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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) Nitrate and nitrite in urine, serum, and peritoneal macrophage supernatant medium from NOS2–modified MRL–lpr/lpr mice.  20-wk-old mice were examined. Serum and urine were collected as  noted in Materials and Methods. Resident peritoneal macrophages were  cultured for 3 d with 10 ng/mL and 50 U/mL of murine IFN-γ present  in the culture for 3 d. Urine values are presented as μmol/day, while Serum and Mac supt. values are presented as μM. The bars display the  means and one standard deviation from mice of the designated groups. (B)  NOS activity in extracts from cultured peritoneal macrophages from  NOS2-modified MRL–lpr/lpr mice. 20-wk-old mice were examined. The  bars display the means and one standard deviation from mice of the designated groups. Peritoneal macrophages were cultured 3 d with no additives  (no addition), or with 10 ng/mL of LPS and 50 U/mL of murine IFN-γ  (LPS/IFN) present in the culture for 3 d.
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Figure 1: (A) Nitrate and nitrite in urine, serum, and peritoneal macrophage supernatant medium from NOS2–modified MRL–lpr/lpr mice. 20-wk-old mice were examined. Serum and urine were collected as noted in Materials and Methods. Resident peritoneal macrophages were cultured for 3 d with 10 ng/mL and 50 U/mL of murine IFN-γ present in the culture for 3 d. Urine values are presented as μmol/day, while Serum and Mac supt. values are presented as μM. The bars display the means and one standard deviation from mice of the designated groups. (B) NOS activity in extracts from cultured peritoneal macrophages from NOS2-modified MRL–lpr/lpr mice. 20-wk-old mice were examined. The bars display the means and one standard deviation from mice of the designated groups. Peritoneal macrophages were cultured 3 d with no additives (no addition), or with 10 ng/mL of LPS and 50 U/mL of murine IFN-γ (LPS/IFN) present in the culture for 3 d.

Mentions: NO production in vivo was first determined by assaying 24-h urinary nitrite/nitrate production. Nitrate and nitrite are direct catabolites of NO (25). When animals are on a nitrite/nitrate-free diet, serum and urine nitrite/nitrate levels reflect total body NO production (26–28). The (+/+) mice excreted large amounts of nitrite/nitrate (Fig. 1 A), confirming our earlier observations (7). The (+/−) mice excreted intermediate levels, whereas (−/−) mice excreted very low levels of nitrite/nitrate (comparable to those of normal BALB/c mice). Nitrite/nitrate levels in sera from 20-wk-old animals were comparable to the urinary measures, with very low levels in the (−/−) mice, high levels in the (+/+) mice, and intermediate levels in the (+/−) mice (Fig. 1 A). Levels of urinary and serum nitrite/nitrate in (+/+) or (+/−) mice were significantly higher than those in the (−/−) mice (P <0.003).


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) Nitrate and nitrite in urine, serum, and peritoneal macrophage supernatant medium from NOS2–modified MRL–lpr/lpr mice.  20-wk-old mice were examined. Serum and urine were collected as  noted in Materials and Methods. Resident peritoneal macrophages were  cultured for 3 d with 10 ng/mL and 50 U/mL of murine IFN-γ present  in the culture for 3 d. Urine values are presented as μmol/day, while Serum and Mac supt. values are presented as μM. The bars display the  means and one standard deviation from mice of the designated groups. (B)  NOS activity in extracts from cultured peritoneal macrophages from  NOS2-modified MRL–lpr/lpr mice. 20-wk-old mice were examined. The  bars display the means and one standard deviation from mice of the designated groups. Peritoneal macrophages were cultured 3 d with no additives  (no addition), or with 10 ng/mL of LPS and 50 U/mL of murine IFN-γ  (LPS/IFN) present in the culture for 3 d.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: (A) Nitrate and nitrite in urine, serum, and peritoneal macrophage supernatant medium from NOS2–modified MRL–lpr/lpr mice. 20-wk-old mice were examined. Serum and urine were collected as noted in Materials and Methods. Resident peritoneal macrophages were cultured for 3 d with 10 ng/mL and 50 U/mL of murine IFN-γ present in the culture for 3 d. Urine values are presented as μmol/day, while Serum and Mac supt. values are presented as μM. The bars display the means and one standard deviation from mice of the designated groups. (B) NOS activity in extracts from cultured peritoneal macrophages from NOS2-modified MRL–lpr/lpr mice. 20-wk-old mice were examined. The bars display the means and one standard deviation from mice of the designated groups. Peritoneal macrophages were cultured 3 d with no additives (no addition), or with 10 ng/mL of LPS and 50 U/mL of murine IFN-γ (LPS/IFN) present in the culture for 3 d.
Mentions: NO production in vivo was first determined by assaying 24-h urinary nitrite/nitrate production. Nitrate and nitrite are direct catabolites of NO (25). When animals are on a nitrite/nitrate-free diet, serum and urine nitrite/nitrate levels reflect total body NO production (26–28). The (+/+) mice excreted large amounts of nitrite/nitrate (Fig. 1 A), confirming our earlier observations (7). The (+/−) mice excreted intermediate levels, whereas (−/−) mice excreted very low levels of nitrite/nitrate (comparable to those of normal BALB/c mice). Nitrite/nitrate levels in sera from 20-wk-old animals were comparable to the urinary measures, with very low levels in the (−/−) mice, high levels in the (+/+) mice, and intermediate levels in the (+/−) mice (Fig. 1 A). Levels of urinary and serum nitrite/nitrate in (+/+) or (+/−) mice were significantly higher than those in the (−/−) mice (P <0.003).

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