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

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.

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.

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.

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.

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.

Figure 3

(A and B) Pathologic features of NOS2-modified MRL–lpr/lpr mice. 20-wk-old mice were examined. The bars show the mean and one standard deviation for the designated groups for renal and joint scores (A), and vessel scores (B).

Figure 3

(A and B) Pathologic features of NOS2-modified MRL–lpr/lpr mice. 20-wk-old mice were examined. The bars show the mean and one standard deviation for the designated groups for renal and joint scores (A), and vessel scores (B).

Figure 4

(A, B, C, and D) Histology of tissues from NOS2-modified MRL–lpr/lpr mice. 20-wk-old mice were examined. A shows a representative section from kidney of a (−/−) MRL–lpr/lpr mouse, demonstrating glomerulonephritis with inflammation, sclerosis, and crescent formation. Comparable lesions were observed in (+/+) mice and in (+/−) mice. B shows a representative section of a knee joint from a (−/−) MRL–lpr/lpr mouse. There is synovial proliferation and inflammation. Comparable lesions were observed in (+/+) mice and in (+/−) mice. C and D show kidney sections containing medium-sized arterioles from MRL–lpr/lpr (+/+) (C) and (−/−) (D) mice. That from the (+/+) a mouse in C demonstrates vasculitis, whereas that from a (−/−) mouse in D is essentially normal with no evidence of vasculitis. (Hematoxylin and eosin stain; original magnifications 200×).

Figure 5

(A and B) Anti-DNA and anti-Ig rheumatoid factor antibody activity in sera from NOS2-modified MRL–lpr/lpr mice. 20-wk-old mice were examined. A shows results from experiments testing sera for anti-ss-DNA antibody and anti-dsDNA antibody activity, and B shows results from experiments testing sera for IgG and IgM anti-Ig (rheumatoid factor) activity. The bars display the means and one standard deviation.

Figure 5

(A and B) Anti-DNA and anti-Ig rheumatoid factor antibody activity in sera from NOS2-modified MRL–lpr/lpr mice. 20-wk-old mice were examined. A shows results from experiments testing sera for anti-ss-DNA antibody and anti-dsDNA antibody activity, and B shows results from experiments testing sera for IgG and IgM anti-Ig (rheumatoid factor) activity. The bars display the means and one standard deviation.