IL-8 mediates idiopathic pulmonary fibrosis mesenchymal progenitor cell fibrogenicity

Abstract

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease, but the mechanisms driving progression remain incompletely defined. We previously reported that the IPF lung harbors fibrogenic mesenchymal progenitor cells (MPCs), which serve as a cell of origin for IPF fibroblasts. Proliferating IPF MPCs are located at the periphery of fibroblastic foci in an active cellular front at the interface between the myofibroblast-rich focus core and adjacent normal alveolar structures. Among a large set of genes that distinguish IPF MPCs from their control counterparts, we identified IL-8 as a candidate mediator of IPF MPC fibrogenicity and driver of fibrotic progression. IPF MPCs and their progeny displayed increased steady-state levels of IL-8 and its cognate receptor CXCR1 and secreted more IL-8 than did controls. IL-8 functioned in an autocrine manner promoting IPF MPC self-renewal and the proliferation and motility of IPF MPC progeny. Secreted IL-8 also functioned in a paracrine manner stimulating macrophage migration. Analysis of IPF lung tissue demonstrated codistribution of IPF MPCs with activated macrophages in the active cellular front of the fibroblastic focus. These findings indicate that IPF MPC-derived IL-8 is capable of expanding the mesenchymal cell population and recruiting activated macrophages cells to actively evolving fibrotic lesions.

Keywords: IL-8; IPF mesenchymal progenitor cell; fibrotic front; macrophage.

Fig. 1.

IL-8 expression is increased in idiopathic pulmonary fibrosis (IPF) mesenchymal progenitor cells (MPCs). A and B: IL-8 mRNA expression in IPF and control MPCs (A) and IPF and control MPC progeny (B). Shown are relative expression levels of each mRNA by quantitative (Q)-PCR. Data shown represents the mean mRNA levels in IPF MPCs and IPF MPC progeny and control MPCs and control MPC progeny (derived from 4 cell lines each). C and D. IL-8 protein levels were quantified in cell lysates of IPF and control MPCs (C) and IPF and control MPC progeny (D) by ELISA (n = 4 cell lines each). Data are expressed as means ± SE. P values were determined by two-tailed Student’s t-test.

Fig. 2.

IPF MPCs and IPF MPC progeny actively secrete IL-8. A and B: IPF and control MPCs (A) or IPF and control MPC progeny (B) were seeded on tissue culture dishes in fresh DMEM and cultured for 2 days. The medium was collected, and IL-8 protein levels were quantified in cell culture medium by ELISA. Data shown represent the mean protein levels in IPF MPCs and IPF MPC progeny and control MPCs and control MPC progeny (derived from 4 cell lines each). Data are expressed as means ± SE. P values were determined by two-tailed Student’s t-test.

Fig. 3.

IL-8 promotes IPF MPC self-renewal. A: IPF and control MPC (n = 4 cell lines each) CXCR1 mRNA expression levels were quantified by Q-PCR. B and C: to analyze the effect of IL-8 on IPF and control MPC self-renewal, 5,000 cells in a single cell suspension were seeded per well in 24-well dishes containing methylcellulose and recombinant IL-8 as indicated. The cells were cultured for 7 days. B: colony number per 5,000 cells was quantified microscopically (n = 3). C: graph depicting colony size and phase contrast image of IPF colony formation. Data are expressed as means ± SE. P values were determined by two-tailed Student’s t-test.

Fig. 4.

IL-8 promotes the proliferation and motility of IPF MPC progeny. A: CXCR1 mRNA expression levels were quantified in IPF and control MPC progeny (n = 4 cell lines each) by Q-PCR. B: shown is proliferation of IPF and control MPC progeny in response to recombinant IL-8. Cell number was quantified using the MTT Proliferation Assay Kit (n = 3). OD, optical density. C: shown is the migration of IPF and control MPC progeny in response to recombinant IL-8. Migration was quantified using the QCM Chemotaxis Cell Migration Assay Kit (n = 3). Data are expressed as means ± SE. P values were determined by two-tailed Student’s t-test.

Fig. 5.

Paracrine function of IL-8 secreted by IPF MPCs. A: CXCR1 and CXCR2 mRNA expression in macrophages. B: macrophage migration in response to fresh medium (control), IPF MPC progeny-derived conditioned medium containing DMSO (CM), IPF MPC progeny-derived conditioned medium containing the CXCR2 inhibitor SB332235 (1 µM) (CM + SB), and IPF MPC progeny-derived conditioned medium containing the CXCR1/2 inhibitor reparixin (100 µM) (CM + R) were analyzed using the QCM Chemotaxis Cell Migration Assay Kit (n = 3). Data are expressed as means ± SE. P values were determined by two-tailed Student’s t-test.

Fig. 6.

IPF MPCs colocalize with macrophages in the peripheral region of the fibroblastic focus. Immunohistochemical (IHC) was performed on human IPF lung tissue (n = 10 IPF patient specimens). A: shown are 2 fibroblastic foci. Hematoxylin and eosin (H&E) staining was used to identify the fibroblastic focus and IHC was performed using CD163 (macrophage marker) and SSEA4 and S100A4 (MPC markers) antibodies to assess distribution of macrophages and IPF MPCs in the focus. Asterisk denotes focus core. Bar = 50 μm (left) and 20 μm (middle and right). B: shown are serial 4-μm sections stained for H&E, procollagen, SSEA4, S100A4, and CD163. Shown are two fibroblastic foci. Procollagen expression was used to delineate the myofibroblast-rich focus core. IHC analysis of the foci demonstrated S100A4- and SSEA4-positive cells at the periphery of the focus. CD163-positive cells codistributed with SSEA4- and S100A4-expressing cells at the focus perimeter. Asterisk denotes focus core. Fibroblastic Focus 1: bar = 50 μm (top and middle) and 20 μm (bottom); Fibroblastic Focus 2: bar = 50 μm (top) and 20 μm (bottom). C: IHC analysis of IPF lung tissue for CXCR1 and CXCR2 expression and CD163. Left: dual staining for CXCR1 (brown) and CD163 (red). Shown in the inset is a CD163-positive macrophage (red) with CXCR1 labeling (brown) on the perimeter of the cell. Right: dual staining for CXCR2 (brown) and CD163 (red). Bar = 100 μm (top) and 50 μm (bottom). Asterisk denotes focus core. D: in situ hybridization for IL-8 was performed on IPF lung tissue. Bar = 50 μm (top and bottom left) and 20 μm (bottom right). E: H&E staining and IHC was performed on human control lung tissue (n = 3 control patient specimens) using antibodies to S100A4, SSEA4, and CD163. Bar = 50 μm (H&E) and 20 μm (IHC).