Effects of Active Chronic Cigarette-Smoke Exposure on Circulating Fibrocytes

Abstract

Purpose: This study aimed to evaluate the hypothesis that active smoking impacts upon mediators and abundance of circulating fibrocyte cells in smoking-related disease characterised by fibrosis.

Methods: Flow cytometry and enzyme-linked immunosorbent assays were used to investigate blood from five patient groups: healthy never-smokers, healthy current smokers, stable chronic obstructive pulmonary disease (COPD) active smokers, idiopathic pulmonary fibrosis (IPF) never-smokers, and IPF active smokers.

Results: A significant inverse dose-response relationship was observed in healthy smokers among cumulative smoking burden (pack-years) and fibrocyte abundance (p = 0.006, r = -0.86). Among serum profibrotic fibrocyte chemokines measured, CCL18 rose significantly alongside fibrocyte numbers in all five subject groups, while having an inverse dose-response relationship with pack-year burden in healthy smokers (p = 0.003, r = -0.89). In IPF, CCL2 rose in direct proportion to fibrocyte abundance irrespective of smoking status but had lower serum levels in those currently smoking (p = < 0.001). For the study population, CXCL12 was decreased in pooled current smokers versus never-smokers (p = 0.03).

Conclusion: The suppressive effect of current, as distinct from former, chronic smoking on circulating fibrocyte abundance in healthy smokers, and modulation of regulatory chemokine levels by active smoking may have implications for future studies of fibrocytes in smoking-related lung diseases as a potential confounding variable.

Keywords: Chronic obstructive pulmonary disease; Circulating fibrocyte; Idiopathic pulmonary fibrosis; Smoking.

Fig. 1

Expression of CD45, collagen I (Col I), and CXCR4 in peripheral circulating leucocytes—Representative flow cytometric analysis from a patient with COPD. A The total cell population was analysed for PerCP stained CD45 expression. B This gate was then applied to the CD45 positive cells which are live, i.e. 99.9%. C Live CD45 positive cell population was further evaluated for APC-stained Col I positive cells. D Live CD45, Col-I positive cell population was further gated down to BV450 stained CXCR4 positive cells, i.e. 3776 events of 957,460 CD45+ cells are alive Col1+ and CXCR4+ (0.39%) called fibrocytes. E The isotope control for APC-stained Col I cells. F The isotype control for BV450 stained CXCR4 cells

Fig. 2

Effect of chronic smoke exposure on circulating fibrocyte abundance. A Relationship between cumulative burden of tobacco (expressed as cigarette pack-years) and fibrocyte abundance (% of total circulating leukocytes) in healthy smokers. B Prevalence of fibrocytes (% of total circulating leukocytes) in healthy subjects assessed by smoking status. C Prevalence of fibrocytes (% of total circulating leukocytes) in IPF non-smokers and IPF smokers

Fig. 3

Role of Serum CCL18 in smoking-induced fibrocyte regulation in healthy subjects. A Relationship between serum CCL18 and circulating fibrocytes as a percentage of total leukocytes among healthy non-smokers. B Relationship between serum CCL18 and circulating fibrocytes as a percentage of total leukocytes among healthy smokers. C Serum CCL18 levels in healthy non-smokers versus healthy smokers. D Relationship of serum CCL18 levels to cigarette pack-year burden in the healthy smoker group. E Relationship between serum CCL18 and circulating fibrocytes as a percentage of total leukocytes among COPD smokers

Fig. 4

Association of serum CCL2 with circulating fibrocyte abundance and smoking status in idiopathic pulmonary fibrosis. A Concentration of serum CCL2 among healthy smokers, IPF smokers, and COPD smokers. B Relationship of serum CCL2 to circulating fibrocyte prevalence among IPF non-smokers and IPF smokers. C Concentration of serum CCL2 in IPF non-smokers and IPF smokers

Fig. 5

Role of Serum CXCL12 in smoking-induced fibrocyte regulation in healthy subjects, IPF and COPD patients. A Concentration of serum CXCL12 in IPF and non-IPF groups. B Relationship between serum CXCL12 and circulating fibrocytes in IPF-Non Smokers. C Relationship between serum CXCL12 and circulating fibrocytes in COPD smokers. D Concentration of serum CXCL12 among IPF non-smokers VS IPF smokers. E Concentration of serum CXCL12 in healthy non-smokers and healthy smokers. F Concentration of serum CXCL12 in all smokers VS all non-smokers