Circulating monocytes from systemic sclerosis patients with interstitial lung disease show an enhanced profibrotic phenotype

Abstract

Profibrotic cells derived from circulating CD14+ monocytes include fibrocytes and alternatively activated macrophages. These cells are associated with interstitial lung disease (ILD) and are implicated in the pathogenesis of systemic sclerosis (SSc); however, the simultaneous presence of profibrotic cells and their associated mediators in the circulation of these patients has not been defined. We hypothesized that monocytes from patients with SSc-related ILD (SSc-ILD) would show profibrotic characteristics when compared with normal controls. We recruited patients with SSc-ILD (n=12) and normal controls (n=27) and quantified circulating collagen-producing cells by flow cytometry for CD45 and pro-collagen I. The in vitro activation potential of CD14+ monocytes in response to lipopolysaccharide was assessed using flow cytometry for CD163, and by ELISA for CCL18 and IL-10 secretion. Profibrotic mediators in plasma were quantified using Luminex-based assays. The concentration of circulating collagen-producing cells was increased in the SSc-ILD patients when compared with controls. These cells were composed of both CD34+ fibrocytes and a population of CD34+CD14+ cells. Cultured CD14+ monocytes from SSc-ILD patients revealed a profibrotic phenotype characterized by expression of CD163 and by enhanced secretion of CCL18 and IL-10 in response to proinflammatory activation. Plasma levels of IL-10, MCP-1, IL-1RA, and TNF levels were significantly elevated in the plasma of the SSc-ILD cohort. Subgroup analysis of the normal controls revealed that unlike the subjects < or =35 years, subjects > or =60 years old showed higher levels of circulating CD34+CD14+ cells, collagen-producing CD14+ monocytes, CD163+ monocytes, IL-4, IL-10, IL-13, MCP-1, and CCL18. These data indicate that the blood of patients with SSc-ILD and of healthy aged controls is enriched for fibrocytes, profibrotic monocytes, and fibrosis-associated mediators. Investigations defining the factors responsible for this peripheral blood profile may provide new insight into SSc-ILD as well as the pathophysiology of aging.

Figure 1. CD45⁺/pro-Col-1α⁺ cells in SSc-ILD

A: CD45-PE vs. intracellular isotype control + anti-rat-FITC analyses on blood from a representative normal subject. This control was used to set negative gates. X-axis: rat IgGk intracellular isotype. Y-axis: anti-CD45-PE. B: CD45⁺/pro-Col-1α⁺ cells in normal blood. X-axis: pro-Col-1α stained with anti-rat secondary antibody. Y-axis: anti-CD45-PE. C: Anti CD45-PE vs. intracellular isotype control and anti-rat FITC performed on blood from patient with SSc-ILD. This control was used to set negative gates for the SSc-ILD sample. X-axis: rat IgGk intracellular isotype. Y-axis: anti-CD45-PE. D: CD45+/pro-Col-1α⁺ cells in SSc-ILD blood, same axes as 1B. E: FSC vs. SSC analysis of PBMCs from the sample in (D). F: FSC vs. SSC analysis of PBMCs from the double positive population in (D). G. Comparison of fibrocyte percentages (left axis) and quantities (right axis) in normal (n = 27, gray bar) and SSc-ILD subjects (n=12, black bar). Fibrocyte quantities but not percentages are increased in SSc-ILD. H: Comparison of lymphocytes (dotted bar), monocytes (checked bar) and fibrocytes (black bar) fibrocyte concentrations in normal control (left) and SSC-ILD blood (right). Monocyte quantities are increased in patients with SSc-ILD. Data are expressed as mean ± SEM. *p<0.05, *p<0.01.

Figure 2. Alternative macrophage activation in SSc-ILD

A, B: Light microscopy of CD14+ peripheral blood monocytes that were cultured in the absence (A) and presence (B) of LPS. C: Overlay of histograms of CD163 staining on cultured CD14+ monocytes. Brown: young control, no LPS. Dark blue: young control +LPS. Green: aged control, no LPS. Red: aged control, +LPS. Light blue: SSc-ILD, no LPS. Purple: SSc-ILD, + LPS. The pink bar indicates the positive gate. D: Percentage of CD14+ cells that express CD163 following 48 hours in culture. White bar: control, black bar: SSc-ILD. E: CCL-18 secretion following 48 hours in culture. White bar: control, black bar: SSc-ILD. *p<0.05, **p<0.01, ***p<0.001. F: IL-10 secretion following 48 hours in culture. White bar: control (n = 27). Black bar: SSc-ILD (n = 12).*p<0.05, **p<0.01, ***p<0.001. All data are expressed as mean ± SEM.

Figure 3

Multianalyte ELISA comparisons of plasma cytokines in control (n = 27, left) vs. SSc-ILD subjects (n = 11, right). A: IL-13 B: IL-4. C: IFN-γ. D: IL-10 E: MCP-1. F: TNFα. G: IL-1 RA. H: CCL18. I: M-CSF. All data are expressed as pg/ml (mean ± SEM). *p<0.05. **p<0.01. ***p<0.001.

Figure 4

Subgroup analysis of peripheral blood monocyte subpopulations and fibrogenic mediators in young vs. aged controls. A: Staining with anti-CD45-PE vs. intracellular isotype control antibody used to set negative gates. B:CD45⁺/pro-Col-1α⁺ staining (CD45-PE vs. pro-Col-Iα) in young healthy control. C: Anti-CD45-PE vs intracellular isotype control antibody used to set negative gates. D: CD45+/pro-Col-1α⁺ staining (CD45-PE vs. pro-Col-Iα) in aged healthy control. E: Fibrocyte percentages (left axis) and quantities (right axis) in young subjects (white bar), aged subjects (black bar) and SSc-ILD patients (slanted lines). F: Comparison of lymphocytes (dotted line), monocytes (checked bar) and CD45⁺/pro-Col-Iα⁺ cells (black bar) in (left to right) young, aged, and SSc-ILD subjects. G: CD14+ monocyte expression of CD163 after 48 hours in culture. H-O: Concentrations of the plasma cytokines IL-4 (H), IL-10 (I), IL-13 (J), IL-1 RA (K), MCP-1 (L), TNFα (M), CCL-18 (N) and M-CSF (O) in young controls (n = 10, white bar), aged controls ( n = 12, black bar) and patients with SSc-ILD (n = 11, slanted bar). All data are expressed as mean ± SEM. *p<0.05. **p<0.01. ***p<0.001.

Figure 5

Phenotype of CD45⁺/pro-Col-1α⁺ cells in SSc-ILD and aged, healthy controls A. PerCP isotype control (X axis) vs. Anti-CD45-APC (Y axis).This control was used to set the negative gate for PerCP. B. Anti-CD45-PerCP isotype control (X axis) vs. APC isotype control (Y axis). This control was used to set the negative gate for APC. C. Anti-pro-Col-Iα (X axis) vs. anti-CD45 (Y axis) on PBMCs from a subject > 60 years old. D. Anti-CD34 (X axis) vs anti-CD14 (Y axis) on the double positive population seen in C. E. Anti-pro-Col-1α (X axis) vs. anti-CD45 (Y axis) on PBMCs from a patient with SSc-ILD. F. Anti-CD34 (X axis) vs anti-CD14 (Y axis) on the double positive population seen in E. G-J: Comparison of CD34+ CD14- (G), CD14+ CD34+ (H), CD34-CD14+ (I) and CD14-CD34- (J) Populations in the pro-Col-1α⁺ cells obtained from (left to right) subjects greater than 60 years old (n = 10, black bar) and those with SSc-ILD (n = 7, slanted bar). Data are expressed as mean ± SEM. *p<0.05, **p<0.01.