Cigarette smoke inhibits macrophage sensing of Gram-negative bacteria and lipopolysaccharide: relative roles of nicotine and oxidant stress

Abstract

Background and purpose: Smoking cigarettes is a major risk factor for the development of cardiovascular and respiratory disease. Moreover, smokers are more prone to infections. This has been associated with a suppression of the immune system by smoke. However, it is not clear how cigarette smoke affects the ability of immune cells to sense pathogens. Cigarette smoke contains a large number of molecules which may mediate responses on immune cells and of these, nicotine and oxidants have both been identified as inhibitory for the sensing of bacterial lipopolysaccharide (LPS). Nitric oxide synthase (NOS) and tumour necrosis factor (TNF)-alpha are both induced in macrophages on stimulation with Gram negative bacteria or LPS.

Experimental approach: We used murine macrophages stimulated with whole heat-killed bacteria or LPS. We measured output of NO (as nitrite) and TNFalpha, NOS protein by Western blotting and cellular oxidant stress.

Key results: Cigarette smoke extract suppressed the ability of murine macrophages to release NO, but not TNFalpha in response to whole bacteria. Cigarette smoke extract also inhibited nitric oxide synthase II protein expression in response to LPS. The effects of cigarette smoke extract on nitrite formation stimulated by LPS were unaffected by inhibition of nicotinic receptors with alpha-bungarotoxin (100 units ml(-1)). However, the effects of cigarette smoke extract on LPS-induced nitrite formation were mimicked by hydrogen peroxide and reversed by the anti-oxidants N-acetyl cysteine and glutathione.

Conclusions and implications: We suggest that cigarette smoke exerts its immunosuppressive effects through an oxidant-dependent and not a nicotine-dependent mechanism.

Figure 1

Effect of Gram-negative P. aeruginosa and E. coli on nitrite and TNFα formation by J774.2 macrophages. A range of concentrations of P. aeruginosa or E. coli were added to the cells for 24 h and (a) nitrite and (b) TNFα levels were measured. The data are the mean±s.e.mean; n=12.

Figure 2

Effect of cigarette smoke extract (CSE) on nitrite formation induced by Gram-negative bacteria or LPS. (a) E. coli (10⁸ CFU ml⁻¹), (b) P. aeruginosa (10⁸ CFU ml⁻¹) or (c) LPS (1 μg ml⁻¹) were added to J774 macrophages for 24 h in the presence or absence of CSE (1.25–5%). The data are the mean±s.e.mean; n=3–9 experiments. Analysis was carried out using one-way ANOVA followed by a Bonferroni post-test. ^*P<0.05 vs stimulus alone.

Figure 3

Effect of cigarette smoke extract (CSE) on NOSII protein expression. (a) A typical western blot for NOSII expression in J774.2 macrophages stimulated with LPS (1 μg ml⁻¹) for 24 h in the presence or absence of CSE (5%). (b) Pooled data of the relative absorbance from four separate western blots. The data are normalized to the absorbance seen in cells treated with LPS (100%). The data were analysed using a one-sample t-test for normalized data. ^*P<0.05 vs LPS alone.

Figure 4

Effect of cigarette smoke extract (CSE) on TNFα release from cells stimulated with (a) E. coli or (b) S. aureus. Bacteria (10⁸ CFU ml⁻¹) in the presence or absence of CSE (0.16–5%) were added to cells for 24 h. The data are the mean±s.e.mean; n=5–9 experiments. Analysis was carried out using one-way ANOVA followed by a Bonferroni post-test.

Figure 5

Effects of nicotine-related drugs on LPS (1 μg ml⁻¹) and cigarette smoke extract (CSE) induced responses in macrophages. (a) Cells were treated with LPS in the presence or absence of nicotine (Nic; 10 nM), ACh (Ach; 100 μM) or α-bungarotoxin (Bung; 100 U ml⁻¹) for 24 h and NO formation was measured by the levels of nitrite in culture medium. (b) Cells were treated with LPS (1 μg ml⁻¹) and CSE (2.5%) in the presence and absence of α-bungarotoxin (Bung, 0.01–10 U ml⁻¹) for 24 h. Data are the means±s.e.mean; n=3–12 experiments. Analysis was carried out using one-way ANOVA followed by a Bonferroni post-test.

Figure 6

Role of oxidant stress in the effects of cigarette smoke extract (CSE) on NOSII activity in J774 macrophages. (a) Macrophages were treated with CSE for 10 min before extraction and measurement of rhodamine-123. Data are the mean±s.e.mean; n=3 experiments. Data are normalized to control levels and statistical differences are measured using one-sample t-test for normalized data. ^*P<0.05 vs control. (b) Cells were treated with LPS (1 μg ml⁻¹) in the presence and absence of CSE (2.5%) with and without increasing concentrations of N-acetylcysteine (NAC). The data are the mean±s.e.mean; n=12–21 experiments. Data were analysed by one-way ANOVA followed by a Bonferroni post-test. ^*P<0.05 vs (LPS+CSE).

Figure 7

Effect of oxidant stress and antioxidants on LPS-induced NO release (measured as nitrite) by J774.2 macrophages. (a) Rhodamine-123 was measured as in Figure 6 under basal conditions and after 10 min stimulation with H₂O₂ (1 mM). Data are normalized to control levels and statistical differences are measured using one-sample t-test for normalized data. (b) J774.2 macrophages were treated with increasing concentrations of H₂O₂ (0.3–10 mM) and LPS (1 μg ml⁻¹) for 24 h in the presence or absence of N-acetylcysteine (NAC 1 mM). The data are the means±s.e.mean for three experiments. Data were analysed by two-way ANOVA. ^*P<0.05 between LPS alone and LPS+NAC.

Figure 8

Comparison of units of assessment of the ‘strength' of CSE. Cigarette smoke extract (CSE) was prepared in Dulbecco's modified Eagle's medium as described in the Methods section. The absorbance at 320 nm (a), the nitrite content (b) or the oxidant potential (c) of solutions of CSE over the range of dilutions shown were measured according to protocols described in the Methods section. The data shown are means±s.e.mean; n=3.