Respiratory syncytial virus induces interleukin-10 by human alveolar macrophages. Suppression of early cytokine production and implications for incomplete immunity.

Respiratory syncytial virus (RSV) causes repeated infections thought to be due to an ineffective immune response. We examined the hypothesis that incomplete immunity may result, in part, from RSV-infected alveolar macrophage production of IL-10 which can interfere with the production of immunoregulatory cytokines. We also assessed whether RSV induced the expression of the 2',5' oligoadenylate (2-5A)-dependent RNase L, an endoribonuclease involved in the antiviral activities of interferons. Human alveolar macrophages were exposed to medium (uninfected control), RSV, LPS, and RSV + LPS then were assessed for expression of the cytokines TNF-alpha, IL-1 beta, IL-8, IL-10, as well as 2-5A-dependent RNase L. LPS up-regulated the expression of protein and mRNA for all cytokines. RSV stimulated the protein levels of TNF-alpha, did not alter IL-1 beta, and decreased IL-8. RSV markedly stimulated protein expression of IL-10 and 2-5A-dependent RNase L. RSV had minor effects on the steady state mRNA levels of TNF-alpha, IL-1 beta, and IL-8, yet potently induced IL-10. Cells costimulated with RSV + LPS demonstrated reduced protein and mRNA levels of TNF-alpha, IL-1 beta, IL-8 but synergistically increased IL-10 levels compared to RSV- or LPS-activated cells. Kinetic analysis indicated that RSV induced a delayed and sustained increase in IL-10 transcripts. Furthermore, RSV-infected alveolar macrophage supernatants suppressed IL-1 beta and IL-8 production by LPS-stimulated alveolar macrophages as did recombinant IL-10. Anti-IL-10 neutralized these effects. These studies indicate that RSV is capable of suppressing production of early immunoregulatory cytokines through induction of IL-10 perhaps mediated by 2-5A-dependent RNase L (or other endoribonucleases) accounting for the ineffective immune response to this virus.

Respiratory syncytial virus replication in human lung epithelial cells: inhibition by tumor necrosis factor alpha and interferon beta.

Respiratory syncytial virus (RSV) is the major pathogen causing severe lung disease in children. RSV initially replicates efficiently in the respiratory tract but becomes undetectable by 7 to 21 d after infection in normal children, suggesting that intrinsic cellular mechanisms, as yet undefined, may restrict virus replication. To provide an in vitro model to examine mechanisms that restrict RSV replication, three human lung epithelial cell lines were exposed to RSV in vitro and virus replication proceeded in a dose- and time-dependent manner, although less efficiently than the highly permissive CV-1 cell line (monkey kidney epithelial cell). Tumor necrosis factor alpha (TNF alpha) and/or interferon beta (IFN beta) markedly inhibited RSV replication in a dose- and time-dependent manner. TNF alpha combined with IFN beta essentially aborted RSV replication in A549 epithelial cells. TNF alpha and/or IFN beta did not induce cell membrane damage, cause cell lysis, or inhibit cellular protein synthesis. RSV-infected human alveolar macrophages, which produce TNF alpha, failed to productively infect lung epithelial cells in co-culture. Together these studies suggest that endogenous TNF alpha coupled with exogenous IFN beta could restrict RSV replication in lung epithelium.

Acidosis stimulates nitric oxide production and lung damage in rats.

Systemic hypotension during sepsis is thought to be due to nitric oxide (NO) overproduction, but it may also be due to acidosis. We evaluated in healthy rats the consequences of acid infusion on NO and blood pressure. Sprague-Dawley rats were anesthetized, and ventilated with room air. The animals were randomized into four groups. Group 1 (C, n = 10) received only normal saline at rates comparable to the other groups. Group 2 (A1, n = 10) received hydrochloric acid at 0.162 mmol in the first 15 to 30 min, followed by a continuous infusion of 0.058 mmol/h for 5 h. Group 3 (AG+A1, n = 6) was pretreated with aminoguanidine (AG, 50 mg/kg), and HCl was infused as above. Group 4 (A2, n = 7) received HCl at twice the rate used in A1. Nitric oxide concentration in the exhaled gas (ENO), blood gases, and mean arterial pressure were measured every 30 min. Acid infusion in A1 caused the pH to fall gradually from 7.43 +/- 0. 01 to 7.13 +/- 0.05. This moderate decrease in pH was associated with a marked increase in ENO (1.6 +/- 0.3 to 114.2 +/- 22.3 ppb), an increase in plasma nitrite/nitrate (17.3 +/- 3.7 to 35.2 +/- 4.3 microM), and a significant decrease in blood pressure (110.5 +/- 6.3 to 63.3 +/- 15.0 mm Hg). Furthermore, acidosis caused lung inflammation, as suggested by the increase in lung myeloperoxidase activity (282.2 +/- 24.7 to 679.3 +/- 57.3 U/min/g) and lung injury score (1.7 +/- 0.2 to 3.5 +/- 0.6). Acidosis after AG pretreatment was associated with a similar change in pH, but the increase in ENO, nitrite/nitrate, and systemic hypotension were prevented. Furthermore, lung injury was attenuated by AG, as suggested by a lower myeloperoxidase activity, though lung injury score was not altered. In this model, moderate acidosis causes increases in NO, hypotension, and lung inflammation. Lung inflammation and injury are due in part to acidosis and NO production. This is the first report to show a direct effect of chronic acidosis on NO production and lung injury. These results have profound implications on the role of acidosis on NO production and lung injury during sepsis.

Treatment of septic shock in rats with nitric oxide synthase inhibitors and inhaled nitric oxide.

OBJECTIVE: To evaluate the effect of treatment with a combination of nitric oxide synthase inhibitors and inhaled nitric oxide on systemic hypotension during sepsis. DESIGN: Prospective, randomized, controlled study on anesthetized animals. SETTING: A cardiopulmonary research laboratory. SUBJECTS: Forty-seven male adult Sprague-Dawley rats. INTERVENTIONS: Animals were anesthetized, mechanically ventilated with room air, and randomized into six groups: a) the control group (C, n=6) received normal saline infusion; b) the endotoxin-treated group received 100 mg/kg i.v. of Escherichia coli lipopolysaccharide (LPS, n=9); c) the third group received LPS, and 1 hr later the animals were treated with 100 mg/kg i.v. Nw-nitro-L-arginine (LNA, n=9); d) the fourth group received LPS, and after 1 hr, the animals were treated with 100 mg/kg i.v. aminoguanidine (AG, n=9); e) the fifth group received LPS and 1 hr later was treated with LNA plus 1 ppm inhaled nitric oxide (LNA+NO, n=7); f) the sixth group received LPS and 1 hr later was treated with aminoguanidine plus inhaled NO (AG+NO, n=7). Inhaled NO was administered continuously until the end of the experiment. MEASUREMENTS AND MAIN RESULTS: Systemic mean blood pressure (MAP) was monitored through a catheter in the carotid artery. Mean exhaled NO (ENO) was measured before LPS (T0) and every 30 mins thereafter for 5 hrs. Arterial blood gases and pH were measured every 30 mins for the first 2 hrs and then every hour. No attempt was made to regulate the animal body temperature. All the rats became equally hypothermic (28.9+/-1.2 degrees C [SEM]) at the end of the experiment. In the control group, blood pressure and pH remained stable for the duration of the experiment, however, ENO increased gradually from 1.3+/-0.7 to 17.6+/-3.1 ppb after 5 hrs (p< .05). In the LPS treated rats, MAP decreased in the first 30 mins and then remained stable for 5 hrs. The decrease in MAP was associated with a gradual increase in ENO, which was significant after 180 mins (58.9+/-16.6 ppb) and reached 95.3+/-27.5 ppb after 5 hrs (p< .05). LNA and AG prevented the increase in ENO after LPS to the level in the control group. AG caused a partial reversal of systemic hypotension, which lasted for the duration of the experiment. LNA reversed systemic hypotension almost completely but only transiently for 1 hr, and caused severe metabolic acidosis in all animals. The co-administration of NO with AG had no added benefits on MAP and pH. In contrast, NO inhalation increased the duration of the reversal in MAP after LNA, alleviated the degree of acidosis, and decreased the mortality rate (from 55% to 29%). CONCLUSIONS: In this animal model, LPS-induced hypotension was alleviated slightly and durably after AG, but only transiently after LNA. Furthermore, co-administration of NO with AG had no added benefits but alleviated the severity of metabolic acidosis and mortality after LNA. We conclude that nitric oxide synthase (NOS) inhibitors, given as a single large bolus in the early phase of sepsis, can exhibit some beneficial effects. Administration of inhaled NO with NOS inhibitors provided more benefits in some conditions and therefore may be a useful therapeutic combination in sepsis. NO production in sepsis does not seem to be a primary cause of systemic hypotension. Other factors are likely to have a major role.