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*
Department of Immunology, University of Glasgow, Glasgow, United Kingdom; and
Department of Bacteriology, Glasgow Royal Infirmary, Glasgow, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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synthesis
in vivo and in vitro and enhanced IFN-
compared with IL-4 production
in vitro in iNOS-mutant mice demonstrated exaggerated Th1 polarization
of the host response. These data indicate that high output NO
production is not a prerequisite for severe articular destruction and
imply that NO is of importance in synovial defense against
staphylococcal infection. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-1ß,
and IFN- (9, 10), or by bacterial products, such as LPS and
staphylococcal enterotoxin (SE) B (4, 9, 10, 11), and iNOS expression has
now been demonstrated in many cell types, including macrophages,
endothelial cells, chondrocytes, and synovial fibroblasts (10, 11, 12, 13). Although NO clearly has been shown to mediate cytotoxicity against intracellular pathogens, including protozoa, viruses, bacteria, and fungi (14, 15, 16, 17), its role in extracellular Gram-positive bacterial infections is less well understood. NO has been implicated in S. aureus killing by cytokineplasts from human neutrophils (18, 19), and in a cell-free system, NO donors are bactericidal for S. aureus, although the time course of bacterial killing is delayed compared with that mediated by reactive oxygen intermediates (20). In vivo, injection of certain staphylococcal exotoxins, such as toxic shock syndrome toxin (TSST)-1 or SEB, leads to a T lymphocyte-mediated shock syndrome in BALB/c mice, which can be exacerbated with exogenous NOS inhibitors (21). However, staphylococcal cell wall components, such as peptidoglycan and lipoteichoic acid, synergistically induce multiple organ failure in rats by an NO-dependent mechanism (22). NO production may therefore have potential deleterious, as well as beneficial, effects during S. aureus infection in vivo.
Intravenous inoculation of Swiss mice with an exotoxin-producing strain of S. aureus leads to development of severe septic arthritis, but rarely induces septicemia (23, 24). The articular lesion exhibits synovial hyperplasia, containing neutrophils, macrophages, and a prominent T lymphocyte infiltrate, with associated erosion of underlying cartilage and bone. Anti-CD4 or anti-Vß11 Ab administration to delete superantigen-responsive T cell subsets improves disease outcome following infection (25, 26), confirming a central role of T cell activation in pathogenesis. Moreover, amplification of this T cell response by infection of Vß3 transgenic mice with a strain of TSST-1-producing S. aureus leads to significantly enhanced disease severity, with systemic involvement and increased mortality (27). This indicates that regulatory pathways normally operate to limit immunologically mediated systemic complications following S. aureus infection. Such pathways include the production of glucocorticoids, anti-inflammatory cytokines, principally IL-10, and perhaps, the generation of NO (28).
We previously generated mice lacking iNOS, which exhibit normal
constitutive endothelial NOS and neuronal NOS expression (29). This
provides an ideal model in which to address the role of high output NO
synthesis on the clinical and immunologic sequelae to S.
aureus infection. We now report that i.v. inoculation with
S. aureus induced significantly increased clinical severity
of septic arthritis, with attendant septicemia, in iNOS-deficient mice
compared with similarly infected heterozygous or wild-type mice. These
observations were associated with enhanced production of IFN- and
TNF- in vivo and in vitro, indicating Th1 polarization of the
cell-mediated immune response. Our data define an important net
protective role for NO in the host response to infections with S.
aureus. They also strongly caution against the clinical use of
selective NOS-inhibitor therapy in the management of septic
arthritis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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iNOS-deficient mice were constructed as previously described
(29). Disruption of the murine iNOS gene was achieved by homologous
recombination in 129sv embryonic stem cells. The recombinant allele was
passed through the germline following mating of embryonic stem cell
chimeras with MF1 mice (Harlan Ltd., Oxon, UK). The homozygous,
heterozygous, and wild-type mice thus generated were backcrossed to MF1
for three generations. All of the mice used were from littermate
matings, and should therefore have similar MF1 genetic background. In
later experiments, homozygous, heterozygous, and wild-type littermates
of the 129svx129sv strain were used which were derived from mating the
chimeras with 129 mice (Harlan). Data from spleen cell cultures from
these mice were identical to those obtained in 129svxMF1 mice. In some
experiments, spleen cells from mice maintained in a specific
pathogen-free (SPF) environment were also used. Extensive experiments
using a variety of parameters demonstrated phenotypic similarity
between the heterozygous and wild-type littermates. Peritoneal cells
from the mutant mice did not produce iNOS protein following activation
with IFN- and LPS in vitro, as judged by Western blot. They also did
not produce detectable amounts of nitrate up to 48-h culture with
IFN- and LPS. By 72 h, however, low levels of nitrite were
detected in culture supernatants of cells from the mutant mice, which
may have reflected the accumulation of nitrite produced by constitutive
NOS, or the induction of constitutive NOS (29).
Induction and assessment of septic arthritis
This was performed as previously described (23, 24). Briefly,
LS-1, a TSST-1-producing S. aureus originally isolated from
a spontaneous outbreak of murine septic arthritis (gift of Dr. T.
Bremell, University of Lund, Lund, Sweden), was stored in nutrient agar
at 4°C, and before each experiment was cultured on 5% blood agar for
24 h at 37°C before resuspension in sterile PBS. The cell
suspension was standardized spectrophotometrically to contain 5 x
108 CFU/ml. Male and female mice aged 3 to 4 wk received
either 5 x 107 CFU S. aureus in 100 µl
PBS injected i.v. via the tail vein, or 100 µl PBS alone. Wild-type
or iNOS-deficient heterozygote mice served as controls for comparison
with iNOS-deficient homozygote mice (29). Individual mice were observed
daily for up to 14 days, blind to genotype or infection status.
Incidence of arthritis, number of limbs involved, and
footpad/intermalleolar diameter (calliper measurement; Kroeplin,
Metallex, Paris, France)were measured, and an articular index was
derived, 
3 points/limb: 1, erythema alone; 2, swelling and erythema;
and 3, erythema, swelling, and extension/loss of function. In one
experiment, arthritic limbs (articular index = 3) from five
iNOS-deficient and five heterozygote mice were fixed in 10% neutral
buffered Formalin, and 5-µm sections were subsequently stained with
hematoxylin and eosin (Sigma Chemical Co., Poole, U.K.) for assessment
in a genotype-blinded manner. The severity of septicemia was judged
clinically by characteristic changes in coat appearance (1), posture
(1), presence of spontaneous movement (1), and mucocutaneous abscess
formation (1), from which a septic index (maximum score 4/mouse) was
derived for each animal by a blinded observer and also by daily weight
measurement (g). Autopsy was performed on every animal to determine
internal skeletal or large organ involvement. Viable S.
aureus tissue distribution was estimated by whole organ culture,
as described (27). Mice (n = 3/group) were
killed before, or 3, 7, and 11 days after i.v. staphylococcal
injection. Spleens and kidneys were dissected, homogenized, then
diluted in 10-fold steps in sterile PBS. Blood (200 µl) was similarly
diluted in sterile PBS. Each dilution (100 µl) was plated on to
prewarmed 5% blood agar and cultured for 24 h at 37°C to
determine the number of CFU present, expressed per ml of blood, or per
whole organ. Serum was also collected and stored at -70°C until
assay either for cytokine concentration by ELISA, or for estimation of
nitrite/nitrate concentration using a chemiluminescence method (11, 29). Briefly for the latter, individual sera were incubated with
nitrate reductase (Sigma Chemical Co.) at 37°C for 2 h, then
introduced through boiling glacial acetic acid, with 6% sodium iodide,
into a chemiluminescence NO analyzer (Dasibi Environmental Corporation,
Osaka Japan).
Spleen cell culture
Spleen single cell suspensions, obtained by gently mincing spleens, followed by filtration through Nytex membrane, were cultured at 2 x 106 cells/ml for up to 96 h in RPMI (Life Technologies, Paisley, U.K.) supplemented with 2 mM L-glutamine, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 10% FCS (all Life Technologies) at 37°C. Cells were stimulated with either the staphylococcal exotoxins SEA and TSST-1 (both Sigma Chemical Co.), or heat-killed S. aureus at concentrations indicated in figure legends. Proliferation assays were performed in triplicate in U-bottom 96-well culture plates (Nunclon microwell; Nunc, Roskilde, Denmark), with addition of 1 µCi of [3H]thymidine (Amersham Life Sciences, Bucks, U.K.) in 25 µl during the final 6 h of culture before harvesting onto a glass fiber filter (Packard, Wallac, Milton Keynes, UK) using a Micromate 196 Harvester (Packard). [3H]Thymidine incorporation was measured using a Matrix 96, direct beta counter (Packard). In some experiments, duplicate 500-µl cultures were performed for up to 96 h in 48-well plates (Costar Corning Costar, Burks, UK), and supernatants were stored at -70°C until estimation of cytokine content by ELISA. Spleens from SPF mice aged 4 to 6 wk were obtained from Dr. Charles Lam (Sandoz, Vienna, Austria).
FACS phenotyping
Spleen cells were harvested from the above cultures after 48 h by gentle pipetting, washed once in serum-free RPMI, then 105 cells were incubated with 5 µl of Ab for 20 min at room temperature in the dark. Anti-murine CD4 (phycoerythrin), anti-murine CD8 (FITC), and IgG1 negative control Abs were obtained from PharMingen (San Diego, CA). One milliliter of FACS brand lysing solution (Becton Dickinson, Oxford, U.K.) was added for 10 min, spun at 300 x g for 5 min, then washed once in 1 ml of FACSFlow (Becton Dickinson) at 200 x g for 5 min. Cells within the lymphocyte region, gated by forward and side light scatter, were analyzed using Lysis II software (Becton Dickinson).
Cytokine estimation
Murine TNF-, IFN-, IL-4, and IL-6 were detected in
supernatants or serum by ELISA, with paired capture and biotinylated
detection mAbs for each cytokine (PharMingen). The lower limit of
detection in each assay was as follows: murine TNF-, 10 pg/ml;
murine IFN-, 30 pg/ml; murine IL-4, 40 pg/ml; and murine IL-6, 20
pg/ml.
Statistical analysis
This was performed using Minitab software for Macintosh.
Analysis was by Log-Rank, 
2, Mann-Whitney, or
Student’s t tests, as indicated.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We first investigated the incidence and severity of septic
arthritis in iNOS-deficient mice. Homozygous iNOS-deficient
(n = 33), heterozygous (n
= 28), or wild-type (n = 20) mice received
5 x 107 CFU/ml S. aureus i.v. in three
independent experiments. Clinical signs of arthritis were observed
within 2 days of infection. After 10 days, whereas articular
inflammation was evident in only 50% of heterozygous and wild-type
mice, up to 93% of iNOS-deficient mice developed arthritis
(p < 0.005; Fig. 1
a). Moreover, the mean
articular index was significantly higher in arthritic homozygous mice
than in arthritic control mice from day 5 (p <
0.05 to p < 0.01; Fig. 1b). The
peripheral distribution of arthritis was similar between groups, but
iNOS-deficient mice showed a higher incidence of paraspinal abscess
formation, often with hind-limb paralysis and urinary incontinence
(39% vs 10.7%; p < 0.05). Paraspinal abscess
formation was usually a premorbid presentation, but in 9% of mutant
mice was asymptomatic and found only at postmortem. Histologic
examination of arthritic joints of equivalent severity from
iNOS-deficient and control mice confirmed the presence of cartilage and
bone erosion by synovial pannus, but revealed no difference in
appearance between groups (data not shown). Thus, in contrast to murine
autoimmune arthritis models, in which inhibition of NO synthesis is
protective (6, 7, 8), mice lacking the capability for high output NO
synthesis display an increased incidence and severity of septic
arthritis following S. aureus infection.
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Clinical evidence of septicemia was evident in 65% of
iNOS-deficient mice within 48 h, compared with 14% of controls,
rising to 83% compared with 20%, respectively, after 4 days
(p < 0.005; Fig. 2a). Disease severity
in animals developing sepsis was greater in homozygous than control
mice during the acute phase up to day 6 (p <
0.001 to p < 0.04; Fig. 2b). These
observations were reflected in significantly greater weight loss in
iNOS-deficient mice following infection compared with controls
(p < 0.01; Fig. 2c), or with
uninfected iNOS-deficient littermates (data not shown). Increased
mortality was observed in mutant mice, which reached 30% after 8 days
(p < 0.005 to p < 0.03; Fig. 2d). No significant clinical differences between
heterozygote and wild-type mice were observed. Serum nitrite/nitrate
concentrations in wild-type (day 0, 38 ± 5.4 µM vs day 1,
101 ± 7.4 µM (mean SD); n = 3,
p < 0.001) and heterozygous (day 0, 25 ± 6.3
µM vs day 1, 43 ± 2.9 µM; n = 3,
p < 0.05) mice were elevated significantly 24 h
after inoculation. In contrast, basal levels of serum nitrite/nitrate
were lower in iNOS-deficient mice and remained unaltered by
staphylococcal infection (day 0, 19 ± 1.3 µM vs day 1,
16.4 ± 1.6 µM; n = 3). Thus, increased NO
generation rapidly followed staphylococcal infection in vivo, and this
response was absent in homozygous mice.
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a). High numbers of
bacteria were present in the spleens of both groups after day 3, but
persisted to days 7 and 11 only in mutant mice (Fig. 3b). These data indicated that although both groups
similarly localized staphylococci to spleen, subsequent bacterial
viability within, and dissemination from, the spleen were increased in
iNOS-deficient mice. This was relevant to end-organ disease since
greater numbers of viable staphylococcal CFU were also detected in
kidneys of mutant mice (Fig. 3c).
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We next investigated whether the altered clinical phenotype
in iNOS-deficient mice was reflected in the immune response following
staphylococcal infection. TNF- has been implicated previously in the
pathogenesis of Gram-positive bacterial shock (30). Serum TNF-
levels were significantly higher at day 3 (p <
0.05) and day 7 (p < 0.009) in mutant compared
with control mice (Fig. 4a), corresponding to
that period of maximal clinical sepsis and weight loss (Fig. 2
).
Moreover, spleen cells from iNOS-deficient donors cultured with
heat-killed staphylococci in vitro generated significantly higher
levels of TNF- (p < 0.005, Fig. 4b) than did spleen cells from controls. Elevated
levels of IL-6 were also present in serum, but in contrast to TNF-,
there was no significant difference observed between the groups at any
time point (range 710–824 pg/ml, days 3–7 postinfection).
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nor IL-4 was detected in serum in any infected group at
days 0, 3, 7, or 11. IFN- and IL-4 production were therefore
compared in spleen cell suspensions, obtained from infected mutant or
heterozygous mice, cultured in the presence or absence of heat-killed
S. aureus. Spontaneous IFN- production was significantly
higher in cultures from iNOS-deficient mice at each time point
(p < 0.005 to p < 0.001; Fig. 4c). Addition of heat-killed S. aureus led
to significant up-regulation of IFN- synthesis in cultures from
control mice, to levels similar to those spontaneously produced by
iNOS-deficient spleen cells (Fig. 4d), showing that
diminished spontaneous IFN- production was not due to some inherent
deficit in control mice. No further up-regulation of IFN- synthesis
in cultures from mutant mice was observed, indicating that maximal
induction of IFN- production had been achieved in vivo (Fig. 4d). No IL-4 was produced in vitro by cultures from
any mice, even after S. aureus stimulation (data not shown).
These observations are consistent with the presence of a dominant Th1
response during staphylococcal infection, which is enhanced in
iNOS-deficient mice. Superantigen responses in mice lacking iNOS
The diverse immunoregulatory properties of NO raised the
possibility that iNOS-deficient mice generated altered immune responses
a priori, thereby contributing to T lymphocyte-mediated pathology,
irrespective of bacterial distribution (Fig. 3a–c).
Spleen cells from uninfected mutant or control mice were cultured with
TSST-1, the exotoxin secreted by the S. aureus LS-1.
Significantly enhanced proliferation (p <
0.005; Fig. 5a) and
IFN- production (p < 0.05,
p < 0.005; Fig. 5c) to TSST-1 were
observed in cultures from iNOS-deficient mice compared with cultures
from control mice. Low levels of IL-4 were detected after 96 h,
but in contrast to IFN-, no significant difference was observed
between groups (Fig. 5e). Similar data were obtained
using SEA (Fig. 5, b, d, and
f), indicating that this was not a unique effect of
TSST-1. FACS analysis confirmed the presence of similar
CD4+ and CD8+ populations in both
iNOS-deficient (mean ± SD; 19.67 ± 2.5% CD4+,
7 ± 0.7% CD8+) and control (21 ± 2%
CD4+, 7.5 ± 0.5% CD8+) spleen cell
cultures, suggesting that enhanced IFN- production was not due to
altered T cell subset proportions in vitro. Since iNOS-deficient mice
might have different pathogen/commensal exposure during development,
the effect of TSST-1 and SEA-induced spleen cell proliferation and
cytokine production was further investigated in SPF mice. Proliferation
and IFN- production were enhanced significantly in cultures from
mutant mice compared with those from controls (Fig. 6a–d). IL-4 was not
detected in any cultures up to 72 h (data not shown). Together
these data clearly demonstrate enhanced Th1 polarization in responses
to superantigens in iNOS-deficient mice.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and TNF-, which should have ensured maximal phagocytic
activity within the reticuloendothelial system. This is in accordance
with in vitro observations indicating that NO is an important cofactor
in staphylococcal phagocytosis and subsequent killing by
polymorphonuclear cells (18, 19, 31). Studies indicating that NO
promotes multiple organ failure following injection of staphylococcal
cell wall products (22) do not address either phagocytosis and killing
of viable bacteria, nor the crucial regulatory role of NO in
exotoxin-mediated pathogenesis. The potential for articular and
systemic pathology resulting from superantigen-mediated T cell
activation has been documented clearly in studies in which
superantigen-specific T cell subsets have been targeted using mAbs (25, 26). Such responses mediate a predominantly Th1-lead process (27, 32, 33). Our data demonstrate that failure to regulate such Th1 activity in
iNOS-deficient mice is likely to contribute significantly to
pathology. NO appears critical in down-regulating immune responses capable of potential damage to the host. Since uninfected iNOS-deficient mice exhibited enhanced Th1 polarization in responses to superantigens in vitro, it seems likely that the exaggerated Th1 response observed during staphylococcal infection in vivo reflected an inherent failure to properly regulate T cell responses. That this was due to altered susceptibility to infection with other pathogens during development was unlikely since similar observations were made in SPF animals. These data are consistent with our earlier finding that iNOS-deficient mice infected with Leishmania major develop elevated Th1 responses (29), and with observations in vitro that proliferation and cytokine production by Th1 clones are inhibited by high concentrations of NO donors (34). Moreover, macrophage-mediated suppression of SEA- or SEB-induced T cell activation in vitro has been attributed to NO production (35). The mechanism whereby NO influences such immunoregulation is currently unknown. It is possible that NO preferentially regulates the production of cytokines implicated in Th1/Th2 differentiation through modification of cytokine gene transcription (36). As NO has also been implicated in apoptotic pathways (37), activation-induced cell death, which normally follows superantigen stimulation, could be impaired in iNOS-deficient mice. Endothelial expression of P-selectin, which selectively mediates Th1 recirculation to inflammatory sites, is down-regulated by peroxynitrite (38), providing a further potential regulatory role for NO during Th1-mediated staphylococcal arthritis. Other immunoregulatory circuits operate following SEB injection in mice, including production of IL-10 and granulocyte-macrophage CSF and of endogenous glucocorticoids (28). The interaction of NO with such pathways is currently unclear.
Previous studies of autoimmune inflammatory arthritis in rodents
indicated that lack of NO is protective since NOS inhibitors retard or
abrogate collagen-induced, streptococcal cell wall or adjuvant-induced
arthritis (6, 7, 8). Our data clearly indicate that the opposite is the
case in infectious arthritis. NO up-regulates metalloproteinase and
cyclooxygenase function, impairs chondrocyte chemotaxis and
biosynthetic activity, and promotes bone resorption (39, 40, 41, 42). Such
proinflammatory effects for NO mediated locally, as demonstrated in
other arthritis models (6, 7, 8), cannot be ruled out in septic arthritis.
However, it appears that enhanced systemic Th1 cytokine production,
leading to increased TNF- synthesis, dominates the host response to
S. aureus with resultant articular destruction, and in this
context NO is a necessary protective regulator. Thus, there appears to
be a clear distinction between arthritis caused by live organisms
compared with that induced by either nonliving organisms or their
secreted/cell wall components. It will be important to establish
whether other cytokines, such as IL-15 (43), which are thought to be
important in articular inflammation, are also up-regulated in
iNOS-deficient mice.
We have demonstrated that NO is an important component of the host
response to extracellular Gram-positive bacteria. In the absence of
high output NO synthesis, mice developed an exaggerated Th1 response
and exhibited increased frequency and severity of septic arthritis,
with associated septicemia. This increased susceptibility to sepsis may
reflect a role in staphylococcal killing for NO, either directly, or
through the synthesis of peroxynitrite together with superoxide. Other
pathogenetic mechanisms, including the altered immunoregulation
described in this work, which led to enhanced TNF- production, may
also influence systemic disease manifestations. These findings suggest
that selective inhibitors of iNOS, keenly sought for the treatment of
septic shock, are unlikely to be of benefit in septic arthritis.
Moreover, the potential for reduced synovial defense against
staphylococcal infection, and the removal of an important Th1
regulatory element, suggests that iNOS inhibitors may perhaps be of
reduced value in the management even of noninfectious inflammatory
arthropathies, which are believed to be Th1 mediated.
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Acknowledgments |
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Footnotes |
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2 Current address: Arthritis & Rheumatism Branch, NIAMS, National Institutes of Health, Bethesda, MD 20892.
3 Address correspondence and reprint requests to Dr. F. Y. Liew, Department of Immunology, University of Glasgow, Glasgow, G11 6NT, U.K. E-mail address:
4 Abbreviations used in this paper: NO, nitric oxide; iNOS, inducible nitric oxide synthase; NOS, nitric oxide synthase; SEA, staphylococcal enterotoxin A; SEB, staphylococcal enterotoxin B; SPF, specific pathogen-free; TSST, toxic shock syndrome toxin.
Received for publication May 13, 1997. Accepted for publication September 18, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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receptor deficiency on experimental Staphylococcus aureus septicaemia and arthritis. J. Immunol. 155:5736.[Abstract]
production in rheumatoid arthritis. Nat. Med. 3:189.[Medline]
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D. M. McKay, J. Lu, S. Jedrzkiewicz, W. Ho, and K. A. Sharkey Nitric Oxide Participates in the Recovery of Normal Jejunal Epithelial Ion Transport Following Exposure to the Superantigen, Staphylococcus aureus Enterotoxin B J. Immunol., October 15, 1999; 163(8): 4519 - 4526. [Abstract] [Full Text] [PDF]
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X.-q. Wei, B. P. Leung, W. Niedbala, D. Piedrafita, G.-j. Feng, M. Sweet, L. Dobbie, A. J. H. Smith, and F. Y. Liew Altered Immune Responses and Susceptibility to Leishmania major and Staphylococcus aureus Infection in IL-18-Deficient Mice J. Immunol., September 1, 1999; 163(5): 2821 - 2828. [Abstract] [Full Text] [PDF]
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D. Piedrafita, D. Xu, D. Hunter, R. A. Harrison, and F. Y. Liew Protective Immune Responses Induced by Vaccination with an Expression Genomic Library of Leishmania major J. Immunol., August 1, 1999; 163(3): 1467 - 1472. [Abstract] [Full Text] [PDF]
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A. E. Millar, J. Sternberg, C. McSharry, X.-Q. Wei, F. Y. Liew, and C. M. R. Turner T-Cell Responses during Trypanosoma brucei Infections in Mice Deficient in Inducible Nitric Oxide Synthase Infect. Immun., July 1, 1999; 67(7): 3334 - 3338. [Abstract] [Full Text] [PDF]
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 F. Facchetti, W. Vermi, S. Fiorentini, M. Chilosi, A. Caruso, M. Duse, L. D. Notarangelo, and R. Badolato Expression of Inducible Nitric Oxide Synthase in Human Granulomas and Histiocytic Reactions Am. J. Pathol., January 1, 1999; 154(1): 145 - 152. [Abstract] [Full Text] [PDF]
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S. L. James, A. W. Cheever, P. Caspar, and T. A. Wynn Inducible Nitric Oxide Synthase-Deficient Mice Develop Enhanced Type 1 Cytokine-Associated Cellular and Humoral Immune Responses after Vaccination with Attenuated Schistosoma mansoni Cercariae but Display Partially Reduced Resistance Infect. Immun., August 1, 1998; 66(8): 3510 - 3518. [Abstract] [Full Text] [PDF]
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 C. Bogdan The Multiplex Function of Nitric Oxide in (Auto)immunity J. Exp. Med., May 4, 1998; 187(9): 1361 - 1365. [Full Text] [PDF]
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R. J. van't Hof, K. J. Armour, L. M. Smith, K. E. Armour, X. Q. Wei, F. Y. Liew, and S. H. Ralston Requirement of the inducible nitric oxide synthase pathway for IL-1-induced osteoclastic bone resorption PNAS, July 5, 2000; 97(14): 7993 - 7998. [Abstract] [Full Text] [PDF]
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)
exhibited significantly higher incidence of arthritis from day 2 vs
wild-type (p < 0.005, n = 20,
) and from day 3 vs heterozygote controls (p
< 0.003, n = 28, 
). b, Comparison of
arthritic mice in each group demonstrated higher articular indices in
iNOS-deficient mice (*p < 0.01, **p <
0.05 vs control groups, mean ± SEM). Data are pooled from three
independent experiments.
