The effects of repeated Toll-like receptors 2 and 4 stimulation in COPD alveolar macrophages

Abstract

Background: COPD is a progressive inflammatory airway disease characterized by increased numbers of alveolar macrophages in the lungs. Bacterial colonization of the lungs is a common feature in COPD and can promote inflammation through continual and repeated Toll-like receptor (TLR) stimulation. We have studied the response of COPD alveolar macrophages to repetitive stimulation with TLR2 and TLR4 ligands. We investigated the effect of sequential stimulation with different ligands to determine whether this results in tolerance or amplification of the immune response.

Methods: We stimulated alveolar macrophages from COPD patients (n=9) and smokers (n=8) with the TLR4 agonist lipopolysaccharide (LPS) or the TLR2 agonist Pam3CSK4 for 24 hours before restimulating again for 24 hours. Cytokine protein release and gene expression were investigated.

Results: Repetitive stimulation of COPD and smokers macrophages with LPS for both 24-hour periods caused a reduction in tumor necrosis factor α, CCL5, and IL-10 production compared to cells that were not exposed initially to LPS. IL-6 and CXCL8 production were not significantly altered following repetitive LPS stimulation. The same pattern was observed for repeated stimulation with Pam3CSK4. Using COPD macrophages, LPS followed by Pam3CSK4 stimulation increased the levels of all cytokines compared to media followed by Pam3CSK4.

Conclusion: TLR tolerance in COPD alveolar macrophages occurs after repetitive stimulation with the same TLR ligand, but this only occurs for selected cytokines. CXCL8 production is not reduced after repetitive TLR stimulation with the same ligand; this may be an important mechanism for the increased CXCL8 levels that have been observed in COPD. We showed that TLR4 stimulation followed by TLR2 stimulation does not cause tolerance, but enhances cytokine production. This may be a relevant mechanism by which bacteria cause excessive inflammation in COPD patients.

Keywords: COPD; alveolar macrophages; tolerance.

Figure 1

LPS pre-stimulation results in differential desensitization of cytokine and chemokine release following subsequent LPS stimulation. Alveolar macrophages from smoking controls (n=7) and COPD patients (n=9) were cultured in media (M) or LPS (L) (1 μg/mL) for 24 hours before washing and restimulating in the presence or absence of LPS for further 24 hours. Supernatants were removed and TNFα (A), CCL5 (B), IL-10 (C), CXCL8 (D), and IL-6 (E) were quantified. Paired t-tests were carried out to compare conditions ML versus LL and LL versus LM. Bars indicate statistical significance was reached. *p<0.05 and **p<0.01. Abbreviations: LPS, lipopolysaccharide; TNF, tumor necrosis factor.

Figure 2

TLR2 pre-stimulation results in differential desensitization of cytokine and chemokine release following subsequent TLR2 stimulation. Alveolar macrophages from COPD (n=8) patients were cultured in media (M) or Pam3CSK4 (P) (0.1 μg/mL) for 24 hours before washing and restimulating in the presence or absence of Pam3CSK4 (0.1 μg/mL) for further 24 hours. Supernatants were removed and TNFα (A), CCL5 (B), IL-10 (C), CXCL8 (D), and IL-6 (E) were quantified. Paired t-tests were carried out to compare PP versus MP and PP versus PM. Bars indicate statistical significance was reached. *p<0.05 and **p<0.01. Abbreviations: TLR, Toll-like receptor; TNF, tumor necrosis factor.

Figure 3

LPS tolerance results in the differential desensitization of TNFα and CXCL8 gene expression. COPD alveolar macrophages were cultured in media or LPS (1 μg/mL) for 24 hours before washing and restimulating with LPS (1 μg/mL) for further 4 or 24 hours as indicated. Cells were harvested in TRIzol and TNFα (A) and CXCL8 (B) gene expression was measured by qPCR and normalized to GAPDH levels (n=6). Data show mean ± SEM fold induction compared to non-stimulated time-matched controls. Paired t-tests were carried out to compare each condition to ML. **p<0.01. Abbreviations: LPS, lipopolysaccharide; TNF, tumor necrosis factor.

Figure 4

The effects of LPS, Pam3CSK4, and UPLPS on TLR2 and TLR4 expression. COPD alveolar macrophages were left untreated or stimulated with LPS (1 μg/mL; n=6) (A), Pam3CSK4 (0.1 μg/mL) (B), or UPLPS (0.1 μg/mL; n=5 different donors) (C) for 4, 6, 24, and 48 hours. TLR2 and TLR4 gene expression was measured by qPCR and fold change was normalized to GAPDH. Paired t-tests were carried out to compare fold induction to unstimulated time-matched controls. *,**Indicates significantly increased above unstimulated time-matched control (p<0.05 and p<0.01, respectively). ^#,##Indicates significantly decreased below unstimulated time-matched control (p<0.05 and p<0.01, respectively). Abbreviations: LPS, lipopolysaccharide; TLR, Toll-like receptor; NS, no stimulation.

Figure 5

LPS pre-stimulation enhances the effect of Pam3CSK4 on the production of IL-6, IL-10, CXCL8, and CCL5. COPD (n=8) alveolar macrophages were cultured in media (M), Pam3CSK4 (P) (0.1 μg/mL), or LPS (L) (1 μg/mL) for 24 hours before washing and restimulating in the presence or absence of M or Pam3CSK4 (0.1 μg/mL) for further 24 hours. Supernatants were removed and TNFα (A), CCL5 (B), IL-10 (C), CXCL8 (D) and IL-6 (E) were quantified. Paired t-tests were carried out to compare MP versus LP and LM versus LP. Bars indicate statistical significance was reached, *p<0.05. Abbreviations: LPS, lipopolysaccharide; TNF, tumor necrosis factor.