By
* From the Ralph H. Johnson Veterans Affairs Medical Center and the Medical University of South
Carolina, Charleston, South Carolina 29425; Merck Research Laboratories, Rahway, New Jersey
07065; § University of California Davis, Davis, California 95616;
University of Miami Medical
Center, Miami, Florida 33136; and the ¶ Veterans Affairs and Duke University Medical Centers,
Durham, North Carolina 27705
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.
MRL-lpr/lpr mice develop a systemic autoimmune
disease characterized by autoantibody production in
association with a variety of inflammatory manifestations
(1). Although MRL-lpr/lpr mice develop glomerulonephritis, arthritis, and vasculitis, genetic studies suggest that
these clinical manifestations result from different pathogenic mechanisms (5, 6). In these mice, there is overproduction of nitric oxide (NO)1, and this overproduction
parallels the development of clinical disease manifestations
(7). Compared with control mice, MRL-lpr/lpr mice have
enhanced expression of nitric oxide synthase type 2 (NOS2)
in their spleens and kidneys. In addition, they have increased levels of nitrosylated proteins in their kidneys, indicating enhanced NO production within diseased organs
(8). The importance of NO to disease manifestations is further supported by observations that treatment of these mice
with NG-monomethyl-L-arginine inhibits NO production
and prevents glomerulonephritis, arthritis, and vasculitis (7, 9).
NO plays a key role in a number of physiologic processes
(10, 11). As shown in recent studies, three enzyme isoforms
produce NO: a neuronal NOS (NOS1), an endothelial NOS
(NOS3), and NOS2. The inducible form (NOS2) is found
in many cell types, including mononuclear phagocytes, hepatocytes, and chondrocytes. Enhanced NOS2 expression with
increased NO production plays an important role in host
resistance to microbes and neoplasia (12, 13), and in various
forms of inflammation including that seen in MRL-lpr/lpr and
SJL mice (7, 14). Also, patients with rheumatoid arthritis overexpress NOS2 and overproduce NO (17).
Mice lacking detectable NOS2 activity have been derived using recombinatorial techniques (20). To define
the role of NOS2 in the pathogenesis of disease in MRL-
lpr/lpr mice, mice with genetically disrupted NOS2 genes
(NOS2 knockout mice) were bred onto the MRL-lpr/lpr
background for four generations. Littermates were derived
that were homozygous for disrupted NOS2 [( Mice.
The derivation of mice with genetically disrupted
NOS2 has been previously described (20). MRL-lpr/lpr mice
were obtained from the Jackson Laboratories (Bar Harbor, ME)
and bred to the NOS2 modified mice. Derived F1 mice were
bred to MRL-lpr/lpr mice to the N3 generation, and then Fas
homozygotes were identified by PCR analysis of DNA (23), and
used for breeding for the N4 generation to assure Fas homozygosity. N4 MRL-lpr/lpr mice heterozygotic for NOS2 [(+/ Specimen Collection and Nitrate/Nitrite Measures.
Beginning at
12 wk of age, the mice were fed a nitrate/nitrite-free diet (Zeigler Brothers, Gardners, PA). They were placed in murine metabolic cages for 24 h every 2 wk for collection of urine.
/
) mice], heterozygous for disrupted NOS2 [(+/
) mice], or wild-type NOS2 [(+/+) mice]. As shown by pathological and
serological analyses, the absence of NOS2 had varying effects on disease manifestations, with (
/
) mice displaying
equivalent degrees of nephritis and arthritis as compared
with (+/+) mice, while showing markedly reduced vasculitis. These results suggest heterogeneity in mechanisms of
inflammation in MRL-lpr/lpr mice. Furthermore, we demonstrate differences in response to inhibiting NO production by a nonspecific NOS inhibitor in contrast with genetic disruption of the gene for NOS2.
)
mice] were bred to yield the littermates used in these experiments. A total of 6 (+/+) mice, 9 (+/
) mice, and 9 (
/
)
mice were studied. DNA from each individual mouse was examined by Southern blot analysis to confirm the genotype for NOS2
(20). The animals were maintained under pathogen-free conditions at the Merck animal facility (Rahway, NJ) before transfer to
the Durham VA Medical Center animal facility at age 12 wk. Mice
were negative for common murine viral pathogens by sera analyses. Similarly aged female MRL-lpr/lpr and BALB/c mice (Jackson Laboratories) were maintained in the Durham VA Medical
Center animal facility and studied as controls.
Autoantibody Levels. Anti-DNA antibody levels were determined by ELISA as previously described (7). Calf thymus DNA (Sigma Chemical Co., St. Louis, MO) was purified by phenol extraction. Double-stranded (ds) DNA was obtained by treating the DNA with S1 nuclease (Sigma); single-stranded (ss) DNA was derived by boiling the DNA solution for 10 min followed by immediate immersion in ice. The concentrations of DNA were determined by OD260 reading. Purity of the DNA was assessed by OD260/280 ratio with a ratio of >1.9 obtained for the DNA used in this study.
Wells of microtiter plates (Dynatech, McLean, VA) were coated with 0.1 mL of either ds or ss DNA 5 µg/mL. Sera were added in serial dilutions beginning at a 1/100 dilution in PBS containing Tween. Peroxidase-conjugated goat anti-mouse IgG (Histology. Tissues obtained at post mortem dissection were immediately placed in 10% buffered formalin. After fixation and paraffin embedding, sections were cut and stained with hematoxylin and eosin. Slides were read by a pathologist (PR) blinded as to the group of mouse origin. Renal and knee joints were graded according to a scale previously described (6). Vasculitis was noted when present in the renal sections and graded 0-3+.
NOS Enzyme Activity. NOS activity was measured in freshly isolated and cultured peritoneal macrophages by assessing the conversion of L-arginine to L-citrulline using radiolabeled arginine as previously described (7, 19). In brief, cell extracts were derived by 3-5 freeze-thaw cycles in PBS containing protease inhibitors. Lysates were collected after centrifugation and assayed for protein and NOS activity. L-arginine labeled with 14C in the guanido position was added to the lysates; 30 µL of sample were used in a total reaction mixture of 50 µL. Samples were analyzed in duplicate or triplicate. L-arginine to L-citrulline was determined by a lack of adherence of L-citrulline to Dowex AG 50W-X8 cation exchange resin.
Immunoblot Analyses. Immunoblots were performed as described before (19, 24) using a mouse monoclonal anti-mouse NOS2 antibody (Transduction Laboratories, Lexington, KY). Protein extracts from spleen cells were electrophoresed through a denaturing polyacrylamide gel and transferred to nitrocellulose and processed using the enhanced chemiluminescence reagents (ECL, Amersham).
Statistical Analyses. Statistical analysis for significance was performed using the Mann-Whitney U test (two-tailed) unless otherwise noted.
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). 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).
To assess NO production in vitro by cells, peritoneal
macrophages were cultured without additives and with IFN-
(50 U/mL) and LPS (10 ng/mL) (Fig. 1 B). Nitrite/nitrate
levels were significantly lower in the tissue culture supernatant media of macrophages from (
/
) mice than (+/+)
mice or (+/
) mice, both without and with treatment
with LPS and IFN-
. Similarly, NOS2 enzyme activity, as
measured by the conversion of L-arginine to L-citrulline,
was significantly less in the cells from (
/
) mice than
those from (+/
) or (+/+) mice (Fig. 1 B). NOS activities were reduced by more than 90% by inclusion of 2 mM
NG-monomethyl-L-arginine in the reaction mixtures (data
not shown). Differences in peritoneal macrophage NOS
activity between (
/
) and the other two groups were
significant at P <0.0003. These studies confirm a lack of
significant NOS activity in (
/
) mice.
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.
Based on the effect of in vivo administration of NG-monomethyl-L-arginine on renal disease and arthritis in
MRL-lpr/lpr mice (7), we predicted that (/
) mice
would develop less renal disease and arthritis than mice of
the other two groups. However, pathologic examination
indicated that glomerulonephritis in the (
/
) mice was
similar in severity to that of the other two groups. Proliferative glomerulonephritis was present in all mice regardless of NOS2 genotype, with overall glomerular scores similar
between the groups (Figs. 3 A and 4 A). Crescentic glomerulonephritis and interstitial disease were present in a small
number of mice in each group. 24-h urinary protein excretion at 20 wk of age was less in the (
/
) mice than in the
other two groups (4.5 ± 2.8 [mean ± SD] mg/day for
[+/+], 2.6 ± 3.0 for [+/
], and 1.9 ± 0.5 for [
/
]),
but these were not statistically different. Synovitis was
present in the majority of the mice with overall synovial
scores similar in the three groups. Synovial hypertrophy, synovial inflammation, and erosive disease were present to
a similar degree in the MRL-lpr/lpr mice regardless of
NOS2 genotype (Figs. 3 A and 4 B).
In contrast with the findings with glomerulonephritis
and arthritis, there was a significant difference in the presence and severity of vasculitis observed in renal vessels depending on NOS2 genotype (Figs. 3 B, 4, C and 4 D).
Four of six (+/+) mice had prominent vasculitis in the
kidney, whereas one of nine of the (+/) mice and zero
of nine of (
/
) mice had vasculitis (Fig. 4 C and 4 D).
The incidence of vasculitis in the (+/+) mice was similar
to that in 20-wk-old female MRL-lpr/lpr control mice
(80%). The difference in the occurrence of vasculitis between the (+/+) mice and the (
/
) mice was significant
at P <0.015.
Histological examination of the brain, liver, lymph nodes,
spleen, and lung revealed similar mild lymphocytic infiltration in all three groups. Although the combined spleen and
lymph node weights were less in the (/
) mice compared with the (+/+) mice, the differences were not significant (Table 1). Hematocrits were slightly higher in the
(
/
) mice than in the other two groups, but these differences were not significant (Table 1).
|
Autoimmune disease in MRL-lpr/lpr mice is associated
with production of a variety of autoantibodies that are observed in both human rheumatoid arthritis and systemic lupus erythematosus (1). Therefore, we determined whether
MRL-lpr/lpr mice of various NOS2 genotypes displayed
qualitative or quantitative differences in autoantibody production. As shown in Fig. 5 A, serum levels of antibodies
to ss or dsDNA did not differ among the various mice, although there was a shift in the isotype of anti-DNA antibodies produced. The IgG1/IgG3 ratio of anti-DNA antibodies was higher in the (/
) than in (+/+) mice
(1.31 versus 0.39). In contrast with anti-DNA antibody
levels, RF levels differed among the MRL-lpr/lpr mice of
various NOS2 genotypes. The (
/
) mice produced significantly less IgG RF and IgM RF than did (+/+) mice
(Fig. 5 B). IgG3 rheumatoid factor activity (known to be
associated with small vessel, but not medium vessel vasculitis in MRL-lpr/lpr mice; reference 29), was similar in the
three groups of mice [(+/+), 0.733 ± 0.215; (+/
),
0.633 ± 0.362; and (
/
), 0.781 ± 0.278]. Total serum
IgG and IgM were similar in the three groups as were serum levels of anti-Sm and anti-La antibodies (results not
shown).
Our results indicate that absence of a functional gene for
NOS2 has variable effects on disease manifestations in autoimmune MRL-lpr/lpr mice. Thus, although (/
) mice
developed glomerular and synovial pathology of similar severity to that noted in (+/
) or (+/+) mice, they had reduced vasculitis. There was a trend toward reduced renal
disease (manifested by proteinuria) with decreasing numbers of NOS2 genes. However, the differences were not
statistically significantly different. This finding could indicate that lack of NOS2 lessens some manifestations of renal disease in these mice, and that if the mice had been examined at a later timepoint, significant differences in proteinuria and renal histology might have been noted. The
(
/
) mice had significantly lower serum levels of IgG
RF levels than did (+/+) or (+/
) littermates, but total
Ig levels and levels of other autoantibodies were unaltered. The varying effects of a lack of functional NOS2 on autoimmune disease likely reflect differences in the pathogenesis of these abnormalities. Indeed, studies in MRL-lpr/lpr
mice indicate that clinical manifestations of autoimmunity
can be genetically separated by backcross breeding to other
strains. Mapping of susceptibility loci in MRL-lpr/lpr mice
indicate that the expression of glomerulonephritis, arthritis,
and vasculitis are dependent on separate genetic loci (1, 5,
6). Of note, susceptibility to vasculitis is distinct from the
susceptibility to glomerulonephritis (5). This result implies
that in MRL-lpr/lpr mice different pathogenic mechanisms for these two manifestations exist, and our results suggest
that vasculitis is dependent on NOS and NO, whereas
glomerulonephritis and synovitis are not.
Results of the current study suggest that the development
of vasculitis in MRL-lpr/lpr mice requires the presence of a
functional NOS2 gene and NOS2-generated NO. Supporting this notion, analysis of samples from our earlier
study (7) showed that NG-monomethyl-L-arginine treatment
also decreased the incidence of vasculitis in MRL-lpr/lpr
mice (4 of 10 untreated controls developed vasculitis, compared with 1 of 9 mice treated with NG-monomethyl-
L-arginine) (Gilkeson and Weinberg, unpublished data)]. Thus,
it appears that while NOS2 overexpression and NO overproduction contribute to glomerulonephritis, arthritis, and
vasculitis in MRL-lpr/lpr mice (7), vasculitis requires an intact NOS2 response. The role of NOS2 and NO in vasculitis has been demonstrated in other studies. Pulmonary vasculitis in rats, induced by infusion of immune complexes, is
characterized by enhanced local production of NO. Furthermore, inhibiting NO production with L-arginine analogues prevents the vasculitis despite deposition of immune
complexes in the pulmonary vessels (30, 31). In this regard,
(+/) mice as well as (
/
) mice have reduced vasculitis.
These findings, while observed with only a limited number
of mice, raise the possibility that even partial inhibition of
NOS2 activity pharmacologically would be beneficial. We
do not fully understand the cause of this heterozygote effect, but it could indicate a threshold effect for NO and
vasculitis. For vasculitis, there may be a certain threshold of
NO production (a level greater than 50% of that noted in
[+/+] mice) that is required for disease manifestation. Alternatively, there may be more complex mechanisms at
work to explain this. These would include interactions of
NO with other factors involved in inflammation (e.g., cyclooxygenase 2 and arachidonic acid metabolites).
In our study, (/
) mice (despite equivalent levels of
anti-DNA antibodies and total IgG) showed significantly
reduced levels of IgG RF compared with (+/
) and (+/+)
mice when tested with heterologous IgG. The reduction of
these RF may be related to a direct effect of NO on B cells
since, as shown by Genaro and others, NO protects B cells
from apoptosis through a Bcl-2-dependent mechanism (32).
In the absence of NOS2-derived NO, RF-producing B
cells may undergo apoptosis (presumably by a Fas-independent mechanism; reference 33). In this regard, the effects of
NOS2 gene disruption on this type of RF production may
be more pronounced than the effects on other autoantibodies because of differences in the activation requirements
in this response. Thus, as shown in other studies, IgG RF
occurs more sporadically in MRL-lpr/lpr mice than do
anti-DNA antibodies; furthermore, RF levels are not directly related to the extent of hyperglobulinemia, suggesting antigen-specific regulatory interactions (34). These interactions may confer an increased sensitivity to the effects
of NO.
Although the basis for the reduced RF production in
(/
) mice is not clear, this reduction may be linked to
the decrease in the observed frequency and intensity of vasculitis. IgG RF production occurs prominently in humans
with vasculitis, as well as in MRL-lpr/lpr mice. These autoantibodies most likely promote vasculitis because of the
formation of immune complexes that deposit in the vessel
and stimulate local inflammation. In MRL-lpr/lpr mice,
IgG3 RF have been implicated in small vessel disease (35). However, RF of this isotype were found in (
/
) mice,
despite the absence of vasculitis and overall reduction of
RF autoantibodies. Together with studies on anti-DNA
antibody levels, these finding suggest that the effects of NO
on pathogenic autoantibody responses are complex and influence the overall magnitude of these responses as well as
the expression of particular antibody isotypes.
The mechanism(s) for the contrasting effects on renal and
synovial diseases of a genetically disrupted NOS2 as opposed to pharmacological inhibition of NOS activity with
NG-monomethyl-L-arginine is not clear. It is unlikely that
there were compensatory increases in NOS1 or NOS3 in
the (/
) mice, because we noted very low total body NO
production in the (
/
) mice. NG-monomethyl-L-arginine is an isoform-nonspecific NOS inhibitor, blocking all
three isoforms of the NOS enzymes (36). Inhibiting all NOS
isoforms (and hence potentially all NO production) with NG-monomethyl-L-arginine may be more effective in disease prevention than genetically disrupting only NOS2. Alternative inflammatory pathways may not be active when
NO production is acutely blocked by NG-monomethyl-
L-arginine; however, these pathways might become active
over time when NOS2 is genetically disrupted and absent the entire life of the animal.
Studies using NG-monomethyl-L-arginine in MRL-lpr/lpr mice indicate that NO is important in the pathogenesis of glomerulonephritis, arthritis, and vasculitis (7, 9). However, the work reported here studying inflammation in MRL- lpr/lpr mice with genetically disrupted NOS2 highlights the heterogeneity and complexity of the role NOS2 and NO.
Address correspondence to J. Brice Weinberg, MD, VA and Duke University Medical Centers, 508 Fulton Street, Durham, NC 27705. Phone: 919-286-6833; FAX: 919-286-6891; E-mail: brice{at}acpub.duke.edu
Received for publication 7 April 1997 and in revised form 3 June 1997.
The work was supported in part by the Veterans Affairs Research Service, the James R. Swiger Hematology Research Fund, an Arthritis Foundation Biomedical Research Grant, a grant from the Lupus Foundation of America, and National Institutes of Health award AR-39162.1. | Cohen, P.L., and R.A. Eisenberg. 1991. Lpr and gld: single gene models of systemic autoimmunity and lymphoproliferative disease. Annu. Rev. Immunol. 9: 243-269 [Medline]. |
2. | Andrews, B.S., R.A. Eisenberg, A.N. Theofilopoulos, S. Izui, C.B. Wilson, P.J. McConahey, E.D. Murphy, J.B. Roths, and F.J. Dixon. 1978. Spontaneous murine lupus-like syndromes. Clinical and immunological manifestations in several strains. J. Exp. Med. 148: 1198-1215 [Abstract]. |
3. | Hang, L., A.N. Theofilopoulos, and F.J. Dixon. 1982. A spontaneous rheumatoid arthritis-like disease in MRL-l mice. J. Exp. Med. 155: 1690-1701 [Abstract]. |
4. | Moyer, C.F., J.D. Strandberg, and C.L. Reinisch. 1987. Systemic mononuclear-cell vasculitis in MRL/Mp-lpr/lpr mice. A histologic and immunocytochemical analysis. Am. J. Pathol. 127: 229-242 [Abstract]. |
5. | Nose, M., M. Nishimura, and M. Kyogoku. 1989. Analysis of granulomatous arteritis in MRL/Mp autoimmune disease mice bearing lymphoproliferative genes. The use of mouse genetics to dissociate the development of arteritis and glomerulonephritis. Am. J. Pathol. 135: 271-80 [Abstract]. |
6. | Watson, M.L., J.K. Rao, G.S. Gilkeson, P. Ruiz, E.M. Eicher, D.S. Pisetsky, A. Matsuzawa, J.M. Rochelle, and M.F. Seldin. 1992. Genetic analysis of MRL-lpr mice: relationship of the Fas apoptosis gene to disease manifestations and renal disease-modifying loci. J. Exp. Med. 176: 1645-1656 [Abstract]. |
7. | Weinberg, J.B., D.L. Granger, D.S. Pisetsky, M.F. Seldin, M.A. Misukonis, S.N. Mason, A.M. Pippen, P. Ruiz, E.R. Wood, and G.S. Gilkeson. 1994. The role of nitric oxide in the pathogenesis of spontaneous murine autoimmune disease: increased nitric oxide production and nitric oxide synthase expression in MRL-lpr/lpr mice, and reduction of spontaneous glomerulonephritis and arthritis by orally administered NG-monomethyl-L-arginine. J. Exp. Med. 179: 651-660 [Abstract]. |
8. | Privalle, C.T., T. Keng, G.S. Gilkeson, and J.B. Weinberg. 1996. The role of nitric oxide and peroxynitrite in the pathogenesis of spontaneous murine autoimmune disease. In The Biology of Nitric Oxide. Vol. 5. J. Stamler, S.S. Gross, and S. Moncada, editors. Portland Press, London. 20. |
9. | Oates, J.C., P. Ruiz, A. Alexander, A.M.M. Pippen, and G.S. Gilkeson. 1997. Effect of late modulation of nitric oxide production on murine lupus. Clin. Immunol. Immunopath. 83: 86-92 [Medline]. |
10. |
Nathan, C..
1992.
Nitric oxide as a secretory product of
mammalian cells.
FASEB J.
6:
3051-3064
|
11. |
Moncada, S., and
A. Higgs.
1993.
The L-arginine-nitric oxide pathway.
N. Eng. J. Med.
329:
2002-2012
|
12. | Nathan, C.F., and J.B. Hibbs Jr.. 1991. Role of nitric oxide synthesis in macrophage antimicrobial activity. Curr. Opin. Immunol. 3: 65-70 [Medline]. |
13. | Hibbs, J. Jr.. 1991. Synthesis of nitric oxide from L-arginine: a recently discovered pathway induced by cytokines with antitumour and antimicrobial activity. Res. Immunol. 142: 565-569 [Medline]. |
14. |
Clancy, R.M., and
S.B. Abramson.
1995.
Nitric oxide![]() |
15. | Tamir, S., T. Derojaswalker, A. Gal, A.H. Weller, X.T. Li, J.G. Fox, G.N. Wogan, and S.R. Tannenbaum. 1995. Nitric oxide production in relation to spontaneous B-cell lymphoma and myositis in SJL mice. Cancer Res. 55: 4391-4397 [Abstract]. |
16. | Huang, F.P., G.J. Feng, G. Lindop, D.I. Stott, and F.Y. Liew. 1996. The role of interleukin 12 and nitric oxide in the development of spontaneous autoimmune disease in MRL: MP-lpr:lpr mice. J. Exp. Med. 183: 1447-1459 [Abstract]. |
17. | Farrell, A.J., D.R. Blake, R.M. Palmer, and S. Moncada. 1992. Increased concentrations of nitrite in synovial fluid and serum samples suggest increased nitric oxide synthesis in rheumatic diseases. Ann. Rheum. Dis. 51: 1219-1222 [Abstract]. |
18. | Sakurai, H., H. Kohsaka, M.F. Liu, H. Higashiyama, Y. Hirata, K. Kanno, I. Saito, and N. Miyasaka. 1995. Nitric oxide production and inducible nitric oxide synthase expression in inflammatory arthritides. J. Clin. Invest. 96: 2357-2363 [Medline]. |
19. | St. Clair, E.W., W.E. Wilkinson, T. Lang, L. Sanders, M.A. Misukonis, G.S. Gilkeson, D.S. Pisetsky, D.L. Granger, and J.B. Weinberg. 1996. Increased expression of blood mononuclear cell nitric oxide synthase type 2 in rheumatoid arthritis patients. J. Exp. Med. 184: 1173-1178 [Abstract]. |
20. | Macmicking, J.D., C. Nathan, G. Hom, N. Chartrain, D.S. Fletcher, M. Trumbauer, K. Stevens, Q.W. Xie, K. Sokol, N. Hutchinson, H. Chen, and J.S. Mudgett. 1995. Altered responses to bacterial infection and endotoxic shock in mice lacking inducible nitric oxide synthase. Cell. 81: 641-650 [Medline]. |
21. | Wei, X.Q., I.G. Charles, A. Smith, J. Ure, C.J. Feng, F.P. Huang, D.M. Xu, W. Muller, S. Moncada, and F.Y. Liew. 1995. Altered immune responses in mice lacking inducible nitric oxide synthase. Nature (Lond.). 375:408-411. |
22. | Laubach, V.E., E.G. Shesely, O. Smithies, and P.A. Sherman. 1995. Mice lacking inducible nitric oxide synthase are not resistant to lipopolysaccharide-induced death. Proc. Natl. Acad. Sci. USA. 92: 10688-10692 [Abstract]. |
23. |
Mixter, P.F.,
J.Q. Russell,
F.H. Durie, and
R.C. Budd.
1995.
Decreased CD4-CD8-TCR-alpha beta + cells in lpr/
lpr mice lacking beta 2-microglobulin.
J. Immunol.
154:
2063-2074
|
24. | Anstey, N.M., J.B. Weinberg, M. Hassanali, E.D. Mwaikambo, D. Manyenga, M.A. Misukonis, D.R. Arnelle, D. Hollis, M.I. McDonald, and D.L. Granger. 1996. Nitric oxide in Tanzanian children with malaria. Inverse relationship between malaria severity and nitric oxide production/nitric oxide synthase type 2 expression. J. Exp. Med. 184: 557-567 [Abstract]. |
25. | Stamler, J.S., D.J. Singel, and J. Loscalzo. 1992. Biochemistry of nitric oxide and its redox-activated forms. Science (Wash. DC). 258: 1898-1902 [Medline]. |
26. |
Granger, D.L.,
J. Hibbs Jr., and
L.M. Broadnax.
1991.
Urinary
nitrate excretion in relation to murine macrophage activation. Influence of dietary L-arginine and oral NG-monomethyl-L-arginine.
J. Immunol.
146:
1294-1302
|
27. | Granger, D.L., W.C. Miller, and J.B. Hibbs, Jr. 1996. Methods of analyzing nitric oxide production in the immune response. In Methods in Nitric Oxide Research. M. Feelisch and J.S. Stamler, editors. John Wiley & Sons, Ltd., New York. 603-617. |
28. | Hibbs, J.B. Jr., C. Westenfelder, R. Taintor, Z. Vavrin, C. Kablitz, R.L. Baranowski, J.H. Ward, R.L. Menlove, M.P. McMurry, J.P. Kushner, and W.E. Samlowski. 1992. Evidence for cytokine-inducible nitric oxide synthesis from L-arginine in patients receiving interleukin-2 therapy (erratum published 90:295). J. Clin. Invest. 89: 867-877 [Medline]. |
29. |
Takahashi, S.,
M. Nose,
J. Sasaki,
T. Yamamoto, and
M. Kyogoku.
1991.
IgG3 production in MRL/lpr mice is responsible for development of lupus nephritis.
J. Immunol.
147:
515-519
|
30. | Mulligan, M.S., S. Moncada, and P.A. Ward. 1992. Protective effects of inhibitors of nitric oxide synthase in immune complex-induced vasculitis. Brit. J. Pharmacol. 107: 1159-62 [Abstract]. |
31. |
Mulligan, M.S.,
J.S. Warren,
C.W. Smith,
D.C. Anderson,
C.G. Yeh,
A.R. Rudolph, and
P.A. Ward.
1992.
Lung injury after deposition of IgA immune complexes. Requirements for CD18 and L-arginine.
J. Immunol.
148:
3086-3092
|
32. | Genaro, A.M., S. Hortelano, A. Alvarez, C. Martineza, and L. Bosca. 1995. Splenic B lymphocyte programmed cell death is prevented by nitric oxide release through mechanisms involving sustained Bcl-2 levels. J. Clin. Invest. 95: 1884-1890 [Medline]. |
33. | Zheng, L., G. Fisher, R.E. Miller, J. Peschon, D.H. Lynch, and M.J. Lenardo. 1995. Induction of apoptosis in mature T cells by tumour necrosis factor. Nature (Lond.). 377: 348-351 [Medline]. |
34. | Warren, R.W., S.A. Caster, J.B. Roths, E.D. Murphy, and D.S. Pisetsky. 1984. The influence of the lpr gene on B cell activation: differential antibody expression in lpr congenic mouse strains. Clin. Immunol. Immunopath. 31: 65-77 [Medline]. |
35. | Berney, T., T. Fulpius, T. Shibata, L. Reininger, J. Van Snick, H. Shan, M. Weigert, A. Marshak-Rothstein, and S. Izui. 1992. Selective pathogenicity of murine rheumatoid factors of the cryoprecipitable IgG3 subclass. Int. Immunol. 4: 93-99 [Abstract]. |
36. | Southan, G.J., and C. Szabo. 1996. Selective pharmacological inhibition of distinct nitric oxide synthase isoforms. Biochem. Pharmacol. 51: 383-394 [Medline]. |