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Interferon (IFN)-alpha activation of human blood mononuclear cells in vitro and in vivo for nitric oxide synthase (NOS) type 2 mRNA and protein expression: possible relationship of induced NOS2 to the anti-hepatitis C effects of IFN-alpha in vivo.

Sharara AI, Perkins DJ, Misukonis MA, Chan SU, Dominitz JA, Weinberg JB - J. Exp. Med. (1997)

Bottom Line: Treatment of normal monocytes or mononuclear cells in vitro with IFN-alpha caused a dose-dependent increase in monocyte NOS2 activity and NO production, and increased expression of NOS2 protein and mRNA expression.Untreated patients with chronic hepatitis C virus infection had levels of NOS activity and NOS2 antigen in freshly isolated mononuclear cells similar to those of healthy subjects, and they expressed minimal or no NOS2 mRNA.The degree of IFN-alpha-enhanced mononuclear cell NOS2 antigen content correlated significantly with the degree of reduction in serum alanine aminotransferase levels.

View Article: PubMed Central - PubMed

Affiliation: Division of Gastroenterology, Department of Medicine, Veterans Affairs and Duke University Medical Centers, Durham, North Carolina 27705, USA.

ABSTRACT
Although researchers have noted high level activation of rodent mononuclear phagocytes for nitric oxide (NO) synthase type 2 (S2) expression and NO production with a variety of agents such as interferon (IFN) gamma and endotoxin, it has been difficult to demonstrate activation of human mononuclear phagocytes. The purpose of this study was to determine if IFN-alpha serves as an activator in vitro and in vivo in humans. Treatment of normal monocytes or mononuclear cells in vitro with IFN-alpha caused a dose-dependent increase in monocyte NOS2 activity and NO production, and increased expression of NOS2 protein and mRNA expression. To determine if in vivo administration of IFN-alpha also modulated NOS2, we studied blood cells from patients with hepatitis C before and after IFN-alpha therapy. Untreated patients with chronic hepatitis C virus infection had levels of NOS activity and NOS2 antigen in freshly isolated mononuclear cells similar to those of healthy subjects, and they expressed minimal or no NOS2 mRNA. However, IFN-alpha treatment of patients with hepatitis C infection was associated with a significant elevation in mononuclear cell NOS activity, NOS2 antigen content, and NOS2 mRNA content. IFN-alpha-treated patients had significant decreases in levels of serum alanine aminotransferase and plasma hepatitis C mRNA. The degree of IFN-alpha-enhanced mononuclear cell NOS2 antigen content correlated significantly with the degree of reduction in serum alanine aminotransferase levels. Thus, IFN-alpha treatment of cells in vitro or administration of IFN-alpha to hepatitis C patients in vivo increases expression of mononuclear cell NOS2 mRNA expression, NOS activity, NOS2 antigen expression, and NO production. Since NO has been reported to have antiviral activity for a variety of viruses, we speculate that induced NO production may be related to the antiviral action(s) of IFN-alpha in hepatitis C infection.

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Correlation between mononuclear cell NOS2  immunoblot band density and  degree of reduction of serum  ALT in response to IFN-α treatment. Immunoblots were processed using an antibody against  NOS2. The NOS2 density was  calculated by densitometry and  expressed as the ratio of NOS2  band density/background density using a densitometry. The  “fold increase post-IFN” for  band densities and “fraction of pre-IFN” ALT values were calculated by  dividing the pretreatment values by the posttreatment values. The ALT  values before IFN-α treatment are shown for each point. A normal ALT  level was 10–60 U/liter.
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Figure 5: Correlation between mononuclear cell NOS2 immunoblot band density and degree of reduction of serum ALT in response to IFN-α treatment. Immunoblots were processed using an antibody against NOS2. The NOS2 density was calculated by densitometry and expressed as the ratio of NOS2 band density/background density using a densitometry. The “fold increase post-IFN” for band densities and “fraction of pre-IFN” ALT values were calculated by dividing the pretreatment values by the posttreatment values. The ALT values before IFN-α treatment are shown for each point. A normal ALT level was 10–60 U/liter.

Mentions: There was a statistically significant correlation between the fold increase in NOS2 antigen immunoblot band density and the degree of reduction of the serum ALT level (fraction of pretreatment ALT level after IFN-α therapy); with increasing NOS2 antigen expression, there was a greater treatment-associated decrease in serum ALT (Fig. 5). The correlation was statistically significant (P <0.001 by ANOVA, and <0.05 by Spearman testing). Because of a lack of certain simultaneous determinations, we could analyze data from only five of the eight subjects before and after treatment. While the NOS2 antigen immunoblot band density and the degree of reduction of the serum ALT level correlated, the correlation between NOS activity (pico moles citrulline/mg protein) and ALT reduction was not significant. Nevertheless, these data suggest that the augmentation in NOS2 levels may be causally related to the observed decreases in serum ALT levels and decrease in liver inflammation associated with IFN-α therapy.


Interferon (IFN)-alpha activation of human blood mononuclear cells in vitro and in vivo for nitric oxide synthase (NOS) type 2 mRNA and protein expression: possible relationship of induced NOS2 to the anti-hepatitis C effects of IFN-alpha in vivo.

Sharara AI, Perkins DJ, Misukonis MA, Chan SU, Dominitz JA, Weinberg JB - J. Exp. Med. (1997)

Correlation between mononuclear cell NOS2  immunoblot band density and  degree of reduction of serum  ALT in response to IFN-α treatment. Immunoblots were processed using an antibody against  NOS2. The NOS2 density was  calculated by densitometry and  expressed as the ratio of NOS2  band density/background density using a densitometry. The  “fold increase post-IFN” for  band densities and “fraction of pre-IFN” ALT values were calculated by  dividing the pretreatment values by the posttreatment values. The ALT  values before IFN-α treatment are shown for each point. A normal ALT  level was 10–60 U/liter.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199112&req=5

Figure 5: Correlation between mononuclear cell NOS2 immunoblot band density and degree of reduction of serum ALT in response to IFN-α treatment. Immunoblots were processed using an antibody against NOS2. The NOS2 density was calculated by densitometry and expressed as the ratio of NOS2 band density/background density using a densitometry. The “fold increase post-IFN” for band densities and “fraction of pre-IFN” ALT values were calculated by dividing the pretreatment values by the posttreatment values. The ALT values before IFN-α treatment are shown for each point. A normal ALT level was 10–60 U/liter.
Mentions: There was a statistically significant correlation between the fold increase in NOS2 antigen immunoblot band density and the degree of reduction of the serum ALT level (fraction of pretreatment ALT level after IFN-α therapy); with increasing NOS2 antigen expression, there was a greater treatment-associated decrease in serum ALT (Fig. 5). The correlation was statistically significant (P <0.001 by ANOVA, and <0.05 by Spearman testing). Because of a lack of certain simultaneous determinations, we could analyze data from only five of the eight subjects before and after treatment. While the NOS2 antigen immunoblot band density and the degree of reduction of the serum ALT level correlated, the correlation between NOS activity (pico moles citrulline/mg protein) and ALT reduction was not significant. Nevertheless, these data suggest that the augmentation in NOS2 levels may be causally related to the observed decreases in serum ALT levels and decrease in liver inflammation associated with IFN-α therapy.

Bottom Line: Treatment of normal monocytes or mononuclear cells in vitro with IFN-alpha caused a dose-dependent increase in monocyte NOS2 activity and NO production, and increased expression of NOS2 protein and mRNA expression.Untreated patients with chronic hepatitis C virus infection had levels of NOS activity and NOS2 antigen in freshly isolated mononuclear cells similar to those of healthy subjects, and they expressed minimal or no NOS2 mRNA.The degree of IFN-alpha-enhanced mononuclear cell NOS2 antigen content correlated significantly with the degree of reduction in serum alanine aminotransferase levels.

View Article: PubMed Central - PubMed

Affiliation: Division of Gastroenterology, Department of Medicine, Veterans Affairs and Duke University Medical Centers, Durham, North Carolina 27705, USA.

ABSTRACT
Although researchers have noted high level activation of rodent mononuclear phagocytes for nitric oxide (NO) synthase type 2 (S2) expression and NO production with a variety of agents such as interferon (IFN) gamma and endotoxin, it has been difficult to demonstrate activation of human mononuclear phagocytes. The purpose of this study was to determine if IFN-alpha serves as an activator in vitro and in vivo in humans. Treatment of normal monocytes or mononuclear cells in vitro with IFN-alpha caused a dose-dependent increase in monocyte NOS2 activity and NO production, and increased expression of NOS2 protein and mRNA expression. To determine if in vivo administration of IFN-alpha also modulated NOS2, we studied blood cells from patients with hepatitis C before and after IFN-alpha therapy. Untreated patients with chronic hepatitis C virus infection had levels of NOS activity and NOS2 antigen in freshly isolated mononuclear cells similar to those of healthy subjects, and they expressed minimal or no NOS2 mRNA. However, IFN-alpha treatment of patients with hepatitis C infection was associated with a significant elevation in mononuclear cell NOS activity, NOS2 antigen content, and NOS2 mRNA content. IFN-alpha-treated patients had significant decreases in levels of serum alanine aminotransferase and plasma hepatitis C mRNA. The degree of IFN-alpha-enhanced mononuclear cell NOS2 antigen content correlated significantly with the degree of reduction in serum alanine aminotransferase levels. Thus, IFN-alpha treatment of cells in vitro or administration of IFN-alpha to hepatitis C patients in vivo increases expression of mononuclear cell NOS2 mRNA expression, NOS activity, NOS2 antigen expression, and NO production. Since NO has been reported to have antiviral activity for a variety of viruses, we speculate that induced NO production may be related to the antiviral action(s) of IFN-alpha in hepatitis C infection.

Show MeSH
Related in: MedlinePlus