Bacterial meningitis is a disease worsened by neutrophil-induced damage in the subarachnoid space. In this study, the A2A adenosine receptors on human neutrophils were characterized, and the role of A2A receptors on the trafficking of leukocytes to the cerebrospinal fluid and on blood-brain barrier permeability (BBBP) was assessed in a rat meningitis model. Neutrophils bind the A2A selective antagonist, 125I-ZM241385 (Bmax=843 receptors/neutrophil; KD=0.125 nM). A selective A2A receptor agonist, WRC-0470 (2-cyclohexylmethylidene-hydrazinoadenosine; 0.03-1 microM), alone and synergistically with the type IV phosphodiesterase inhibitor, rolipram, increased neutrophil [cAMP]i and reduced cytokine-enhanced neutrophil adherence, superoxide release, and degranulation. These effects of WRC-0470 were reversed by ZM241385 (100 nM). In a lipopolysaccharide-induced rat meningitis model, WRC-0470 (0-0.9 microgram/kg/h), with or without rolipram (0-0.01 microgram/kg/h), inhibited pleocytosis and reduced the lipopolysaccharide-induced increase in BBBP, indicative of decreased neutrophil-induced damage.

The role of nitric oxide (NO) radicals in killing the intracellular bacterial pathogen Legionella pneumophila (Lp) was examined in infected macrophages. Murine (RAW 264.7) and human (HL-60) cell monolayers were treated with 100 U/ml gamma-interferon (IFN) and cocultured with Lp in the presence and absence of NGMMA, a specific inhibitor of NO production. Viable Lp in IFN-treated RAW 264.7 cells decreased from 3.8 to 0.7 +/- 0.12 log CFU/ml after 24 h incubation, whereas in IFN+NGMMA-treated RAW 264.7 cells, viable Lp persisted at 2.2 +/- 0.2 log CFU/ml after 24 h. This increased survival corresponded with an inhibition of NO production (5.65 +/- 2.99 microM with NGMMA vs. 58.6 +/- 5.36 microM without NGMMA). Viable Lp were susceptible to killing, in a dose-dependent fashion, by 0, 2.5, and 5.0 mM sodium nitroprusside, a source of NO radicals. IFN-treated RAW 264.7 cells also had significantly decreased levels of intracellular iron (below assay limit) when compared to IFN+NGMMA-treated cells (72.0 +/- 0.78% of control). Normally permissive HL-60 cells treated with IFN were bacteriostatic rather than bactericidal, and NO production was not detected above background. Thus, NO radicals play a critical role in the bactericidal activity against Lp by IFN-treated RAW 264.7 cells, but the absence of NO production limits IFN-treated HL-60 cells to bacteriostasis.

We have investigated the possible role of nitric oxide (NO) in the pathophysiology of bacterial meningitis (BM) by using the rat model of experimental BM. The nitrite concentration in cerebrospinal fluid (CSF) was used as a measure of NO production in vivo since NO rapidly degrades to nitrite and nitrate. Rats were inoculated intracisternally with live bacteria (5 ؋ 10 6 CFU of Haemophilus influenzae type b strain DL42 or Rd ؊ /b ؉ /O2), with bacterial endotoxin (20 ng of DL42 lipooligosaccharide [LOS] or 200 ng of Escherichia coli lipopolysaccharide), or with a saline control vehicle. CSF samples were collected preinoculation and at the time of maximal alteration in blood-brain barrier permeability (BBBP). CSF [nitrite] was quantified by measuring A 550 after addition of the Greiss reagent and comparison to a standard curve of sodium nitrite. Rats inoculated with either DL42, Rd ؊ /b ؉ /O2, LOS, or lipopolysaccharide demonstrated a significantly elevated mean peak CSF [nitrite] (8.34, 15.62, 10.75, and 10.44 mM, respectively) versus the concentration prior to treatment and/or those in saline-treated animals (5.29 and 5.33 mM, respectively; P < 0.05 for each comparison). We then determined if there was a correlation between CSF [nitrite] and percent BBBP (%BBBP) at various time points postinoculation with Rd ؊ /b ؉ /O2. %BBBP was defined as the concentration of systemically administered 125

Under certain circumstances, soluble antigens, particulate antigens, and/or microorganisms have been shown to bind to primate erythrocytes via complement receptor 1 (CR1) in the presence of specific antibodies and complement. This immune adherence reaction, specific for CR1, can lead to neutralization of antigens in the circulation and their subsequent clearance from the blood. The present experiments utilized cross-linked monoclonal antibody complexes (heteropolymers) with specificity for both CR1 and either 35 S-labeled herpes simplex virus capsid or Haemophilus influenzae as prototype viral and bacterial particulate antigens, respectively. In each case, the respective specific heteropolymers facilitated binding of the target antigens (Ն70 to 90%) in vitro to erythrocytes in the absence of complement. Several experimental protocols were employed to demonstrate that heteropolymers mediate specific, rapid (Ն30 s), and quantitative binding of prototypical particulate pathogens to human and monkey erythrocytes but not to sheep erythrocytes, which lack CR1. These results extend the potential use of the erythrocyte-heteropolymer system to the neutralization and clearance of particulate viral and bacterial pathogens from the blood.