-10 OE mice. Both control and IL-10 OE mice were fed with tetracycline chow for 1 mo. Total RNA from both adherent BAL cells and from whole lung tissue was analyzed for IL-13 gene expression. Protein levels of IL-13 in the BAL fluids and from whole lung homogenates were measured by ELISA analysis. Data represent n = 4 per group and were repeated 3 times. C: in vitro activation of M2 macrophages by IL-10 and IL-13. BAL macrophage isolation and culture were performed as described in Materials and Methods. Cells were incubated with recombinant mouse IL-10 or IL-13 (20 ng/ml) for 2 and 4 days and then subjected to Q-PCR analysis for M2 marker gene expression. In each group, results are ± SE of 4 wells and represent 3 independent experiments. D: immunocytochemical staining of Arg1. BAL macrophages were treated with PBS, 20 ng/ml IL-10 or IL-13 for 3 days. Cells were then fixed, permeabilized, and incubated with rabbit anti-Arg1 antibody, followed by horseradish peroxidase-conjugated secondary antibody incubation and subsequent color reaction using DAB as substrate. E: IL-10- and IL-13-induced M2 macrophages express different flow cytometry profiles. BAL macrophages were treated with 20 ng/ml IL-10 or IL-13 for 3 days and then subjected to Fc blocking and staining with different fluorochrome-conjugated flow antibodies. Changes in expression of MHC II, MMR, CD11c, and CD11b were assessed by comparision against PBS-treated control macrophages. A-E represent at least 3 independent experiments.

Fig. 8.

Anti-CCL2 blocking attenuates IL-10 OE-induced fibrotic responses. Both control and IL-10 OE mice received an intraperitoneal injection of 0.5 ml of anti-CCL2 antiserum or normal serum starting at day 3 after tetracycline chow and every 1 wk after that for a total of 3 wk. At 1 mo posttetracycline-chow treatment, mice lungs were harvested and subjected to hydroxyproline assay for measurement of total lung collagen levels. Values are expressed as mean ± SE; n = 5–6 mice/group.