Cigarette smoke inhibits the induction on NOSII activity in murine macrophages stimulated with gram negative bacteria 1M.R. Fleet, 1J. Anandarajah, 1M.J. Walters, 1P.E. Belcher, 2S. Sriskandan & 1J.A. Mitchell. 1Unit of Critical Care, The National Heart and Lung Institute, Imperial College, London, SW3 6LY, UK. 2Dept. of Infectious Diseases, Hammersmith Hospital, Imperial College, London, W12 0NN, UK.

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Macrophages produce nitric oxide (NO) in response to many stimulants, including lipopolysaccharide (LPS) (Shultz, et al., 1991). The production of NO is believed to have bacteriostatic effects, preventing or limiting infection (Klebanoff, 1993). Bacterial infection is particularly important in chronic obstructive pulmonary disease (COPD). These patients have impairment of their local immune defence which allows bacterial pathogens to gain a foothold in the lower respiratory tract (Sethi, 2000). Since the most important risk factor for developing COPD is smoking, we have looked at the effect of cigarette smoke on NO synthase (NOS) activity induced by Gram negative bacteria or bacterial LPS by murine J774 macrophages.

Cigarette smoke was bubbled from four cigarettes (full strength Marlboro) through 100mls of DMEM, (100% CSE solutions; Walters & Mitchell, 2003) and the 'strength' determined by measuring nitrite levels using the Greiss reaction (Bishop-Bailey, et al., 1997). Each solution was added immediately to the cells. The cells were then stimulated with either Escherichia coli (E. coli) (8.85*10⁸ CFU/ml) or LPS (1µg/ml). In some experiments the mixed NOS inhibitor, L-N^G-nitro-L-arginine (L-NAME; 1mM) or the selective NOSII inhibitor, 1400W (100µM), were added to the cells. Since cigarette smoke contains NO and other oxides of nitrogen, the nitrite values of 'dummy plates' identical, except devoid of cells, were prepared for each protocol and subtracted from those produced by plates containing cells. NOS activity was determined by nitrite release. TNF levels were measured using ELISAs. Incubations were for 24hrs. None of the treatments shown affect cell viability (measured by the conversion of MTT to formazan; Bishop-Bailey et al., 1997).

Cells stimulated with whole heat killed E. coli (8.85*10⁷-2.82*10⁹ CFU/ml) or LPS (0.1-100µg/ml) released detectable nitrite with respective E_max values of 25.0± 8.6µM and 21.7±10.8µM (n=9 wells from 3 experimental days). Nitrite release induced by either LPS (0.1µg/ml) or E. coli (8.85*10⁸CFU/ml) was respectively inhibited by either L-NAME (by 69.2±5.8% & 49.6±3.2%) or 1400W (by 82.1 ±2.4% & 85.8±1.5%), (n=3-6). CSE caused concentration dependent inhibitions of nitrite release induced by either LPS or E. coli (Fig.1).

Figure 1: E. coli (A) or LPS (B) stimulate nitrite release which is inhibited by CSE. The data shown is the mean ± s.e.m. for n= 9 wells from n=3 experimental days. All statistics shown are a One-way ANOVA with Dunnett's multiple comparison test.

Similarly, J774 macrophages released increased levels of TNFafter stimulation with either LPS or E. coli (basal 0±84.6pg/ml; plus LPS, 4,465±2,445pg/ml; plus E. coli, 14,727±209pg/ml). LPS induced TNFrelease was inhibited by smoke extract (by 82.8±23% at 5% CSE, n=9). By contrast, E. coli induced TNF release was not affected by CSE (5%), (111±27% of control, n=9).
It is possible that the inhibitory effect of cigarette smoke on macrophage immune function contributes to bacterial colonisation in the lungs in patients with COPD.

Acknowledgement: This work was supported by the M.R.C.
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