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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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(A) Immunoblot  analyses of extracts of mononuclear cells treated in vitro with  IFN-α. Cells were cultured with  (+) or without (−) 500 U/ml of  IFN-α for 3 d. Extracts were  then made as described in the  Methods section, and equivalent  amounts of cellular protein were  analyzed. Antibody anti-MacNOS was used. Cells from two  separate, normal individuals were  analyzed. Analysis of the first is  shown in lanes 1 and 2, and that  of the second is in lanes 3 and 4.  DLD-1 + signifies extracts from  the human colon cancer cell line DLD-1 treated with IFN-γ, IL-1, and  TNF in vitro. Results demonstrate that IFN-α treatment induces NOS2  antigen expression. (B) RT-PCR analysis of mRNA of mononuclear  cells treated in vitro with IFN-α. Cells from two separate, normal individuals were cultured with (+) or without (−) 500 U/ml of IFN-α for  12 h. RNA was extracted and analyzed as described in the Methods section for NOS2 and glyceraldehyde-3-phosphate dehydrogenase mRNA.  M, molecular weight markers; D, cells of the human colon cancer cell  line DLD-1 treated with IFN-γ, IL-1, and TNF; and W, distilled water.
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Figure 2: (A) Immunoblot analyses of extracts of mononuclear cells treated in vitro with IFN-α. Cells were cultured with (+) or without (−) 500 U/ml of IFN-α for 3 d. Extracts were then made as described in the Methods section, and equivalent amounts of cellular protein were analyzed. Antibody anti-MacNOS was used. Cells from two separate, normal individuals were analyzed. Analysis of the first is shown in lanes 1 and 2, and that of the second is in lanes 3 and 4. DLD-1 + signifies extracts from the human colon cancer cell line DLD-1 treated with IFN-γ, IL-1, and TNF in vitro. Results demonstrate that IFN-α treatment induces NOS2 antigen expression. (B) RT-PCR analysis of mRNA of mononuclear cells treated in vitro with IFN-α. Cells from two separate, normal individuals were cultured with (+) or without (−) 500 U/ml of IFN-α for 12 h. RNA was extracted and analyzed as described in the Methods section for NOS2 and glyceraldehyde-3-phosphate dehydrogenase mRNA. M, molecular weight markers; D, cells of the human colon cancer cell line DLD-1 treated with IFN-γ, IL-1, and TNF; and W, distilled water.

Mentions: Freshly isolated monocytes from eight healthy male volunteers showed an increase in NOS activity in response to IFN-α2b treatment in vitro. The increase peaked at 500 U/ml (Fig. 1 A). Similarly, production of NO (measured as nitrate and nitrite, the stable catabolites of NO) increased with IFN-α2b treatment (Fig. 1 B). Increasing doses of IFN-α2b were associated with significantly increased NOS activity and nitrite/nitrate production (P <0.001). Studies also demonstrated that treatment of monocytes in vitro with IFN-α2a (Roferon®; Roche Laboratories, Nutley, NJ) augmented NOS activity and NO production (data not shown). In immunoblot studies, normal subject monocytes from four out of six treated in vitro with IFN-α2b had increased expression of NOS2 antigen (Fig. 2 A). We did RT-PCR analysis of RNA from blood mononuclear cells to determine if IFN-α induced increased levels of NOS2 mRNA. Mononuclear cells from normal subjects were cultured for 3 d without or with 500 U/ml IFN-α. Untreated cells from zero out of six normal individuals had NOS2 mRNA expression, whereas all those treated with IFN-α in vitro expressed NOS2 mRNA (six out of six) (Fig. 2 B).


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)

(A) Immunoblot  analyses of extracts of mononuclear cells treated in vitro with  IFN-α. Cells were cultured with  (+) or without (−) 500 U/ml of  IFN-α for 3 d. Extracts were  then made as described in the  Methods section, and equivalent  amounts of cellular protein were  analyzed. Antibody anti-MacNOS was used. Cells from two  separate, normal individuals were  analyzed. Analysis of the first is  shown in lanes 1 and 2, and that  of the second is in lanes 3 and 4.  DLD-1 + signifies extracts from  the human colon cancer cell line DLD-1 treated with IFN-γ, IL-1, and  TNF in vitro. Results demonstrate that IFN-α treatment induces NOS2  antigen expression. (B) RT-PCR analysis of mRNA of mononuclear  cells treated in vitro with IFN-α. Cells from two separate, normal individuals were cultured with (+) or without (−) 500 U/ml of IFN-α for  12 h. RNA was extracted and analyzed as described in the Methods section for NOS2 and glyceraldehyde-3-phosphate dehydrogenase mRNA.  M, molecular weight markers; D, cells of the human colon cancer cell  line DLD-1 treated with IFN-γ, IL-1, and TNF; and W, distilled water.
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Figure 2: (A) Immunoblot analyses of extracts of mononuclear cells treated in vitro with IFN-α. Cells were cultured with (+) or without (−) 500 U/ml of IFN-α for 3 d. Extracts were then made as described in the Methods section, and equivalent amounts of cellular protein were analyzed. Antibody anti-MacNOS was used. Cells from two separate, normal individuals were analyzed. Analysis of the first is shown in lanes 1 and 2, and that of the second is in lanes 3 and 4. DLD-1 + signifies extracts from the human colon cancer cell line DLD-1 treated with IFN-γ, IL-1, and TNF in vitro. Results demonstrate that IFN-α treatment induces NOS2 antigen expression. (B) RT-PCR analysis of mRNA of mononuclear cells treated in vitro with IFN-α. Cells from two separate, normal individuals were cultured with (+) or without (−) 500 U/ml of IFN-α for 12 h. RNA was extracted and analyzed as described in the Methods section for NOS2 and glyceraldehyde-3-phosphate dehydrogenase mRNA. M, molecular weight markers; D, cells of the human colon cancer cell line DLD-1 treated with IFN-γ, IL-1, and TNF; and W, distilled water.
Mentions: Freshly isolated monocytes from eight healthy male volunteers showed an increase in NOS activity in response to IFN-α2b treatment in vitro. The increase peaked at 500 U/ml (Fig. 1 A). Similarly, production of NO (measured as nitrate and nitrite, the stable catabolites of NO) increased with IFN-α2b treatment (Fig. 1 B). Increasing doses of IFN-α2b were associated with significantly increased NOS activity and nitrite/nitrate production (P <0.001). Studies also demonstrated that treatment of monocytes in vitro with IFN-α2a (Roferon®; Roche Laboratories, Nutley, NJ) augmented NOS activity and NO production (data not shown). In immunoblot studies, normal subject monocytes from four out of six treated in vitro with IFN-α2b had increased expression of NOS2 antigen (Fig. 2 A). We did RT-PCR analysis of RNA from blood mononuclear cells to determine if IFN-α induced increased levels of NOS2 mRNA. Mononuclear cells from normal subjects were cultured for 3 d without or with 500 U/ml IFN-α. Untreated cells from zero out of six normal individuals had NOS2 mRNA expression, whereas all those treated with IFN-α in vitro expressed NOS2 mRNA (six out of six) (Fig. 2 B).

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