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Clinical and serologic manifestations of autoimmune disease in MRL-lpr/lpr mice lacking nitric oxide synthase type 2.

Gilkeson GS, Mudgett JS, Seldin MF, Ruiz P, Alexander AA, Misukonis MA, Pisetsky DS, Weinberg JB - J. Exp. Med. (1997)

Bottom Line: In previous studies, we showed that inhibiting NO production with the nonselective nitric oxide synthase (NOS) inhibitor NG-monomethyl-arginine reduced glomerulonephritis, arthritis, and vasculitis in MRL-lpr/lpr mice.MRL-lpr/lpr littermates homozygous for disrupted NOS2 (-/-), heterozygous for disrupted NOS2 (+/-), or wildtype (+/+) were derived for this study.There was no detectable NOS2 protein by immunoblot analysis of the spleen, liver, kidney, and peritoneal macrophages of the (-/-) animals, whereas that of (+/+) was high and (+/-) intermediate.

View Article: PubMed Central - PubMed

Affiliation: Ralph H. Johnson Veterans Affairs Medical Center, Medical University of South Carolina, Charleston 29425, USA.

ABSTRACT
Nitric oxide (NO) is an important mediator of the inflammatory response. MRL-lpr/lpr mice overexpress inducible nitric oxide synthase (NOS2) and overproduce NO in parallel with the development of an autoimmune syndrome with a variety of inflammatory manifestations. In previous studies, we showed that inhibiting NO production with the nonselective nitric oxide synthase (NOS) inhibitor NG-monomethyl-arginine reduced glomerulonephritis, arthritis, and vasculitis in MRL-lpr/lpr mice. To define further the role of NO and NOS2 in disease in MRL-lpr/lpr mice, mice with targeted disruption of NOS2 were produced by homologous recombination and bred to MRL-lpr/lpr mice to the N4 generation. MRL-lpr/lpr littermates homozygous for disrupted NOS2 (-/-), heterozygous for disrupted NOS2 (+/-), or wildtype (+/+) were derived for this study. Measures of NO production were markedly decreased in the MRL-lpr/lpr (-/-) mice compared with MRL-lpr/lpr (+/+) mice, with intermediate production by the MRL-lpr/lpr (+/-) mice. There was no detectable NOS2 protein by immunoblot analysis of the spleen, liver, kidney, and peritoneal macrophages of the (-/-) animals, whereas that of (+/+) was high and (+/-) intermediate. The (-/-) mice developed glomerular and synovial pathology similar to that of the (+/-) and (+/+) mice. However, (-/-) mice and (+/-) mice had significantly less vasculitis of medium-sized renal vessels than (+/+) mice. IgG rheumatoid factor levels were significantly lower in the (-/-) mice as compared with (+/+) mice, but levels of anti-DNA antibodies were comparable in all groups. Our findings show that NO derived from NOS2 has a variable impact on disease manifestations in MRL-lpr/lpr mice, suggesting heterogeneity in disease mechanisms.

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(A and B) Anti-NOS2 immunoblot of tissue and cell extracts from NOS2-modified MRL–lpr/lpr mice. 20-wk-old mice were examined. Extracts from kidney, spleen, and  liver of (+/+), (+/−) , (−/−) and BALB/c (B) mice are displayed in A, and that for peritoneal macrophages in B. For negative and positive controls, we used extracts from the mouse  macrophage cell line J774 without (0) or with LPS and IFN-γ (LI). The predominant band appeared at a region corresponding to about 130 kD.
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Figure 2: (A and B) Anti-NOS2 immunoblot of tissue and cell extracts from NOS2-modified MRL–lpr/lpr mice. 20-wk-old mice were examined. Extracts from kidney, spleen, and liver of (+/+), (+/−) , (−/−) and BALB/c (B) mice are displayed in A, and that for peritoneal macrophages in B. For negative and positive controls, we used extracts from the mouse macrophage cell line J774 without (0) or with LPS and IFN-γ (LI). The predominant band appeared at a region corresponding to about 130 kD.

Mentions: Immunoblots were performed on protein extracts from spleens, kidneys, livers, and peritoneal macrophages from the mice using an anti-NOS2 antibody. As shown in Fig. 2, A and B, there was no detectable NOS2 (molecular size ∼130–132 kD) in protein extracts from spleen, kidney, liver, and macrophages of (−/−) mice; extracts from (+/+) and (+/−) mice contained NOS2 protein, with those of the (+/−) mice having ∼25–50% that of the (+/+) mice. As we have shown before (7), extracts from cells and tissues of normal mice (e.g., BALB/c mice) had no detectable NOS2 antigen.


Clinical and serologic manifestations of autoimmune disease in MRL-lpr/lpr mice lacking nitric oxide synthase type 2.

Gilkeson GS, Mudgett JS, Seldin MF, Ruiz P, Alexander AA, Misukonis MA, Pisetsky DS, Weinberg JB - J. Exp. Med. (1997)

(A and B) Anti-NOS2 immunoblot of tissue and cell extracts from NOS2-modified MRL–lpr/lpr mice. 20-wk-old mice were examined. Extracts from kidney, spleen, and  liver of (+/+), (+/−) , (−/−) and BALB/c (B) mice are displayed in A, and that for peritoneal macrophages in B. For negative and positive controls, we used extracts from the mouse  macrophage cell line J774 without (0) or with LPS and IFN-γ (LI). The predominant band appeared at a region corresponding to about 130 kD.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199001&req=5

Figure 2: (A and B) Anti-NOS2 immunoblot of tissue and cell extracts from NOS2-modified MRL–lpr/lpr mice. 20-wk-old mice were examined. Extracts from kidney, spleen, and liver of (+/+), (+/−) , (−/−) and BALB/c (B) mice are displayed in A, and that for peritoneal macrophages in B. For negative and positive controls, we used extracts from the mouse macrophage cell line J774 without (0) or with LPS and IFN-γ (LI). The predominant band appeared at a region corresponding to about 130 kD.
Mentions: Immunoblots were performed on protein extracts from spleens, kidneys, livers, and peritoneal macrophages from the mice using an anti-NOS2 antibody. As shown in Fig. 2, A and B, there was no detectable NOS2 (molecular size ∼130–132 kD) in protein extracts from spleen, kidney, liver, and macrophages of (−/−) mice; extracts from (+/+) and (+/−) mice contained NOS2 protein, with those of the (+/−) mice having ∼25–50% that of the (+/+) mice. As we have shown before (7), extracts from cells and tissues of normal mice (e.g., BALB/c mice) had no detectable NOS2 antigen.

Bottom Line: In previous studies, we showed that inhibiting NO production with the nonselective nitric oxide synthase (NOS) inhibitor NG-monomethyl-arginine reduced glomerulonephritis, arthritis, and vasculitis in MRL-lpr/lpr mice.MRL-lpr/lpr littermates homozygous for disrupted NOS2 (-/-), heterozygous for disrupted NOS2 (+/-), or wildtype (+/+) were derived for this study.There was no detectable NOS2 protein by immunoblot analysis of the spleen, liver, kidney, and peritoneal macrophages of the (-/-) animals, whereas that of (+/+) was high and (+/-) intermediate.

View Article: PubMed Central - PubMed

Affiliation: Ralph H. Johnson Veterans Affairs Medical Center, Medical University of South Carolina, Charleston 29425, USA.

ABSTRACT
Nitric oxide (NO) is an important mediator of the inflammatory response. MRL-lpr/lpr mice overexpress inducible nitric oxide synthase (NOS2) and overproduce NO in parallel with the development of an autoimmune syndrome with a variety of inflammatory manifestations. In previous studies, we showed that inhibiting NO production with the nonselective nitric oxide synthase (NOS) inhibitor NG-monomethyl-arginine reduced glomerulonephritis, arthritis, and vasculitis in MRL-lpr/lpr mice. To define further the role of NO and NOS2 in disease in MRL-lpr/lpr mice, mice with targeted disruption of NOS2 were produced by homologous recombination and bred to MRL-lpr/lpr mice to the N4 generation. MRL-lpr/lpr littermates homozygous for disrupted NOS2 (-/-), heterozygous for disrupted NOS2 (+/-), or wildtype (+/+) were derived for this study. Measures of NO production were markedly decreased in the MRL-lpr/lpr (-/-) mice compared with MRL-lpr/lpr (+/+) mice, with intermediate production by the MRL-lpr/lpr (+/-) mice. There was no detectable NOS2 protein by immunoblot analysis of the spleen, liver, kidney, and peritoneal macrophages of the (-/-) animals, whereas that of (+/+) was high and (+/-) intermediate. The (-/-) mice developed glomerular and synovial pathology similar to that of the (+/-) and (+/+) mice. However, (-/-) mice and (+/-) mice had significantly less vasculitis of medium-sized renal vessels than (+/+) mice. IgG rheumatoid factor levels were significantly lower in the (-/-) mice as compared with (+/+) mice, but levels of anti-DNA antibodies were comparable in all groups. Our findings show that NO derived from NOS2 has a variable impact on disease manifestations in MRL-lpr/lpr mice, suggesting heterogeneity in disease mechanisms.

Show MeSH
Related in: MedlinePlus