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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 blood  mononuclear cells from hepatitis  C patients before and after IFN-α  treatment. Equivalent amounts  of cellular protein were analyzed  in each lane. Antibody 1E8-B8  was used. Samples from patients  one through four were collected  before IFN-α treatment (pre– IFN-α) or after receiving IFN-α  in vivo (post–IFN-α). Controls  for human NOS2 were from the  human colon cancer cell line DLD-1 without (−) or with (+) treatment  with IFN-γ, IL-1, and TNF in vitro. (B) RT-PCR analysis of mononuclear cells from normal subjects, patients with hepatitis C, and patients  with hepatitis C treated in vivo with IFN-α. Cells were isolated, frozen,  extracted, and analyzed as noted in the Methods section. Cells from the  two normal subjects (HEP C − and IFN-α −), one patient with hepatitis  C (HEP C + and IFN-α −), and two patients with hepatitis C on treatment with IFN-α (HEP C + and IFN-α +) were analyzed. 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 4: (A) Immunoblot analyses of extracts of blood mononuclear cells from hepatitis C patients before and after IFN-α treatment. Equivalent amounts of cellular protein were analyzed in each lane. Antibody 1E8-B8 was used. Samples from patients one through four were collected before IFN-α treatment (pre– IFN-α) or after receiving IFN-α in vivo (post–IFN-α). Controls for human NOS2 were from the human colon cancer cell line DLD-1 without (−) or with (+) treatment with IFN-γ, IL-1, and TNF in vitro. (B) RT-PCR analysis of mononuclear cells from normal subjects, patients with hepatitis C, and patients with hepatitis C treated in vivo with IFN-α. Cells were isolated, frozen, extracted, and analyzed as noted in the Methods section. Cells from the two normal subjects (HEP C − and IFN-α −), one patient with hepatitis C (HEP C + and IFN-α −), and two patients with hepatitis C on treatment with IFN-α (HEP C + and IFN-α +) were analyzed. 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: To determine if the increase in NOS activity after IFN-α treatment was accompanied by an increase in NOS2 protein, we analyzed cells for NOS2 antigen content. NOS2 antigen in extracts of blood mononuclear cells was detectable by immunoblot analysis in zero out of seven of the untreated hepatitis C patients and in zero out of five of the healthy control subjects tested. However, eight out of eight samples from hepatitis C patients treated with IFN-α2b had cells with detectable NOS2 antigen. These differences in NOS2 antigen expression among the groups were significant (P <0.00001, Fisher's exact test). Similarly, analysis of matched sets of cells from hepatitis C patients before and after IFN-α2b treatment revealed zero out of four with detectable NOS2 before treatment and four out of four with detectable NOS2 after treatment (Fig. 4 A). Using RT-PCR analyses with cells isolated from six normal individuals and examined without any in vitro culture, we could find no NOS2 mRNA (zero out of six) (Fig. 4 B). In two out of two patients with hepatitis C not on IFN-α treatment, we noted relatively low level expression of NOS2 mRNA, while three out of three hepatitis C patients on IFN-α treatment had relatively higher levels of NOS2 mRNA expression. Fig. 4 B shows representative results.


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 blood  mononuclear cells from hepatitis  C patients before and after IFN-α  treatment. Equivalent amounts  of cellular protein were analyzed  in each lane. Antibody 1E8-B8  was used. Samples from patients  one through four were collected  before IFN-α treatment (pre– IFN-α) or after receiving IFN-α  in vivo (post–IFN-α). Controls  for human NOS2 were from the  human colon cancer cell line DLD-1 without (−) or with (+) treatment  with IFN-γ, IL-1, and TNF in vitro. (B) RT-PCR analysis of mononuclear cells from normal subjects, patients with hepatitis C, and patients  with hepatitis C treated in vivo with IFN-α. Cells were isolated, frozen,  extracted, and analyzed as noted in the Methods section. Cells from the  two normal subjects (HEP C − and IFN-α −), one patient with hepatitis  C (HEP C + and IFN-α −), and two patients with hepatitis C on treatment with IFN-α (HEP C + and IFN-α +) were analyzed. 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 4: (A) Immunoblot analyses of extracts of blood mononuclear cells from hepatitis C patients before and after IFN-α treatment. Equivalent amounts of cellular protein were analyzed in each lane. Antibody 1E8-B8 was used. Samples from patients one through four were collected before IFN-α treatment (pre– IFN-α) or after receiving IFN-α in vivo (post–IFN-α). Controls for human NOS2 were from the human colon cancer cell line DLD-1 without (−) or with (+) treatment with IFN-γ, IL-1, and TNF in vitro. (B) RT-PCR analysis of mononuclear cells from normal subjects, patients with hepatitis C, and patients with hepatitis C treated in vivo with IFN-α. Cells were isolated, frozen, extracted, and analyzed as noted in the Methods section. Cells from the two normal subjects (HEP C − and IFN-α −), one patient with hepatitis C (HEP C + and IFN-α −), and two patients with hepatitis C on treatment with IFN-α (HEP C + and IFN-α +) were analyzed. 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: To determine if the increase in NOS activity after IFN-α treatment was accompanied by an increase in NOS2 protein, we analyzed cells for NOS2 antigen content. NOS2 antigen in extracts of blood mononuclear cells was detectable by immunoblot analysis in zero out of seven of the untreated hepatitis C patients and in zero out of five of the healthy control subjects tested. However, eight out of eight samples from hepatitis C patients treated with IFN-α2b had cells with detectable NOS2 antigen. These differences in NOS2 antigen expression among the groups were significant (P <0.00001, Fisher's exact test). Similarly, analysis of matched sets of cells from hepatitis C patients before and after IFN-α2b treatment revealed zero out of four with detectable NOS2 before treatment and four out of four with detectable NOS2 after treatment (Fig. 4 A). Using RT-PCR analyses with cells isolated from six normal individuals and examined without any in vitro culture, we could find no NOS2 mRNA (zero out of six) (Fig. 4 B). In two out of two patients with hepatitis C not on IFN-α treatment, we noted relatively low level expression of NOS2 mRNA, while three out of three hepatitis C patients on IFN-α treatment had relatively higher levels of NOS2 mRNA expression. Fig. 4 B shows representative results.

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