Gram positive and Gram negative bacteria synergise to cause nitric oxide synthase induction in murine macrophages J. Anandarajah*, M.R. Fleet*, M.J. Walters*, P.E. Belcher*, S. Sriskandan1; J.A. Mitchell*.*Unit of Critical Care, The National Heart and Lung Institute, Imperial College, London, SW3 6LY, UK. 1Dept Infectious Diseases, Imperial College, Hammersmith Hospital W12 0HS.

Print abstract Search PubMed for: Anandarajah J Fleet MR Walters MJ Belcher PE Sriskandan S Mitchell JA

The induction of nitric oxide synthase II (NOS II, iNOS) and subsequent production of free radical gas NO is an important anti-infectious mechanism of innate immunity, in which macrophages play a significant role (Trajkovic, 2001). Murine macrophages exposed to lipopolysaccharide (LPS), a component of Gram negative bacteria, release large amounts of NO which contributes to their bacteriocidal activity (Di Rosa et al, 1990). However the role of NO in the cytolysis of Gram positive organisms is unknown. Furthermore, we have recently shown that by contrast to LPS or Gram negative Escherichia.coli, Gram positive Staphylococcus aureus does not induce murine macrophages to release NO, despite stimulating TNF release (Belcher et al, 2003). Components of S. aureus. (peptidoglycan G and lipoteichoic acid) have been shown to act synergistically with LPS to induce the release of a number of cytokines from murine and human macrophages (Wang et al, 2001). However, the nature of any interaction between whole S. aureus and LPS is not well understood.

Murine derived J774 macrophages were cultured in 96-well plates as described previously (Mitchell et al., 1992) and stimulated for 24hrs with LPS (0.01 to 10µg/ml), heat killed S. aureus (1.21 x 10⁸ to 1.21 x 10¹⁰ cfu/ml), or LPS plus S. aureus. In some experiments the NOS inhibitor L-N^G-nitro-L-arginine (L-NAME; 1mM) or D-NAME (1mM) was added to the cells just prior to LPS or S. aureus respectively. NO was measured by the Greiss reaction.

LPS induced nitrite formation in a concentration dependant manner (Figure 1). Nitrite release was inhibited by L-NAME (by 82.3±4.3% at 10µg/ml LPS; n=12) but not D-NAME (18.2±6.9%). Whole, heat killed S. aureus did not induce nitrite release from J774 cells at concentrations up to 1.21 x 1010 cfu/ml.

However, at 1.21 x 109 CFU, S. aureus synergised with LPS to cause an increase in nitrite release by J774 cells (Figure 1).

Figure 1; S. aureus and LPS synergise to induce nitrite release from J774 macrophages. The figure shows the mean±s.e.m. for n=12 wells from 4 separate experimental days. * represents significant differences between cells treated with or without S. aureus (p<0.05; two-way ANOVA)

Here we show that whilst LPS clearly induces NOS activity and NO release by murine macrophages, S. aureus has no effect. This observation is consistent with our understanding that Gram positive and Gram negative bacteria activate different pathways in the innate immune response (Gottar et al. 2002). However, we also show a profound synergistic relationship between S. aureus and LPS. This data suggests that where Gram positive infections co-incide with sub clinical levels of Gram negative bacteria or LPS, macrophages are activated via both pathways.

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This work was supported by grants from the MRC and BHF.