Nitric oxide inhibits rhinovirus-induced granulocyte macrophage colony-stimulating factor production in bronchial epithelial cells.

Infection of asthmatics with human rhinovirus (HRV) enhances airway eosinophilia and airways hyperreactivity. The current studies were performed to further characterize HRV-induced generation by human bronchial epithelial cells of granulocyte macrophage colony-stimulating factor (GM-CSF), a cytokine that could contribute to airway eosinophilia by increasing the survival and activation of eosinophils, and to determine the effects of the antiviral agent nitric oxide (NO) on HRV-induced GM-CSF production. Maximal levels of messenger RNA (mRNA) for GM-CSF were seen 1 h after HRV infection. Expression was sustained through 24 h and declined by 48 h. GM-CSF protein was detected in cell supernatants by 2 h after infection and reached maximal concentrations by 24 h, with the most rapid rate of production occurring from 2 to 7 h. The NO donor 3-(2-hydroxy-2-nitroso-1-propyl-hydrazino)-1-propanamine (NONOate) inhibited HRV-induced GM-CSF protein production in a time- and dose-dependent fashion. NONOate also inhibited HRV-induced GM-CSF mRNA levels at both times (1 and 4 h) examined. NONOate increased GM-CSF mRNA stability, suggesting that reduced mRNA levels were due to inhibition of transcription. The transcription factor nuclear factor-kappa B was rapidly induced by HRV infection, but was not inhibited by NONOate, implying a role for other transcription factors. Thus, NO may play an important anti-inflammatory role in virally induced exacerbations of diseases such as asthma.

Rhinovirus infection induces expression of type 2 nitric oxide synthase in human respiratory epithelial cells in vitro and in vivo.

BACKGROUND: Human rhinovirus (HRV) infections are the predominant cause of the common cold and are associated with exacerbations of asthma. Nitric oxide (NO) may play an important role in host defense by means of its potent antiviral properties. OBJECTIVE: We sought to determine whether epithelial expression of type 2 nitric oxide synthase (NOS 2), which produces NO, is induced on rhinovirus infection in vitro and in vivo. METHODS: Primary cultures of human airway epithelial cells were infected with HRV-16, and NOS 2 mRNA expression was assessed by conventional and real-time RT-PCR and NOS 2 protein by using Western blot analysis. Human subjects were also infected with HRV-16 in vivo, and mRNA for NOS 2 was assessed in nasal epithelial scrapings obtained before and after infection. RESULTS: NOS 2 mRNA levels increased within 8 hours after HRV-16 infection of cultured cells and remained elevated up to 48 hours after infection. NOS 2 protein was elevated at 24 hours. Induction of NOS 2 did not occur with UV-inactivated HRV-16 but could be reproduced by using double-stranded RNA, indicating that induction was dependent on viral replication. Increased NOS 2 expression was also observed in nasal epithelial scrapings during symptomatic colds. CONCLUSION: Increased epithelial expression of NOS 2 mRNA occurs as part of the host response to HRV infection in vitro and in vivo. Given the antiviral effects of NO, we speculate that increased host production of NO may play an important role in host defense during HRV infections.

Role of NF-kappa B in cytokine production induced from human airway epithelial cells by rhinovirus infection.

Infection of human epithelial cells with human rhinovirus (HRV)-16 induces rapid production of several proinflammatory cytokines, including IL-8, IL-6, and GM-CSF. We evaluated the role of NF-kappaB in HRV-16-induced IL-8 and IL-6 production by EMSA using oligonucleotides corresponding to the binding sites for NF-kappaB in the IL-6 and IL-8 gene promoters. Consistent with the rapid induction of mRNA for IL-8 and IL-6, maximal NF-kappaB binding to both oligonucleotides was detected at 30 min after infection. NF-kappaB complexes contained p65 and p50, but not c-Rel. The IL-8 oligonucleotide bound recombinant p50 with only about one-tenth the efficiency of the IL-6 oligonucleotide, even though epithelial cells produced more IL-8 protein than IL-6. Neither the potent glucocorticoid, budesonide (10-7 M), nor a NO donor inhibited NF-kappaB binding to either cytokine promoter or induction of mRNA for either IL-8 or IL-6. Sulfasalazine and calpain inhibitor I, inhibitors of NF-kappaB activation, blocked HRV-16-induced formation of NF-kappaB complexes with oligonucleotides from both cytokines, but did not inhibit mRNA induction for either cytokine. By contrast, sulfasalazine clearly inhibited HRV-16 induction of mRNA for GM-CSF in the same cells. Thus, HRV-16 induces epithelial expression of IL-8 and IL-6 by an NF-kappaB-independent pathway, whereas induction of GM-CSF is at least partially dependent upon NF-kappaB activation.

Daily variations of serum diamine oxidase and the influence of H1 and H2 blockers: a critical approach to routine diamine oxidase assessment.

OBJECTIVE AND DESIGN: Histamine in food has been shown to induce intolerance reactions mimicking food allergy. These reactions seem to be due to impaired histamine metabolism caused by reduced diamine oxidase activity. To validate routine serum diamine oxidase assessment, daily variations of diamine oxidase were evaluated. METHODS: Blood was drawn from each of 20 healthy volunteers (10 female, 10 male; mean age 32.5 years) every 2 h from 9 a.m. to 5 p.m., and diamine oxidase activity was measured using the C14 putrescine method. To assess possible influences of H1 and H2 blockers on diamine oxidase activity, diphenhydramine, ketotifen, cimetidine, and ranitidine were incubated at pharmacologic concentrations with human placental diamine oxidase (identical to neutrophilic and eosinophilic diamine oxidase). Inhibition of diamine oxidase activity was calculated as the percentage of inhibition versus control. In addition, the known diamine oxidase inhibitors, dihydralazine and aminoguanidine, were used as positive controls. RESULTS: Serum diamine oxidase levels showed no significant daily variations (0.041 +/- 0.025; 0.037 +/- 0.022; 0.041 +/- 0.023; 0.040 +/- 0.023; 0.038 +/- 0.025 nKat/l) and no significant sex differences (female 0.040 +/- 0.028 nKat/l versus male 0.039 +/- 0.019 nKat/l). Antihistamines had no influence on diamine oxidase activity except for cimetidine, which caused 25% inhibition at the highest dose tested ( p < 0.0002) (positive control: aminoguanidine 85% inhibition (p< 0.0001), dihydralazine 68% inhibition (p<0.0001)) and diphenhydramine, which caused 19% increase (p<0.0001) of enzyme activity. CONCLUSION: Serum diamine oxidase levels do not show daily variations allowing assessment anytime during office hours. However, diagnostic interpretation of serum diamine oxidase levels may be difficult.

Nitric oxide inhibits rhinovirus-induced cytokine production and viral replication in a human respiratory epithelial cell line.

To better understand the early biochemical events that occur in human rhinovirus (HRV) infections, we examined the kinetics and mechanisms of interleukin-8 (IL-8) and IL-6 production from infected epithelial cells. Several HRV strains caused IL-8 and IL-6 production, but HRV-16 induced maximal IL-8 and IL-6 mRNA expression and protein production more rapidly than did HRV-14, despite similar rates of replication of the two viral strains. Viral induction of cytokine mRNA does not require new protein synthesis, since it was unaffected by cycloheximide treatment. The potent glucocorticoid budesonide did not affect viral replication or cytokine mRNA induction but modestly inhibited cytokine protein production. Interestingly, the nitric oxide donor 3-(2-hydroxy-2-nitroso-1-propylhydrazino)-1-propanamine (NONOate) inhibited both rhinovirus replication and cytokine production in a dose-dependent fashion without reducing levels of cytokine mRNA. The NONOate effects were due to release of nitric oxide, because NONOate that had been depleted of its nitric oxide content had no effect. Thus, nitric oxide may play an important anti-inflammatory and antiviral role in colds and nitric oxide donors may represent a novel therapeutic approach.

Identification of human lung mast cell kininogenase as tryptase and relevance of tryptase kininogenase activity.

We have described previously the IgE-mediated release of kininogenase activity from purified human lung mast cells. Using supernatant fractions from mast cells stimulated with anti-IgE in the presence of deuterium oxide, we have purified this kininogenase to homogeneity by gel filtration and heparin-agarose chromatography and have demonstrated that it is identical to tryptase, the major neutral protease of human lung mast cells. Thus, tryptase and kininogenase activities co-chromatographed through both purification steps with equivalent yields. The final purified kininogenase was free of detectable chymotryptic and carboxypeptidase activities and was identified as tryptase on the basis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), amino acid composition and inhibition profile. Three such preparations of tryptase were all capable of releasing kinin from each of two different preparations of purified, single-chain, human low molecular weight kininogen. Interestingly, kinin generation was optimal at pH 5.5 and was enhanced by heparin, which has been reported to stabilize tryptase. SDS-PAGE analysis of kininogen hydrolysis by tryptase revealed the formation of a diffusely stained region in the molecular weight range of 60,000-65,000, rather than a discrete heavy chain band. Under optimal conditions, the three tryptase preparations released 10-12 micrograms kinin/hr/mg but released only 2 micrograms kinin/hr/mg at pH 7.2. HPLC analysis revealed that the kinin released was bradykinin. We conclude that the kininogenase activity from human lung mast cells is attributable to tryptase. The unique pH optimum of this reaction of a serine protease, however, raises doubts as to the physiologic significance of this activity.