Influence of age, past smoking, and disease severity on TLR2, neutrophilic inflammation, and MMP-9 levels in COPD

Abstract

Chronic obstructive pulmonary disease (COPD) is a common and serious respiratory disease, particularly in older individuals, characterised by fixed airway obstruction and persistent airway neutrophilia. The mechanisms that lead to these features are not well established. We investigated the contribution of age, prior smoking, and fixed airflow obstruction on sputum neutrophils, TLR2 expression, and markers of neutrophilic inflammation. Induced sputum from adults with COPD (n = 69) and healthy controls (n = 51) was examined. A sputum portion was dispersed, total, differential cell count and viability recorded, and supernatant assayed for CXCL8, matrix metalloproteinase- (MMP-) 9, neutrophil elastase, and soluble TLR2. Peripheral blood cells (n = 7) were stimulated and TLR2 activation examined. TLR2 levels were increased with ageing, while sputum neutrophils and total sputum MMP-9 levels increased with age, previous smoking, and COPD. In multivariate regression, TLR2 gene expression and MMP-9 levels were significant independent contributors to the proportion of sputum neutrophils after adjustment for age, prior smoking, and the presence of airflow obstruction. TLR2 stimulation led to enhanced release of MMP-9 from peripheral blood granulocytes. TLR2 stimulation activates neutrophils for MMP-9 release. Efforts to understand the mechanisms of TLR2 signalling and subsequent MMP-9 production in COPD may assist in understanding neutrophilic inflammation in COPD.

Figure 1

Analysis of the effect of age on markers of neutrophilic inflammation. This analysis used healthy never-smoking controls comparing younger (<55 years of age) with older (>55 years of age) controls. (a) Neutrophil number ×10⁴/mL, (b) CXCL8 protein ng/mL, (c) MMP-9 protein ng/mL, (d) NE protein ng/mL, (e) TLR2 gene expression, and (f) soluble TLR2 ng/mL.

Figure 2

Analysis of the effect of smoking on markers of neutrophilic airway inflammation. This analysis used healthy controls, comparing never smokers with exsmokers. (a) Neutrophil number ×10⁴/mL, (b) CXCL8 protein ng/mL, (c) MMP-9 protein ng/mL, (d) NE protein ng/mL, (e) TLR2 gene expression, and (f) soluble TLR2 ng/mL.

Figure 3

Analysis of the effect of the presence of airflow obstruction on markers of neutrophilic inflammation. This analysis compared older healthy controls with participants with COPD. (a) Neutrophil number ×10⁴/mL, (b) CXCL8 protein ng/mL, (c) MMP-9 protein ng/mL, (d) NE protein ng/mL, (e) TLR2 gene expression, and (f) soluble TLR2 ng/mL.

Figure 4

Analysis of the effect of the severity of airflow obstruction on markers of neutrophilic inflammation. This analysis compared participants with moderate and severe COPD. (a) Neutrophil number ×10⁴/mL, (b) CXCL8 protein ng/mL, (c) MMP-9 protein ng/mL, (d) NE protein ng/mL, (e) TLR2 gene expression, and (f) soluble TLR2 ng/mL.

Figure 5

TLR2 surface and gene expression following TLR2 stimulation using Pam3CysK4: (a) mononuclear cells TLR2 surface expression, (b) mononuclear cells TLR2 gene expression, (c) granulocyte TLR2 surface expression, and (d) granulocyte TLR2 gene expression.

Figure 6

Release of inflammatory mediators from mononuclear cells and granulocytes isolated from patients with COPD. (a) CXCL8 release from mononuclear cells, (b) IL-6 release from mononuclear cells, (c) IL-1β release from mononuclear cells, (d) TNF-α  release from mononuclear cells, (e) CXCL8 release from granulocytes, (f) IL-6 release from granulocytes, (g) MMP-9 release from granulocytes, and (h) NE release from granulocytes.