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Effect of an exogenous trigger on the pathogenesis of lupus in NZB Ф NZWF1 mice.

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Vol. 46, No. 8, August 2002, pp 2235–2244
DOI 10.1002/art.10441
© 2002, American College of Rheumatology
Effect of an Exogenous Trigger on the Pathogenesis of
Lupus in (NZB ⫻ NZW)F1 Mice
Hideo Yoshida,1 Minoru Satoh,1 Krista M. Behney,1 Chee-Gun Lee,2 Hanno B. Richards,1
Victoria M. Shaheen,1 Jun-Qi Yang,3 Ram R. Singh,3 and Westley H. Reeves1
Objective. This study examined the interactions
between exogenous and endogenous factors shaping the
phenotype of lupus in autoimmune (NZB ⴛ NZW)F1 mice
exposed to pristane, a model environmental trigger.
Methods. Frequencies of various autoantibodies
in untreated NZB/NZW mice were determined by various means (immunoprecipitation, enzyme-linked immunosorbent assay [ELISA], Crithidia luciliae kinetoplast
staining). Pristane or saline was administered intraperitoneally to 9–12-week-old NZB/NZW mice, followed
by serial studies of autoantibodies, total Ig levels
(ELISA), and proteinuria (dipstick).
Results. Besides antichromatin/DNA responses,
NZB/NZW mice spontaneously produced novel autoantibodies against the double-stranded RNA binding
protein RNA helicase A (RHA). In contrast, NZB/NZW
mice (n ⴝ 70) did not produce autoantibodies against
the nuclear RNP (nRNP), Sm, Ro, or La antigens.
Pristane exposure synergistically activated the production of antichromatin/DNA antibodies and dramatically
accelerated renal disease. Production of anti-nRNP/Sm
and Su autoantibodies also was induced, indicating that
the unresponsiveness of NZB/NZW mice to these antigens can be overcome. Curiously, pristane treatment did
not enhance the production of anti-RHA, suggesting
that these autoantibodies are regulated differently than
anti-DNA/chromatin and Sm. In contrast to previous
reports that suggest a critical role of deficient
interleukin-12 (IL-12) production in the pathogenesis of
lupus, there was overproduction of IL-12 in the peritoneal cavity of pristane-treated NZB/NZW mice, and
their spleen cells also produced large amounts of IL-12.
Conclusion. These data lead us to propose that
environmental influences exacerbate autoimmune manifestations in genetically lupus-susceptible mice through
their stimulatory effects on proinflammatory cytokines,
such as IL-12.
Lupus is an autoimmune disorder of protean
manifestations thought to result from both hereditary
immunoregulatory defects and poorly characterized environmental factors such as ultraviolet radiation or
chemicals (1–3). Multiple genetic loci contribute to the
pathogenesis of lupus in (NZB ⫻ NZW)F1, (NZB/
NZW3), and other lupus-prone mice (4,5). However,
little is known about the mechanisms by which environmental factors influence the disease.
An inducible lupus syndrome with diseasespecific autoantibodies (anti–double-stranded DNA
[anti-dsDNA], anti–nuclear RNP/Sm [anti-nRNP/Sm],
anti–ribosomal P), arthritis, and nephritis develops in
non–autoimmune-prone mice treated with pristane, a
hydrocarbon derived from the metabolism of chlorophyll (6,7). BALB/c, B6, and virtually all other immunocompetent mice are susceptible to pristane-induced lupus, but the associated autoantibodies and clinical
manifestations differ from strain to strain (8). The
development of lupus following pristane treatment is
cytokine dependent. Induction of anti-DNA/chromatin
antibodies and glomerulonephritis by pristane is abrogated in mice lacking interleukin-6 (IL-6) or
interferon-␥ (IFN␥) (9,10). In contrast, the antinRNP/Sm autoantibody frequency is markedly reduced
in IFN␥-deficient mice, but not in IL-6–deficient strains.
Supported by research grants R01-AR44731, R01-AI44074,
and R01-AR47322 from the USPHS.
Hideo Yoshida, MD, Minoru Satoh, MD, PhD, Krista M.
Behney, BS, Hanno B. Richards, MD, Victoria M. Shaheen, MS,
Westley H. Reeves, MD: Department of Medicine, University of
Florida, Gainesville; 2Chee-Gun Lee, PhD: New Jersey Medical
School, University of Medicine and Dentistry of New Jersey, Newark;
Jun-Qi Yang, PhD, Ram R. Singh, MD: Department of Internal
Medicine, University of Cincinnati, Cincinnati, Ohio.
Address correspondence and reprint requests to Westley H.
Reeves, MD, Division of Rheumatology and Clinical Immunology,
University of Florida, 1600 SW Archer Road, PO Box 100221,
Gainesville, FL 32610-0221. E-mail:
Submitted for publication February 6, 2002; accepted in
revised form April 22, 2002.
The goal of this study was to explore the interactions between exogenous and endogenous factors shaping the phenotype of lupus, using pristane as a model
environmental trigger of disease in autoimmune-prone
NZB/NZW mice. These mice spontaneously develop a
strong autoantibody response against DNA and chromatin antigens as well as polyclonal hypergammaglobulinemia and severe immune complex–mediated glomerulonephritis (4,11,12). It has been suggested that they do
not produce anti-Sm (11,13,14), but other reports are
contradictory (15–17). Since there are surprisingly few
systematic studies of autoantibody specificities in NZB/
NZW mice, we examined both the baseline serologic
phenotype and the effect of pristane treatment on this
phenotype. The present study verifies the absence of
anti-nRNP and Sm, as well as anti-Su, Ro/SSA, and
La/SSB, autoantibody production in NZB/NZW mice.
We show for the first time that NZB/NZW mice produce
autoantibodies against anti–RNA helicase A (RHA) and
that the serologic and clinical phenotype of lupus in this
strain is modified substantially by pristane exposure.
This may prove valuable for modeling the role of
exogenous factors in systemic lupus erythematosus
Mice. Four-week-old female NZB/NZW mice from
The Jackson Laboratory (Bar Harbor, ME) were housed in a
virus-free animal facility in barrier cages at the University of
North Carolina, Chapel Hill. At 9–11 weeks of age, 23 mice
received 0.5 ml of pristane (Sigma, St. Louis, MO) intraperitoneally (IP). Controls (n ⫽ 24) received 0.5 ml of phosphate
buffered saline (PBS) IP. Sera were collected 2 weeks before
injection, and monthly thereafter. Proteinuria was measured
with Albustix (Miles, Elkhart, IN).
Additional female mice (9 pristane- and 9 PBS-treated,
ages 9–11 weeks; The Jackson Laboratory) housed in a virusfree conventional colony in barrier cages at the University of
Florida were studied 3 months after treatment for histologic
features and cytokine production. Untreated female NZB/
NZW mice (n ⫽ 37; The Jackson Laboratory) housed in
nonbarrier cages in a conventional animal facility at the
University of Cincinnati were bled at ages 5–10 months. The
respective institutions’ animal care and use committees approved this study.
Immunoprecipitation. Autoantibodies to cellular proteins in murine sera from all 3 institutions were analyzed at the
University of Florida using a standardized immunoprecipitation procedure (6). 35S-radiolabeled K562 cell extract was
immunoprecipitated using 3 ␮l serum. Specificities were verified by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) using human prototype sera for antinRNP/Sm and anti-Su. Human anti-RHA serum was from a
patient with SLE. The University of Florida Institutional
Review Board approved the studies using human sera.
Double-stranded RNA binding proteins were isolated
using poly(IC) agarose (Pharmacia, Piscataway, NJ). A volume
of 120 ␮l beads (50% [volume/volume] in Tris buffered saline
[TBS]/Tween 20 [150 mM NaCl, 20 mM Tris HCl, 0.1% Tween
20 (pH 7.5)] with 3 mM MgCl2) was washed 3 times. Beads
were then incubated with cell extract from 107 35S-methionine–
labeled K562 cells in TBS/Tween 20 plus phenylmethylsulfonyl
fluoride and aprotinin for 1 hour at 4°C, and washed 6 times
with 1 ml TBS/Tween 20 plus 3 mM MgCl2. Bound proteins
were eluted with 600 ␮l of 1.5M NaCl NET/NP40 (1.5M NaCl,
2 mM EDTA, 20 mM Tris [pH 7.5]) for 20 minutes at 4°C, and
an equal volume of water was added (final concentration
0.75M NaCl). The eluate was immunoprecipitated with 3 ␮l of
human or murine autoimmune sera or normal human serum
for 30 minutes at 4°C, washed 3 times with 0.5M NaCl
NET/NP40, and once with NET before SDS-PAGE and autoradiography (6).
Immunoblotting. IgG from 3 ␮l of human or murine
sera containing possible autoantibodies to RHA, or control
serum, was crosslinked to protein A–Sepharose beads using
dimethyl pimelimidate (18). Proteins immunoprecipitated
from K562 cell extract (2 ⫻ 107 cell equivalents) were fractionated by SDS-PAGE and transferred to nitrocellulose. One
filter was probed with rabbit anti-RHA serum at a dilution of
1:3,000 (19,20). A second was probed with preimmune rabbit
serum. Blots were developed with 1:2,000 alkaline
phosphatase–labeled goat anti-rabbit IgG (␥ and light chain–
specific; Southern Biotechnology, Birmingham, AL) and developed using the Western-Star chemiluminescent system (Tropix,
Bedford, MA).
Immunoblot analysis of the fine specificity of antinRNP/Sm antibodies was carried out using murine sera as
described (21). Monoclonal antibodies (mAb) 2.73 (anti–
U1–70 kd) (22), 7-13 (anti–Sm D) (23), and 9A9 (anti–U1-A
plus U2-B⬙) (24) were used as standards.
Ig and autoantibody levels. Immunologic tests were
performed at the University of Florida using standardized and
previously published protocols. Levels of IgG1, IgG2a, IgG2b,
IgG3, and IgM were determined as described using sera
diluted 1:200,000 and/or 1:500,000 (25). Enzyme-linked immunosorbent assays (ELISAs) for antichromatin and anti–singlestranded DNA (anti-ssDNA) antibodies were performed using
sera diluted 1:500 (7,26). IgM and IgG antichromatin antibodies were considered positive when sample absorbance was
higher than the mean optical density from blank wells plus 3SD
Anti-ssDNA antibodies were considered positive when sample
absorbance was higher than the mean optical density plus 3SD
using sera from 8 healthy female BALB/c mice. Anti-dsDNA
antibodies were detected by Crithidia luciliae kinetoplast staining and quantified by titration emulation (Image Titer; Rhigene, Des Plaines, IL) as described (27).
Cytokine production in vitro and in vivo. Three
months after PBS or pristane treatment (6 months of age), the
NZB/NZW mice were killed and the peritoneal cavity was
lavaged with 5 ml of Dulbecco’s modified Eagle’s medium
(DMEM) containing 10% fetal bovine serum (FBS) and 10
units/ml heparin using a 5-ml plastic syringe and 18-gauge
needle. Single-cell suspensions of peritoneal and spleen cells
were treated with Tris NH4Cl to lyse erythrocytes. Cells were
Table 1.
Autoantibodies in sera obtained from 70 untreated or PBS-treated NZB/NZW mice*
No. of mice
Age range, months
Anti-dsDNA (by Crithidia luciliae assay), no. (%)
Anti-ssDNA (by ELISA), no. (%)
Antichromatin (by ELISA), no. (%)
Anti-nRNP/Sm (by immunoprecipitation)
Anti-Su (by immunoprecipitation)
Anti-Ro/SSA (by immunoprecipitation)
Anti-La/SSB (by immunoprecipitation)
Anti–ribosomal P (by immunoprecipitation)
Anti–RNA helicase A (by immunoprecipitation),
no. (%)‡
University of
North Carolina
University of
University of
Virus free, barrier
7 (29)
24 (100)
23 (96)
6 (25)
MHV free, barrier
4 (44)
5 (56)
9 (100)
30/34 (88)†
33/34 (97)†
33/34 (97)†
4 (11)
* PBS ⫽ phosphate buffered saline; MHV ⫽ mouse hepatitis virus; dsDNA ⫽ double-stranded DNA;
ssDNA ⫽ single-stranded DNA; ELISA ⫽ enzyme-linked immunosorbent assay; nRNP ⫽ nuclear RNP.
† Only 34 of the 37 mice were tested for anti-DNA and antichromatin due to insufficient quantities of 3
‡ The 140-kd autoantigen was shown by immunoprecipitation to be RNA helicase A (see Figure 1).
resuspended at 106/ml in DMEM plus 10% FBS and cultured
for 48 hours in 24-well culture plates (Costar, Cambridge, MA)
without stimulation or in the presence of 10 ␮g/ml lipopolysaccharide (LPS; from Salmonella minnesota Re 595; Sigma).
Culture supernatants were stored at ⫺80°C until assayed.
ELISAs for IL-4, IL-6, IL-12, tumor necrosis factor ␣ (TNF␣),
and IFN␥ were performed using rat mAb pairs (PharMingen,
San Diego, CA) following the manufacturer’s instructions.
After incubation with biotinylated cytokine-specific antibodies,
100 ␮l/well of 1:1,000 streptavidin–alkaline phosphatase
(Southern Biotechnology) was added for 30 minutes at 22°C
and the reaction was developed with p-nitrophenyl phosphate
substrate. Standard curves were generated using recombinant
cytokines (PharMingen). Cytokine concentrations were calculated using a 4-parameter logistic equation as part of the
Softmax Pro 3.0 ELISA plate reader software (Molecular
Devices, Sunnyvale, CA).
Statistical analysis. Levels of Ig and autoantibodies
were compared using the Mann-Whitney test. The development of proteinuria (% of mice with ⱖ300 mg/dl) and the
survival rates were evaluated by a log rank test (LifeTest
Procedure; SAS Institute, Cary, NC).
Serologic phenotype of untreated NZB/NZW
mice. Consistent with prior observations (28,29), serologic studies of 70 untreated or PBS-treated NZB/NZW
mice, ages 5–10 months, housed in 3 different institutions revealed that, depending on the animal colony,
29–88% had anti-dsDNA (Crithidia luciliae assay) and
56–100% had anti-ssDNA (ELISA), whereas virtually all
had anti-chromatin antibodies (ELISA) (Table 1).
When screened for anti-nRNP/Sm antibodies by a sensitive immunoprecipitation technique, all 70 sera from
animals at 3 different institutions were negative (Table
1). In addition, all of the sera were negative for anti-Ro/
SSA, anti-La/SSB, anti-Su, and anti–ribosomal P autoantibodies.
Although many specificities were not produced
by NZB/NZW mice, autoantibodies against a protein of
⬃140 kd were detected in up to 25% of the sera. This
protein comigrated with a 140-kd protein recognized by
certain human lupus sera (Figure 1A). The proteins
immunoprecipitated by human autoimmune serum and
2 NZB/NZW sera were transferred to nitrocellulose
membrane and probed with a rabbit antiserum specific
for RHA (Figure 1B). The rabbit antiserum bound
intensely to the 140-kd protein, whereas control (preimmune) rabbit serum did not. The dsRNA binding protein RHA had a molecular weight of ⬃140 kd (20,30). In
addition to its size and reactivity with rabbit anti-RHA
antibodies, the 140-kd protein bound specifically to
poly(IC)–agarose (Figure 1A), as expected for a dsRNA
binding protein. Although the frequency of these autoantibodies varied from group to group, the production of
anti-RHA was a consistent serologic manifestation in
mice from 4 separate experiments at 2 institutions
(University of North Carolina and University of Cincinnati) (Table 1). Anti-RHA was not detected in mice
housed at the University of Florida, possibly due to the
small number evaluated (n ⫽ 9).
The spectrum of autoantibodies in NZB/NZW
mice differed substantially from that in mice with
pristane-induced lupus. Pristane stimulates the production of anti-nRNP/Sm, Su, and ribosomal P in a wide
variety of mouse strains (8), but anti-RHA, anti-Ro/
SSA, and anti-La/SSB have not been reported. In contrast, anti-DNA and antichromatin antibodies are pro-
Figure 1. Spontaneous production of autoantibodies to RNA helicase
A (RHA) by NZB/NZW mice. A, 35S-labeled K562 cell extract was
immunoprecipitated using human serum containing autoantibodies
against a 140-kd protein (RHA), with human serum containing
anti-NF90/NF45/p110 autoantibodies (NF45/90), and with sera from 2
phosphate buffered saline–treated NZB/NZW mice (B/W #1, B/W
#2). Immunoprecipitation with normal mouse serum (NMS) also is
shown. A protein of ⬃140-kd immunoprecipitated by the 3 sera is
indicated to the left, as are the double-stranded RNA binding proteins
NF90, NF45, and p110. On the right, poly(IC) binding proteins from
whole cell extract eluted from the poly(IC)–agarose affinity matrix at
0.15M and 0.5M NaCl were analyzed by sodium dodecyl sulfate–
polyacrylamide gel electrophoresis. Immunoprecipitation using the
1.5M NaCl poly(IC) eluate (after washing the beads with Tris buffered
saline, 0.1% Tween 20, 3 mM MgCl2) is shown in the last 4 lanes. Three
of the prominent proteins in the 1.5M eluate were immunoprecipitated
by anti-NF90/NF45/p110 antiserum. The fourth was immunoprecipitated by human anti-RHA antiserum and by sera from the NZB/NZW
mice (B/W #1, B/W #2). Positions of the molecular weight standards
are indicated on the right. B, Proteins immunoprecipitated by human
(h-RHA) or murine (B/W #1, B/W #2) sera were transferred to
nitrocellulose and probed with polyclonal rabbit anti-RHA antiserum
(anti-RHA) or with preimmune rabbit serum (control). Position of the
RHA is indicated on the left and that of the molecular weight markers
on the right.
duced in both spontaneous (NZB/NZW) and pristaneinduced lupus. It was of interest, therefore, to see if
pristane could alter the pattern of autoantibody production and clinical features of NZB/NZW mice.
Acceleration of the anti-DNA/chromatin response and nephritis. Consistent with previous reports,
NZB/NZW mice spontaneously developed IgM antissDNA and antichromatin antibodies before they were
treated with PBS or pristane at 9 weeks of age (Figure
2). At that time, the frequencies of IgM anti-ssDNA and
IgM antichromatin autoantibodies were 58% and 33%,
respectively. In contrast, IgG anti-ssDNA and IgG antichromatin antibodies were not produced until after 6
months of age (4 months after PBS treatment). By 8
months of age, their frequencies were 100% and 96%,
respectively. Pristane greatly accelerated the onset and
increased the levels of IgM and IgG anti-ssDNA and
antichromatin autoantibodies at 1–4 months (P ⬍ 0.05
versus PBS-treated group, by Mann-Whitney test).
Although antichromatin is typical of the autoimmune syndrome of NZB/NZW mice, anti-dsDNA is
more closely linked to glomerulonephritis. Consistent
with previous observations, anti-dsDNA antibodies were
detected at high titers (Crithidia luciliae assay) in sera
from 29% of 5-month-old PBS-treated NZB/NZW mice
(Figure 3A). However, the titers were dramatically
higher in pristane-treated mice of the same age (P ⫽
0.0004 versus PBS-treated controls, by Mann-Whitney
test), as was their frequency (78% versus 29%; P ⬍ 0.002
by Fisher’s exact test). Similarly, renal disease was
greatly accelerated by pristane treatment. As early as 3
Figure 2. Anti–single-stranded DNA (anti-ssDNA) and antichromatin autoantibody levels. IgM and IgG anti-ssDNA (top) and IgM and
IgG antichromatin (bottom) antibodies were measured in 1:500 diluted sera from phosphate buffered saline-treated or pristane NZB/
NZW mice as a function of time after treatment. Individual data points
represent the mean ⫾ SEM of 23 or 24 mice. Heat-denatured calf
thymus DNA was used as an antigen for detecting anti-ssDNA
antibodies; chicken chromatin was used as an antigen for detecting
antichromatin antibodies.
Figure 3. Acceleration of anti–double-stranded DNA (anti-dsDNA)
antibody production and nephritis by pristane. A, Anti-dsDNA antibody
titers (Crithidia luciliae assay) in sera from phosphate buffered saline
(PBS)–treated (n ⫽ 24) or pristane-treated (n ⫽ 23) NZB/NZW mice
were quantified using titration emulation. The levels were significantly
different between the 2 groups (P ⫽ 0.0004). B, The frequency of
proteinuria ⱖ300 mg/dl was determined monthly after treating 8-weekold NZB/NZW mice with pristane (}; n ⫽ 12) or PBS (■; n ⫽ 12). There
was a significant difference between the 2 groups (P ⫽ 0.01 by log-rank
test). Data are representative of 2 separate experiments. C, Survival rates
were determined in pristane-treated (}; n ⫽ 12) and PBS-treated (■; n ⫽
12) NZB/NZW mice at ages 9–10 weeks. Survival of the pristane-treated
group was reduced compared with that of the PBS controls (P ⫽ 0.006 by
log-rank test). Data are representative of 2 separate experiments.
months after pristane treatment, an increased frequency
of severe proteinuria (ⱖ300 mg/dl) was observed in a
group of pristane-treated mice compared with PBStreated NZB/NZW controls (Figure 3B). Over the ensuing 3 months, the acceleration of renal disease caused
by pristane became increasingly apparent. The difference was significant 5 months after treatment (P ⫽ 0.01
by log-rank test). Pristane treatment also dramatically
accelerated mortality (Figure 3C). Seventy-five percent
of the pristane-treated mice died within 6 months,
compared with 9% of the PBS-treated control mice (P ⫽
0.006 by log-rank test). These data strongly suggest that
pristane acts synergistically with the NZB/NZW genetic
background to promote the development of anti-DNA/
chromatin antibodies and glomerulonephritis.
Induction of anti-nRNP/Sm and anti-Su. NZB/
NZW mice do not produce anti-nRNP/Sm and Su
autoantibodies spontaneously (Table 1), whereas the
production of anti-RHA has not been reported in
pristane-induced lupus. We next sought to determine 1)
whether the production of “pristane-associated” autoantibodies could be induced in NZB/NZW mice and 2)
whether, like anti-DNA and antichromatin antibodies,
anti-RHA was accelerated by pristane treatment.
Although not found in 70 control mice, antinRNP/Sm or Su autoantibodies were detected by immunoprecipitation in 4 of 32 (12.5%) pristane-treated mice
(P ⫽ 0.008 versus controls, by Fisher’s exact test) (Table
2). Anti-nRNP/Sm autoantibodies appeared at high
levels ⬃3 months after pristane treatment (Figure 4A).
Immunoprecipitation of the U5 small nuclear RNP–
specific 200-kd doublet (Figure 4A) indicated that antiSm antibodies were present. That possibility was supported further by the reactivity of sera with the B⬘/B
and/or D proteins by Western blotting (Figure 4B). In 1
mouse (B/W F1 #1 in Figure 4) anti-nRNP preceded the
onset of anti-Sm (U5–200-kd doublet), a pattern characteristic of pristane-induced lupus (21). The presence
of anti-nRNP autoantibodies also was verified by West-
Table 2.
Frequency of IgG autoantibodies in NZB/NZW mice*
No. positive by
immunoprecipitation assay (%)
No. of
PBS or unmanipulated
4 (12)†
10 (14)
1 (3)
* PBS ⫽ phosphate buffered saline; RHA ⫽ RNA helicase A.
† P ⫽ 0.008 versus controls, by Fisher’s exact test.
Figure 4. Induction of anti–nuclear RNP/Sm/Su (anti-nRNP/Sm/Su)
autoantibodies by pristane. A, 35S-labeled K562 cell extract was
immunoprecipitated with sera from 2 NZB/NZW mice that developed
anti-nRNP/Sm autoantibodies following pristane treatment. Sera were
tested 0, 1, 2, 3, and 4 months after treatment. Immunoprecipitates
using normal mouse serum (NMS; negative control) and human
anti-nRNP prototype serum (nRNP; positive control) are shown on
the left. Positions of the U5–200-kd doublet, U1-A, Sm B⬘/B, U1-C,
and Sm, D, E/F, and G proteins are indicated to the left. B,
Immunoblot analysis of sera from the 2 anti-nRNP/Sm–positive NZB/
NZW mice, using affinity-purified U1 small nuclear RNP antigen.
Positive controls include monoclonal antibodies 2.73 (anti–U1–70 kd),
9A9 (anti–U1-A ⫹ U2-B⬙), and 7-13 (anti–Sm D). The pattern with
normal mouse serum (control) is also shown. Positions of the U1–70kd, U1-A, and Sm B⬘/B and D proteins are indicated to the left.
ern blotting, which showed reactivity with the U1-A and
U1–70-kd proteins (Figure 4B).
In striking contrast to the acceleration of antiDNA/chromatin autoantibodies and the induction of
anti-nRNP/Sm and Su, the production of autoantibodies
to RHA was not enhanced by pristane treatment (Table
2). In control mice, the frequency of these autoantibodies was 14% (10 of 70), whereas in the pristane-treated
group, the frequency was 3% (1 of 32). Together, these
data indicate that pristane accelerates the production of
anti-DNA and antichromatin antibodies and induces
anti-Sm, anti-nRNP, and anti-Su autoantibodies in
NZB/NZW mice. In contrast, pristane did not enhance
the production of anti-RHA.
Alteration of cytokine production in NZB/NZW
mice by pristane. In view of the induction/acceleration
of the production of IFN␥-dependent autoantibodies
such as anti-nRNP/Sm and anti-dsDNA, we examined
whether pristane-treated NZB/NZW mice displayed evidence of Th1 cytokine overproduction (Figure 5). LPSstimulated IL-12 production by spleen cells from PBStreated mice by was relatively low (Figure 5A), although
it was greater than the levels seen in PBS-treated B6 or
BALB/c mice (Satoh M: unpublished observations). In
contrast, spleen cells from pristane-treated mice produced large amounts of IL-12 (P ⬍ 0.05 versus PBS
controls, by Mann-Whitney test) (Figure 5A). IL-4 was
produced in LPS-stimulated spleen cell cultures from
PBS-treated NZB/NZW mice, but was reduced in
pristane-treated mice (Figure 5A).
Peritoneal lavage fluid from pristane-treated
mice also contained large amounts of IL-12 (P ⬍ 0.005
versus controls, by Mann-Whitney test) (Figure 5B),
indicating that pristane enhances the production of this
cytokine in vivo. Peritoneal lavage fluid from both
pristane- and PBS-treated mice contained little IL-6 or
TNF␣. Although there was a trend toward increased
levels of IFN␥ with pristane treatment, this did not reach
statistical significance.
Consistent with the effect of IL-12 in priming T
Figure 5. Alteration of cytokine balance by pristane. A, Increased
lipopolysaccharide (LPS)–stimulated interleukin-12 (IL-12) production. Spleen cells were isolated from pristane-treated or phosphate
buffered saline (PBS)–treated NZB/NZW mice and stimulated in vitro
with LPS (10 ␮g/ml). Levels of IL-12 and IL-4 in the culture supernatants were determined 48 hours later. IL-4 production was lower and
IL-12 higher in cultures of cells from pristane-treated mice (P ⬍ 0.05
by Mann-Whitney test). B, The peritoneal cavities of pristane-treated
mice and PBS-treated controls were lavaged with 5 ml of Dulbecco’s
modified Eagle’s medium, and the fluid was tested for IL-12, IL-6,
tumor necrosis factor ␣ (TNF␣), and interferon-␥ (IFN␥) (by enzymelinked immunosorbent assay-treated ELISA). Higher IL-12 levels were
found in the peritoneal lavage fluid from pristane-treated mice compared with the controls (P ⬍ 0.005 by Mann-Whitney test). C, Levels
of total IgG2a and IgG1 in the sera of pristane-treated and PBStreated mice were measured by ELISA 3 months after treatment, and
the ratio of the levels for each mouse was determined. The mean
IgG2a; IgG1 ratio in pristane-treated mice was higher than that in
PBS-treated mice (P ⬍ 0.001 by Mann-Whitney test). RHA ⫽ RNA
helicase A; nRNP ⫽ nuclear RNP. D, Total IgG1, IgG2a, IgG2b, IgG3,
and IgM levels were determined by ELISA as a function of time after
treatment with pristane or PBS.
cells for IFN␥ production and inhibiting IL-4 (31), the
production of IFN␥ versus IL-4–dependent Ig isotypes
was altered dramatically by pristane treatment. There
was a marked difference in the ratio of total IgG2a to
IgG1 in pristane- versus PBS-treated NZB/NZW mice
(P ⬍ 0.001 by Mann-Whitney test) (Figure 5C). Interestingly, the 7 anti–RHA–positive mice (6 control, 1
pristane-treated) all had low IgG2a/IgG1 ratios, whereas
3 of the 4 anti-nRNP/Sm and Su-positive mice (2
anti-nRNP/Sm, 2 Su) had high ratios (P ⬍ 0.05 by
Mann-Whitney test). As seen in non–autoimmuneprone mice treated with pristane, total levels of the
IFN␥-dependent Ig isotype IgG2a increased dramatically in pristane-treated mice versus controls (P ⬍ 0.05
at 3 and 4 months by Mann-Whitney test) (Figure 5D).
The IL-4–dependent isotype IgG1 also increased, but
less markedly and with different kinetics: IgG2a peaked
at 4 months and then declined precipitously, whereas
IgG1 reached a plateau at 5 months. These data strongly
suggest that pristane skews cytokine production toward
Th1 predominance, which might be expected to promote
the formation of anti-nRNP/Sm and Su autoantibodies
Susceptibility to lupus is thought to result from
the action of poorly defined environmental triggers on a
susceptible genetic background (1,12). Multiple genes
contribute independently to the phenotype of lupus
exhibited by lupus-prone mice, such as NZB/NZW
(5,12). The ability to induce lupus-like manifestations in
a wide variety of mice (BALB/c, B6, SJL, CBA, etc.)
with pristane suggests that nearly any genetic background may be permissive, given a sufficiently powerful
inciting event (7,8). To model how environmental stimuli influence the onset of disease in a susceptible host,
we examined the interaction of pristane with the lupusprone NZB/NZW genetic background. Although in this
mouse model, the hydrocarbon oil was administered into
the peritoneal cavity, human exposure to mineral oil
through the respiratory or gastrointestinal tract also
induces inflammatory disease with pathologic changes
similar to those seen in pristane-treated mice (32–34).
Oil-based adjuvants also are used in human and veterinary vaccines (35).
Hydrocarbon exposure synergistically activated
the production of antichromatin and anti-DNA antibodies and accelerated the onset of renal disease.
However, the serologic phenotype of the disease was
altered with the appearance of anti-nRNP/Sm and Su
autoantibodies, specificities that are not produced spontaneously by NZB/NZW mice. NZB/NZW mice spontaneously produced novel autoantibodies against the
dsRNA binding protein RHA. Curiously, pristane treatment did not enhance anti-RHA production despite
dramatically stimulating the production of other autoantibodies, which suggests that anti-RHA is regulated
differently than anti-DNA/chromatin. The Sle1 locus,
which regulates susceptibility to antichromatin responses (28), may not control anti-RHA autoantibody
production, or else additional loci may be required in
concert, a hypothesis that should be testable using Sle1
congenic strains.
NZB/NZW mice produce autoantibodies primarily against chromatin (DNA and histones) (13). We did
not detect anti-Sm, nRNP, Ro, La, Su, or ribosomal P
autoantibodies in 70 NZB/NZW mice, a finding in
agreement with some studies (14,29) but contradicting
other studies in which these specificities were detected
by ELISA or immunoblot analysis (16,17). Although the
reason for this discrepancy is uncertain, differences in
the housing and/or autoantibody assays used in previous
studies versus those used in the present study may be
responsible. It is difficult to ascribe this to housing
differences because mice housed at 3 different institutions did not spontaneously produce anti-nRNP/Sm
Assay reliability is another possible explanation
for the discrepancy. The specificity of anti-Sm antibodies
for SLE has been established by double immunodiffusion, counterimmunoelectrophoresis, and immunoprecipitation (36–38), whereas the diagnostic specificity of
the antibodies detected by ELISA or immunoblotting
has not been established. Since immunoprecipitation
generally correlates favorably with double immunodiffusion but is more sensitive (39), it seems unlikely that the
absence of anti-nRNP/Sm autoantibodies was a consequence of an insensitivity of the assay. While we cannot
exclude the presence of additional low-affinity or lowtiter autoantibodies, insofar as could be determined
here, the spontaneous autoantibody response of NZB/
NZW mice is restricted to chromatin and RHA.
Pristane is powerfully synergistic with the genetic
background in promoting anti-DNA/chromatin responses and renal disease, and it induces de novo
production of anti-nRNP/Sm and Su. In contrast, antiRHA was not enhanced (and was possibly inhibited),
suggesting that its regulation differs from that of other
autoantibodies. There is mounting evidence that subsets
of autoantibodies are differentially regulated. The absence of functional Fas or Fas ligand promotes anti-
DNA/chromatin responses in B6 mice (40), but unlike
the situation in NZB/NZW mice, pristane treatment has
no effect on these responses (26). Pristane’s synergy with
anti-DNA/chromatin responses in NZB/NZW mice and
lack of effect in lpr or gld mice suggest that there is more
than one pathway to antichromatin production.
The spontaneous production of autoantibodies to
the dsRNA binding protein RHA stands in sharp contrast to the absence of other “typical” lupus autoantibodies in NZB/NZW mice. Double-stranded RNA binding proteins are recently described autoantigens in
pristane-induced lupus and SLE (41,42). The novel
140-kd protein immunoprecipitated by sera from ⬃10–
20% of NZB/NZW mice was identified on the basis of
biochemical and immunologic criteria. RHA is a dsRNA
binding protein controlled by IFN regulatory factor (30)
that interacts with adenovirus VA RNAII (20), plays a
role in the nuclear export of viral RNA (43), and
facilitates Rev-mediated gene expression and nuclear
export of RNA in human immunodeficiency virus–
infected cells (44). Consistent with a recent report (42),
autoantibodies to this protein also were found in certain
human lupus sera (Figure 1A). However, we have not
detected anti-RHA autoantibodies in other murine
models of spontaneous lupus (MRL, BXSB), and have
only rarely encountered anti-RHA either spontaneously
or following pristane treatment in non–autoimmuneprone mice (Satoh M, et al: unpublished observations).
The different frequencies of anti-RHA autoantibodies in untreated NZB/NZW mice housed at different
institutions suggest that undefined environmental factors can affect the spontaneous production of autoantibodies in lupus-prone mice. Although the mice in 2 of
the animal colonies (University of Florida and University of North Carolina) were housed in barrier cages and
were free of conventional murine viruses, including
mouse hepatitis virus, it is difficult to completely control
all environmental variables, which may include lighting,
diet, bedding, stress, and the microbial flora. Indeed,
anti-nRNP/Sm and anti-Su autoantibody production in
pristane-induced lupus is enhanced in conventionally
housed mice compared with mice housed under specific
pathogen–free conditions (25). It will be of interest to
further define these effects, which may imply a greater
role of the environment in the pathogenesis of spontaneous lupus than has been generally supposed. We
hypothesize that environmental variables may influence
the levels of cytokines that promote autoantibody formation in pristane-induced lupus, such as IL-6, IFN␥,
and IL-12 (9,10).
IL-12 was the major cytokine overproduced both
in vitro and in vivo following pristane treatment (Figure
5). Interestingly, NZB/NZW, MRL/lpr, and other lupus
mice exhibit a defect in macrophage IL-12 production
prior to the onset of disease (45). Monocyte-derived
macrophages from SLE patients also have defective
IL-12 production (46). In NZB/NZW mice, a defect in
the initiation, but not the propagation, of Th1 responses
may play a fundamental role in lupus by fostering
polyclonal hyperexpansion of autoreactive lymphocytes
and the establishment of a Th1-deficient environment
promoting autoantibody formation (45). However, large
amounts of IL-12 were detected in the peritoneal cavity
of pristane-treated mice, and their spleen cells produced
high levels of LPS-stimulated IL-12 (Figure 5) in association with a dramatic enhancement of anti-DNA/
chromatin autoantibody formation and glomerulonephritis. Our data suggest that the defective IL-12
production reported in murine and human lupus is not
fundamental to the disease process. Indeed, IFN␥ may
promote autoimmunity in NZB/NZW mice, since IFN␥
receptor–deficient NZB/NZW mice have less severe
disease (47), and its neutralization with anti-IFN␥ mAb
(48) or soluble IFN␥ receptor (49) prevents renal disease.
Pristane does not induce anti-DNA/chromatin
antibodies and nephritis in either IL-6 or IFN␥–
deficient BALB/c mice (9,10). The greatly increased
IL-12 production in pristane-treated NZB/NZW mice
versus controls could promote Th1 responses (31). Two
lines of evidence suggest this is the case: 1) IL-4 production in vitro was reduced following pristane treatment,
presumably due to IFN␥ and/or IL-12 production; and
2) there was greatly increased production of the IFN␥dependent (50) IgG2a isotype. Since IgG2a is the predominant isotype in glomerular deposits of NZB/NZW
mice (13), IL-12 could promote disease by enhancing the
production of IFN␥ and the expression of pathogenic
IgG2a anti-dsDNA antibodies (13). The exacerbation of
nephritis by pristane also might be a direct effect of
IL-12 (and/or IFN␥) on the kidney, leading to the
formation of epithelial crescents (51), which have been
reported in pristane-induced lupus (7).
Alternatively, increased IL-12 production could
promote glomerulonephritis by enhancing nitric oxide
biosynthesis (52). An IFN␥-rich environment seems to
favor the production of anti-nRNP/Sm and anti-dsDNA
antibodies (10,53). Mice that produced anti-nRNP/Sm
and Su had a higher IgG2a/IgG1 ratio than those that
produced anti-RHA (Figure 5C), suggesting that antiRHA antibodies may be produced under the influence
of a different set of cytokines than anti-DNA/chromatin
or anti-nRNP/Sm/Su.
Bacterial DNA stimulates the production of a
spectrum of cytokines similar to that induced by pristane
(54). Like pristane treatment, immunization of NZB/
NZW mice with Escherichia coli dsDNA accelerates the
production of anti-dsDNA antibodies while promoting a
shift toward Th1 cytokine production (54). But in contrast to pristane, bacterial DNA reduces the severity of
glomerulonephritis (55). Moreover, bacterial DNA has
not been reported to induce anti-nRNP/Sm or Su autoantibodies. Thus, despite similarities in the cytokines
induced by these 2 exogenous immune stimulants, there
also may be significant differences in their effects. These
differences could have implications for understanding
how various environmental exposures play a role in
shaping the phenotype of lupus.
We are grateful to Dr. Alan Hutson (Biostatistics
Department, University of Florida, Gainesville) for statistical
assistance and Dr. Michael Mathews (New Jersey Medical
School, University of Medicine and Dentistry of New Jersey,
Newark) for helpful discussions. We thank Drs. Robert A.
Eisenberg and Philip L. Cohen (University of Pennsylvania,
Philadelphia) and Dr. Walther J. van Venrooij (University of
Nijmegen, The Netherlands) for providing monoclonal antibodies.
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