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Hyperexpression of cyclooxygenase 2 in the lupus immune system and effect of cyclooxygenase 2 inhibitor diet therapy in a murine model of systemic lupus erythematosus.

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ARTHRITIS & RHEUMATISM
Vol. 56, No. 12, December 2007, pp 4132–4141
DOI 10.1002/art.23054
© 2007, American College of Rheumatology
Hyperexpression of Cyclooxygenase 2 in the Lupus Immune
System and Effect of Cyclooxygenase 2 Inhibitor Diet Therapy
in a Murine Model of Systemic Lupus Erythematosus
Li Zhang, Anne M. Bertucci, Kimberly A. Smith, Luting Xu, and Syamal K. Datta
and aspirin delayed development of nephritis temporarily, but failed to prolong survival. Indeed, treatment
with aspirin alone increased mortality.
Conclusion. The contributions of the major players in the pathogenic autoimmune response, namely, T
cells, B cells, dendritic cells, and macrophages that are
abnormally hyperactive in lupus, depend on the increased expression and activity of COX-2, similar to
inflammatory cells in target organs. Intermittent pulse
therapy with low doses of select COX-2 inhibitors would
be of value in the treatment of lupus.
Objective. To investigate the role of cyclooxygenase 2 (COX-2) in the functioning of different cell types
involved in the lupus autoimmune response, and to
examine the therapeutic effect of COX-2 inhibitors in
mice prone to spontaneously develop systemic lupus
erythematosus (SLE).
Methods. Lupus-prone (SWR ⴛ NZB)F1 mice
were fed with a diet containing different doses of the
COX-2–specific inhibitor celecoxib or the nonspecific
inhibitor aspirin, or a combination of both, and the
effects of the therapy on autoantibody production, development of lupus nephritis, and mortality were determined. Expression of COX-2 by different cells of the
lupus immune system and the effect of COX-2 inhibitors
on the function of these cells in vitro and in vivo were
assessed.
Results. The immune cells of mice with SLE
spontaneously hyperexpressed COX-2, and COX-2 inhibitors could cause cell apoptosis. Treatment with
COX-2 inhibitors resulted in decreased autoantibody
production and inhibition of the T cell response to the
major lupus autoantigen, nucleosome, and its presentation by antigen-presenting cells. Surprisingly, a significant increase in survival occurred only in mice receiving
intermittent therapy with the lowest dose of celecoxib
(500 parts per million), approximating <100 mg of
celecoxib/day in humans. A continuous diet, but not
intermittent feeding, with the combination of celecoxib
In systemic lupus erythematosus (SLE), hyperactivity of the immune system leads to activation of certain
autoimmune T helper cells, which drive autoimmune B
cells to produce somatically mutated IgG autoantibodies
against apoptotic nuclear antigens (1–6). IgG immune
complexes containing autoantigenic DNA and RNA
bind Fc␥ receptors and Toll-like receptors (TLRs),
which results in stimulation of type I interferon (IFN)
production by hyperactive dendritic cells (DCs); moreover, autoimmune B cells bearing the T helper cell–
driven, high-affinity, somatically mutated B cell receptors are dually stimulated by TLRs (7–9). These events
further amplify the ability of antigen-presenting cells
(APCs) to present autoantigens to pathogenic T helper
cells, which is an essential element of disease development (10–13).
Normally, autoreactive T and B cells are eliminated by functional inactivation (anergy) and by
activation-induced cell death (AICD; apoptosis), via Fas
signaling (14). However, autoimmune T helper cells in
human lupus resist AICD by up-regulating the expression of cyclooxygenase 2 (COX-2) and the antiapoptotic
molecule cFLIP (15,16). Even after activation with full
costimulation using anti-CD28 antibodies, anti-CD3 antibodies, and interleukin-2, normal T cells do not upregulate COX-2 to the same extent as that by lupus T
Supported by the NIH (grants R37-AR-39157 and R01-AI41985) and the Solovy Arthritis Research Society.
Li Zhang, PhD, Anne M. Bertucci, BS, Kimberly A. Smith,
MS, Luting Xu, PhD, Syamal K. Datta, MD: Northwestern University
Feinberg School of Medicine, Chicago, Illinois.
Address correspondence and reprint requests to Syamal K.
Datta, MD, Division of Rheumatology, Northwestern University Feinberg School of Medicine, 240 East Huron Street, McGaw M300,
Chicago, IL 60611. E-mail: skd257@northwestern.edu.
Submitted for publication March 7, 2007; accepted in revised
form August 20, 2007.
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COX-2 AND LUPUS AUTOIMMUNITY
cells stimulated with plastic-bound anti-CD3 alone
(which normally induces anergy) (16). Surprisingly, only
certain COX-2 inhibitors (celecoxib [Celebrex] being
one of them) could cause apoptosis of lupus T cells.
Moreover, in vitro in lupus-prone (SWR ⫻ NZB)F1
(SNF1) mice, celecoxib could also markedly block the
response of T cells to nucleosomes, the major lupus
autoantigen, as well as block pathogenic autoantibody
production induced by the nuclear autoantigen–specific
T helper cells (16).
To determine the mechanism of COX-2 inhibition in vivo, we investigated the expression of COX-2 in
other cells of the lupus immune system in an SNF1
mouse model of lupus. In addition, we examined the
effects of treatment with a COX-2 inhibitor diet on
spontaneously developing SLE in SNF1 mice (17,18).
MATERIALS AND METHODS
Mice. NZB, SWR, and BALB/c mice were purchased
from Jackson Laboratory (Bar Harbor, ME). SNF1 hybrids
were bred. Female mice were used in the present study, with
the approval of the Animal Care and Use Committee of
Northwestern University.
Administration of celecoxib and aspirin. Celecoxib was
provided by Pfizer (Groton, CT). Aspirin was purchased from
Sigma (St. Louis, MO). Groups of 12-week-old SNF1 mice
were given a regular diet of rodent chow (Teklad no. 7912)
either by itself or supplemented (19) with 500, 1,000, or 1,500
parts per million (ppm) (or mg per kg of diet) of celecoxib
(Research Diets, New Brunswick, NJ), which was administered
intermittently in a cycle of 3 weeks of celecoxib diet followed
by 4 weeks of drug-free diet. The regimen was continued
through the remaining lifespan of the animals. Another 4
groups of mice were administered continuously a diet of either
500 ppm of celecoxib, 400 ppm of aspirin, or 500 ppm of
celecoxib plus 200 ppm aspirin (continuous combination treatment), or received the regular (drug-free) diet as control.
Finally, additional groups of SNF1 mice received a combination diet of 500 ppm celecoxib plus 200 ppm aspirin administered in an intermittent regimen of 3 weeks on the drugs and
4 weeks off (intermittent combination treatment), or received
the regular (drug-free) diet as control.
All mice were monitored weekly for the development
of proteinuria, by testing with Albustix (VWR Scientific,
Chicago, IL), determining diet intake (weight of chow left on
cage top), and tracking body weight. Treatments lasted until
the mice were moribund.
To study early immunologic changes after treatment
with celecoxib or aspirin, an additional batch of 12-week-old
SNF1 mice (5 mice/group) was treated with the same regimens
as described above, and killed after 6 weeks. Celecoxib levels in
the plasma of mice receiving different doses of the drug were
measured by high-performance liquid chromatography
(HPLC) (19) conducted in the Clinical Pharmacology Core
Facility at Northwestern University.
4133
Quantitation of autoantibodies. IgG-class autoantibodies to double-stranded DNA (dsDNA), single-stranded
DNA (ssDNA), nucleosomes, and histones in the sera and
culture supernatants were estimated by enzyme-linked immunosorbent assay (ELISA) (13,20).
Measurement of intracellular COX-2 and analysis of
surface marker staining by flow cytometry. Total spleen cells
from 6-week-old unmanipulated SNF1, SWR, and BALB/c
mice were stained with rat or hamster monoclonal antibodies
(mAb) to mouse CD4 (for T cells), mouse CD19 and CD86
(for activated B cells), mouse CD11c (for DCs), and mouse
CD11b (for macrophages), conjugated with peridinin chlorophyll A protein (PerCP) or phycoerythrin (BD PharMingen,
San Jose, CA or eBioscience, San Diego, CA, respectively) at
4°C for 30 minutes. After washing and fixation, cells were
permeabilized and stained with mouse anti–COX-2 mAb or its
isotype control (BD PharMingen) at room temperature (RT)
for 30 minutes. After extensive washing, cells were incubated
with second-step goat anti-mouse fluorescein isothiocyanate
(FITC)–conjugated IgG (Molecular Probes, Carlsbad, CA) at
RT in the dark for 30 minutes. Usually, 200,000 cell events
were collected after live-cell gating with FACSCalibur, with
the results analyzed by CellQuest (BD PharMingen) or FlowJo
(TreeStar; FlowJo, Ashland, OR) software. Isotype-matched
controls were used for marking positive and negative cell
populations.
Western blot for COX-2 expression. Whole cell lysates
were prepared from fractionated spleen cell subsets or whole
splenocytes. CD4 T cells (macrophages plus DCs) were purified from splenocytes using a technique as previously described
(21,22). Western blotting was then carried out (15,16), and
blots were probed with primary goat anti–COX-2 antibody
(sc-1745; Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4°C in 5% nonfat dry milk in TBST (60 mmoles/liter
Tris base, 120 mmoles/liter NaCl, 0.5% Tween 20), and then
incubated with a horseradish peroxidase–conjugated secondary antibody (sc-2020; Santa Cruz Biotechnology) in 5% dry
milk in TBST for 1 hour at RT. Bound antibodies were
detected with enhanced chemiluminescence detection reagents
(Amersham Pharmacia Biotech, Buckinghamshire, UK).
Induction of apoptosis. Spleen cells from 6-month-old
SWR or SNF1 mice were cocultured with the various concentrations of celecoxib for 24 hours. Apoptotic cells were detected by staining with annexin V and propidium iodide (BD
PharMingen), and at the same time, whole splenocytes were
stained with antibodies to CD4-PerCP, B220-allophycocyanin,
CD19-PerCP, CD11c-FITC, and CD11b-FITC to analyze apoptosis of CD4 T cells, B cells, DCs, and macrophages,
respectively. Apoptosis in the specific cell subpopulations
gated by flow cytometry was calculated as (% of experimental
apoptosis ⫺ % of spontaneous apoptosis)/(100 ⫺ % of spontaneous apoptosis) (16).
Enzyme-linked immunospot (ELISpot) assay. A
mouse IFN␥ ELISpot assay was used to detect the T cell
response to nucleosomes, as described previously (21).
Helper assays for IgG autoantibody production.
Splenic T cells (2.5 ⫻ 105/well) from mice treated continuously
for 6 weeks or splenic T cells from control SNF1 mice were
cocultured with treated or control SNF1 splenic B cells (2.5 ⫻
105/well) in 96-well plates for 7 days in the presence of 5 ␮g/ml
nucleosomes, as described previously (13,20). Culture super-
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ZHANG ET AL
natants were collected, freeze-thawed, and assayed by ELISA
for IgG autoantibodies.
Histopathologic analysis. Kidneys were fixed in 10%
buffered formalin and paraffin embedded. Tissue sections
were then stained with hematoxylin and eosin and periodic
acid–Schiff. The sections were graded in a blinded manner for
pathologic changes on a scale of 0–4⫹, as described previously
(21,23).
Statistical analysis. Log rank tests and Student’s
2-tailed t-tests were used for statistical analyses. Results are
expressed as the mean ⫾ SEM.
RESULTS
Significantly prolonged survival of lupus-prone
SNF1 mice following intermittent treatment with lowdose COX-2 inhibitor diet. Because the COX-2 inhibitor
celecoxib can cause apoptosis of human lupus T cells
and also blocks autoantigen recognition and autoantibody production in vitro in the cells of SNF1 mice (16),
we first treated SNF1 mice with serologically active lupus
using intermittent pulses of celecoxib, to determine the
effects of this therapy on lupus in vivo. We treated
12-week-old SNF1 mice with a diet containing high
(1,500 ppm), moderate (1,000 ppm), or low (500 ppm)
doses of celecoxib in the following cycle: 3 weeks of
feeding with the celecoxib diet followed by 4 weeks with
regular diet.
As determined by HPLC, the mean concentrations of celecoxib in the plasma of mice receiving 500
ppm, 1,000 ppm, and 1,500 ppm celecoxib were 0.75 ␮M,
1.3 ␮M, and 2.0 ␮M, respectively (0.27 ␮g/ml, 0.50
␮g/ml, and 0.76 ␮g/ml, respectively). The maximum
plasma concentration (Cmax) of celecoxib (Celebrex)
reaches 0.705 ␮g/ml in human subjects, as has been
determined in patients receiving a single 200-mg dose
(see Physician’s Desk Reference for the pharmacokinetics of Celebrex); this Cmax value in humans is 2.6-fold
higher than that reached in the mice receiving the
low-dose (500 ppm) celecoxib diet.
Although differences in the incidence of severe
proteinuria between the treatment groups were not
marked, intermittent therapy with the lowest dose of
celecoxib had the most beneficial effect in prolonging
survival up to 24 months of age (P ⬍ 0.05 by log rank
test), as shown in Figure 1A. The low-dose intermittent
celecoxib therapy also was the most effective in delaying
the onset of severe proteinuria (Figure 1B), but cumulatively, the between-group differences (by log rank test)
did not reach significance.
Figure 1. Effect of intermittent therapy with 500, 1,000, or 1,500 parts
per million (ppm) dietary doses of celecoxib (CC) in the (SWR ⫻
NZB)F1 (SNF1) mouse model of spontaneous systemic lupus erythematosus. A, Percent survival and B, incidence of severe lupus nephritis
among the treatment groups versus untreated controls. Starting at age
3 months, female SNF1 mice (10 mice/group) received cycles of a
regular rodent chow diet containing celecoxib for 3 weeks, followed by
the regular diet alone for 4 weeks, and the treatment cycles were
continued throughout the observation period. The control mice received the same regular diet without the drug.
Significant delay in onset of severe proteinuria,
but failure to prolong mouse survival time, following
continuous treatment with low-dose COX-2 inhibitor
diet. To examine whether continuous therapy with celecoxib would be more beneficial than intermittent therapy, and also whether adding aspirin would counteract
any cardiovascular side effects, SNF1 mice were started
on the COX-2 inhibitor diet at 12 weeks of age, at a time
when high levels of serum autoantibodies were present
but proteinuria was ⬍2⫹ (⬍100 mg/dl). A total of 40
mice was randomly separated into 4 treatment groups
(10 mice per group). Three treatment groups were fed
continuously with a diet containing 500 ppm of celecoxib, 400 ppm of aspirin, or a combination of 200 ppm
aspirin and 500 ppm celecoxib (continuous combination
COX-2 AND LUPUS AUTOIMMUNITY
Figure 2. Effect of continuous therapy with low doses of celecoxib
(CC), aspirin (ASP), or a combination of celecoxib and aspirin
(Combo), or intermittent therapy with the combination diet (Combo
Intermittent) in the (SWR ⫻ NZB)F1 (SNF1) mouse model of
spontaneous systemic lupus erythematosus. A, Percent survival and B,
incidence of severe lupus nephritis among the treatment groups versus
untreated controls. Three-month-old female SNF1 mice (10 mice/
group) received a diet containing celecoxib (500 parts per million
[ppm]) or aspirin (400 ppm) or received continuous combination or
intermittent combination therapy (combination diet containing 500
ppm celecoxib and 200 ppm aspirin), or the regular diet as control.
treatment). Another treatment group received a diet
containing the combination of 200 ppm aspirin and 500
ppm celecoxib, but this was administered intermittently,
comprising 3 weeks on combination therapy and 4 weeks
off the therapy (intermittent combination treatment).
The control group of mice received the same diet but
without the drugs.
Figure 2B shows that continuous treatment with
the combination of COX-2 inhibitors significantly delayed the onset of severe proteinuria as compared with
that in the control group (P ⬍ 0.05 by log rank test). At
age 26 weeks, 70% of control mice had developed
persistent proteinuria of ⬎2⫹ that rapidly progressed to
4⫹, whereas in the continuous combination treatment
group the incidence of severe proteinuria was only 30%.
4135
However, in mice receiving 500 ppm celecoxib, 400 ppm
aspirin, or intermittent combination treatment, there
was no significant difference in the incidence of severe
proteinuria, as compared with controls, at age 26 weeks.
Moreover, by 9.5 months of age, the incidence of severe
proteinuria, even in the continuous combination treatment group, quickly reached 100% (as in the controls).
Survival in all of the treated groups was not
prolonged as compared with that in the control group
(P ⬎ 0.05 by log rank test) (Figure 2A). Indeed, the
group receiving aspirin alone had accelerated mortality,
although the dietary intake and body weight of the
animals were similar to those in the other groups.
When the 2 sets of results were compared,
namely, those in mice treated intermittently with celecoxib (500 ppm) (Figure 1A) versus those in mice
treated intermittently with the celecoxib plus aspirin
combination therapy (Figure 2A), clearly the intermittent therapy with low-dose celecoxib alone was better in
prolonging overall survival (survival up to age 24 months
versus survival up to age 13.5 months; P ⬍ 0.05 by log
rank test) and was more effective in decreasing the
incidence of nephritis, especially after 9 months of age
(Figures 1B and 2B). At age 38 weeks, 100% of the mice
in the intermittent combination therapy group had developed severe nephritis, as compared with only 65% of
those treated intermittently with celecoxib alone (at
9.5–15 months of age; P ⬍ 0.05 by log rank test). The
only difference between the 2 treatment groups was the
presence of aspirin (200 ppm) in the diet of the intermittent combination treatment group, which appeared
to result in a worse outcome.
Significant reductions in serum antinuclear
autoantibody levels following treatment with low-dose
COX-2 inhibitor diet. To determine the effect of in vivo
inhibition of COX-2, we measured serum levels of IgG
antinuclear autoantibodies after short-term treatment of
SNF1 mice for 6 weeks. All 3 low-dose treatment groups
(500 ppm celecoxib, 400 ppm aspirin, and continuous
combination treatment) showed significantly reduced
serum concentrations of IgG anti-dsDNA, anti-ssDNA,
antinucleosome, and antihistone autoantibodies as compared with the control group (P ⬍ 0.01 to P ⬍ 0.05)
(Figure 3). In the SNF1 mouse model of lupus, it is
known that nephritogenic autoantibodies have high affinity for ssDNA and nucleosomes (9,24).
Inhibition of early glomerulonephritis by lowdose COX-2 inhibitor treatment. Persistent, severe proteinuria (2⫹ to 4⫹, or 100 mg/dl to ⬎2,000 mg/dl) in
SNF1 mice is always associated with a 3⫹ to 4⫹ grade of
glomerulonephritis, as determined by histopathology
4136
ZHANG ET AL
Figure 3. Effects of low doses of celecoxib (CC), aspirin (ASP), or
both (Combo) given continuously on serum levels of IgG-class autoantibodies (autoAb) and development of glomerulonephritis. A,
Twelve-week-old mice (5 mice/group) were continuously treated with
the celecoxib diet (500 parts per million [ppm]), aspirin (400 ppm),
combination diet, or control (Ctrl) diet for 6 weeks. Blood samples
were assessed for IgG autoantibodies to double-stranded DNA
(dsDNA), single-stranded DNA (ssDNA), nucleosomes, and histone. ⴱ
⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍ 0.01, versus controls. B, Another group of mice
was treated with regimens identical to those in A and evaluated, after
termination, for renal pathologic features of lupus nephritis, with
scoring of 5 mice per group on 2 sections of each kidney. ⴱⴱ ⫽ P ⬍
0.005 versus controls. Results in A and B are the mean and SEM. C,
Representative examples of kidney sections from the animals described in B (stained with hematoxylin and eosin; original magnification ⫻ 200). In contrast to the kidney from an animal treated with the
celecoxib (500 ppm) diet (right), the kidney from an animal receiving
the control diet (left) shows marked enlargement and hypercellularity
of glomeruli with crescent formation, hyalinization, sclerosis, basement
membrane and mesangial thickening, and interstitial infiltrates of
mononuclear cells.
(21,25), Therefore, we wanted to assess renal pathologic
features at the earliest ages of the mice. Kidney sections
from control mice and from age-matched, low-dose
COX-2 inhibitor–treated mice were examined at the age
of 18 weeks, at which time the mice had been treated for
6 weeks. In contrast to the treated mice, the control
group had markedly higher histopathology scores (P ⬍
0.005), with typical lesions of severe lupus glomerulonephritis, including glomerular enlargement, hypercellularity, crescents, mesangial thickening, and glomerulosclerosis (Figures 3B and C). In addition, perivascular
and interstitial infiltration with mononuclear cells was
evident in the control mice (Figures 3B and C).
Hyperexpression of intracellular COX-2 in T
cells, B cells, DCs, and macrophages of lupus-prone
SNF1 mice. Human lupus T cells resist AICD by upregulating COX-2 expression, and high doses (50 ␮M) of
celecoxib in vitro can cause apoptosis of T cells (16).
However, intermittent therapy with the lowest dose of
celecoxib had the most beneficial effect in vivo in the
lupus-prone SNF1 mice (Figure 1), without diminishing
the expression of intracellular COX-2 (results not
shown). Therefore, celecoxib might be affecting other
COX-2–expressing cells that are involved in lupus
pathogenesis, by inhibiting the enzymatic function of
COX-2.
As indicated in Figures 4A–C, intracellular
COX-2 levels in CD4 T cells, as well as in DCs,
macrophages,
and
spontaneously
activated
(CD19⫹,CD86high) B cells, of SNF1 mice were a mean
2–3.5-fold higher than those in control SWR or BALB/c
mice (P ⬍ 0.05), indicating that COX-2 is important in
the functioning of various lupus cells. Although Western
blotting involved fractionation of cell subsets, which
might have caused some cellular activation during in
vitro manipulations, the results for COX-2 expression
were nevertheless consistent with those obtained by flow
cytometry of gated splenocyte subsets.
Induction of apoptosis of T cells, B cells, DCs,
and macrophages in vitro following treatment with
COX-2 inhibitors. Because SNF1 mouse T cells, activated B cells, DCs, and macrophages have higher basal
COX-2 AND LUPUS AUTOIMMUNITY
4137
Figure 4. Hyperexpression of cyclooxygenase 2 (COX-2) in cells of autoimmune (SWR ⫻ NZB)F1 (SNF1) mice, and apoptosis of CD4 T cells, B
cells, macrophages (M⌽), and dendritic cells (DCs) in vitro following treatment with celecoxib. A, Intracellular COX-2 expression in gated CD4 T
cells, spontaneously activated (CD86high) B cells, macrophages, and DCs, after staining of whole splenocytes from 6-week-old unmanipulated SNF1,
SWR, or BALB/c mice and analysis by flow cytometry. ⴱ ⫽ P ⬍ 0.05 versus SNF1 strain. B, Representative histograms (determined using FlowJo
software; Y-axis scale indicates the percent maximum), showing expression of COX-2high cells (thick lines) within a marker, versus isotype control
(shaded gray areas). Percentage values in A are the mean and SEM of 5 mice per group from 5 experiments. Values shown over the horizontal bars
in B are the percentage values within the range (⫾ SEM) of the mean values shown in A. C, Western blotting for COX-2 expression in fractionated
splenocytes (upper panel) or whole splenocytes (lower panel) from SWR, NZB, SNF1, and BALB/c mice. Results are representative of 2 separate
experiments. D, Frequency of specific apoptosis induced in vitro by celecoxib in splenocyte subsets of SNF1 mice. Splenocytes from 6-month-old
SNF1 mice with overt nephritis or age-matched SWR mice were cultured with celecoxib in the indicated concentrations for 24 hours, and then stained
for analysis by flow cytometry. Apoptotic (annexin V–positive, propidium iodide–negative) cells were analyzed in gated cell subsets (n ⫽ 5 per strain).
Values are the mean and SEM. ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍ 0.01, versus the same cell subsets in SWR mice.
4138
levels of COX-2 proteins as compared with those in
nonautoimmune SWR or BALB/c strains, we checked
whether inhibition of COX-2 enzymatic activity could
cause the apoptosis of these cells. Figure 4D shows that
treatment with celecoxib at 25 ␮M induced the apoptosis
of T cells, B cells, DCs, and macrophages of SNF1 mice,
after 24 hours’ incubation, to a greater extent than that
in the cells from SWR mice. Since the plasma concentration of celecoxib in mice that received the low-dose
(500 ppm) celecoxib diet was 0.75 ␮M, it was interesting
that this concentration of celecoxib could induce splenic
DCs, but not other cells, to undergo some apoptosis in
this short-term assay. Indeed, SNF1 and human lupus T
cells are similar in terms of requiring high levels of
COX-2 to undergo apoptosis in vitro (16).
Suppression of the T cell response to nucleosomes and inhibition of autoantigen presentation by
APCs in vivo following treatment with low-dose COX-2
inhibitors. SNF1 mice at 12 weeks of age were started on
the lowest dose of COX-2 inhibitor–containing diet for a
10-week period. T cells from treated mice were cocultured with APCs from age-matched (22-week-old) untreated control SNF1 mice or, conversely, APCs from
treated mice were cocultured with T cells from untreated control SNF1 mice in the presence of nucleosomes. The IFN␥ production response by T cells to the
autoantigen was measured. T cells from the 3 treated
groups, especially the low-dose (500 ppm) celecoxib and
continuous combination treatment groups, showed a
significantly reduced autoantigen presentation and T
cell response (P ⬍ 0.01), even at the higher doses of
autoantigen (Figures 5A–C).
Suppression of autoantibody-inducing T helper
cell function and reduced capacity of autoimmune B
cells to receive help from T cells following low-dose
COX-2 inhibitor therapy. To determine whether the
reduction in serum autoantibodies by COX-2 inhibition
in vivo was due to suppression of autoimmune T cells or
B cells, or both, we tested T cells and B cells from both
control and COX-2 inhibitor–treated mouse spleens
after 6 weeks of the COX-2 inhibitor diet. Crisscross
helper assays were done in the presence of nucleosomes,
and autoantibody levels were measured in the supernatants of cell cultures.
We found that the levels of IgG-class antidsDNA, anti-ssDNA, antinucleosome, and antihistone
autoantibodies in culture supernatants of T cells from
the 3 different treatment groups of SNF1 mice cocultured with B cells of untreated control SNF1 mice were
significantly reduced in comparison with the levels in
culture supernatants of T cells from untreated control
ZHANG ET AL
Figure 5. Effect of low-dose cyclooxygenase 2 inhibitor diet therapy on
the nucleosome-induced interferon-␥ (IFN␥) response in lupus T cells
and the autoantigen presentation function of lupus antigen-presenting
cells (APCs). Three-month-old (SWR ⫻ NZB)F1 (SNF1) mice were
treated with the lowest doses of A, celecoxib (CC) or B, aspirin (ASP), or
C, with a low-dose combination of both (Combo), or given a regular diet
without drugs as control (Ctrl), continuously for 10 weeks. Splenic T cells
and APCs were then separated and crisscross cocultures were done as
follows: untreated T cells (Ctrl-T) ⫹ untreated APCs (Ctrl-APC) (serving
as the untreated control in A–C), untreated T cells (Ctrl-T) ⫹ treated
APCs (CC-APC, ASP-APC, or Combo-APC), treated T cells (CC-T,
ASP-T, or Combo-T) ⫹ treated APCs (CC-APC, ASP-APC, or ComboAPC), or treated T cells (CC-T, ASP-T, or Combo-T) ⫹ untreated APCs
(Ctrl-APC), all in the presence of various doses of nucleosomes. IFN␥
responses on enzyme-linked immunospot assay are expressed as the mean
and SEM number of IFN␥-positive spots per 106 T cells (n ⫽ 4 per
group). Values are the mean and SEM. ⴱ ⫽ P ⬍ 0.01 versus untreated
control. The mean ⫾ SEM baseline number of IFN␥ spots in SNF1 T cells
cultured with APCs without nucleosomes was 6 ⫾ 2 spots per 106 T cells.
COX-2 AND LUPUS AUTOIMMUNITY
Figure 6. Effect of low-dose cyclooxygenase 2 (COX-2) inhibitor diet
therapy in vivo on autoantigen-specific T helper cell and B cell
function, assessed as IgG autoantibody (autoAb) production. Twelveweek-old (SWR ⫻ NZB)F1 (SNF1) mice received the lowest doses of
COX-2 inhibitor–containing diet for 6 weeks prior to termination. T or
B cells from mice fed continuously with 500 parts per million (ppm)
celecoxib (CC T or CC B), 400 ppm aspirin (ASP T or ASP B), or a
low-dose combination of both drugs (Combo T or Combo B) were
used. IgG autoantibody production was determined in T cells from
treated mice cocultured for 7 days with B cells from control (Ctrl)
SNF1 mice in the presence of 5 ␮g/ml of nucleosomes (top) or in B
cells from treated mice cocultured for 7 days with T cells from control
SNF1 mice in the presence of nucleosomes (bottom). Levels of IgG
autoantibodies produced in the culture supernatants are the mean and
SEM from 5 experiments. ⴱⴱ ⫽ P ⬍ 0.001 versus control. Baseline
levels of IgG autoantibodies produced by B cells cultured alone were
as follows: for anti–double-stranded DNA (anti-dsDNA), 0.004 ⫾
0.001 mg/dl, for anti–single-stranded DNA (anti-ssDNA), 0.003 ⫾
0.0006 mg/dl, for antinucleosome, 0.003 ⫾ 0.001 mg/dl, and for
antihistone, 0.003 ⫾ 0.0005 mg/dl.
animals cocultured with B cells of untreated control
animals (P ⬍ 0.001) (Figure 6). A similar extent of
suppression could be seen in cocultures of B cells from
treated animals and T cells from untreated animals when
compared with cocultures of B and T cells from untreated animals. These results show that COX-2 inhibitors can suppress both the function of T helper cells and
the ability of autoimmune B cells to receive autoantigenspecific help for autoantibody production.
DISCUSSION
Based on our studies on lupus T cells in vitro in
humans (16), we designed the present study to test the
4139
effects of deletion of autoimmune T helper cells in vivo
by intermittent pulse therapy with celecoxib in mice with
lupus. Even when the therapy was administered intermittently, the beneficial effects of celecoxib were evident
in mice that had clinically overt lupus autoimmunity at
the start of treatment. Surprisingly, the lowest dose of
celecoxib diet (500 ppm) was the most effective in
delaying the onset of severe nephritis and in increasing
survival. Plasma levels of celecoxib were, on average,
0.27 ␮g/ml (0.72 ␮M) at the lowest dose, which is
2.6-fold less than that achieved by intake of 200 mg of
celecoxib/day in humans. Although such low doses are
less likely to cause adverse cardiovascular events (26), to
avoid complications, we fed mice with lupus continuously with a combination of low doses of celecoxib and
aspirin (27–29). This regimen, when given continuously
but not intermittently, did delay lupus nephritis temporarily, but failed to prolong survival, probably due to
some long-term adverse effects of continuous therapy in
vivo. Indeed, addition of aspirin led to a worsened
outcome following intermittent celecoxib therapy, and
aspirin given by itself accelerated mortality in the mice
with lupus, although aspirin is an approved drug for
patients with lupus and is not toxic at the low dosage
used in this study in other strains of mice (28).
In the early weeks of therapy, the combination
regimen with both celecoxib and aspirin was effective in
reducing serum levels of potentially pathogenic autoantibodies and in ameliorating the histopathologic changes
of lupus glomerulonephritis (Figure 3). Moreover, therapy with the COX-2 inhibitors in the short term, even at
the lowest doses, could also suppress the functions of
autoimmune T helper cells, B cells, and APCs, reducing
autoantibody production in response to the major lupus
autoantigen, nucleosome, and also its presentation (Figures 5 and 6). Although the low doses of celecoxib did
not cause apoptosis of T or B cells in the short term in
vitro, these autoimmune cells could have been inhibited
by other mechanisms, such as the blocking of NF-kB
activation (30). Indeed, activation markers, such as
CD86 and CD40, were decreased overall in the B cells
and DCs of SNF1 mice after low-dose celecoxib therapy
(results not shown).
Nevertheless, due to some adverse effects in vivo
over the long term, there was no overall benefit, in terms
of survival, with continuous administration of celecoxib
and/or aspirin. It could be that generation of regulatory
T cells, which are important in controlling lupus
(21,31,32), was impaired by continuous inhibition of
COX-2 (33). Thus, for this or additional reasons, intermittent, but not continuous, therapy with low doses of
4140
celecoxib alone is better in prolonging survival in these
lupus-prone mice.
It is interesting that celecoxib in the lowest dose
range could cause modest apoptosis of SNF1 DCs in a
short-term in vitro assay, but low-dose celecoxib diet
therapy significantly suppressed autoantigen presentation by lupus APCs. Up-regulation of COX-2, and
consequently of prostaglandin E2, is needed for survival,
maturation, and activation of human DCs (34,35), and
hyperactivity of DCs in lupus leads to immunogenic
presentation of autoantigens (7,36). Indeed, not only T
cells, but also DCs and macrophages of SNF1 mice
constitutively hyperexpressed COX-2 (Figure 4), and
NF-␬B activation, which is needed for functioning of
these cells of the lupus immune system, could have been
inhibited by celecoxib (30).
The hyperactive B cells of lupus not only produce
pathogenic autoantibodies, but also costimulate and
present autoantigens to pathogenic T cells, and may
stimulate themselves (1,37–39). We have found that
spontaneously activated lupus B cells constitutively express high levels of COX-2, as do mitogenically stimulated, normal human B cells (40,41). Interestingly,
CD86high (activated) B cells from SNF1 mice expressed
more COX-2 than did the same subset from nonautoimmune strains, indicating a lupus-intrinsic defect (Figure 4). Indeed, COX-2 inhibitor therapy could suppress
both autoantigen-specific T helper function and the ability
of autoimmune B cells to receive autoantigen-specific help
for autoantibody production (Figures 5 and 6).
Finally, COX-2 is also up-regulated in inflammatory macrophages and glomerular mesangial cells of the
kidney in lupus (42,43). Thus, major players in the
pathogenic autoimmune response that are abnormally
hyperactive and resistant to apoptosis in lupus depend
on increased activity of COX-2, similar to inflammatory
cells in the target organs. In addition, our observations
show that intermittent therapy with low doses of a
COX-2 inhibitor increases survival irrespective of the
severity of nephritis, by an additional mechanism(s).
In relation to human lupus, this study shows the
importance of COX-2 as a target for lupus therapy, using
more benign COX-2 inhibitors that lack adverse cardiovascular effects. It would be interesting to explore
whether intermittent pulse therapy with low doses of a
selective COX-2 inhibitor in combination with cardioprotective statins, which also have beneficial effects in
lupus models (44), would provide added benefit. Moreover, with a currently permissible COX-2–specific inhibitor such as celecoxib, a low-dose intermittent administration might have a more favorable outcome.
ZHANG ET AL
ACKNOWLEDGMENTS
We thank Dr. Hee-Kap Kang and Michael Liu for their
critical comments and advice regarding the ELISpot assay and
ELISA. We also thank Dr. David Pisetsky (Duke University)
for suggesting the use of aspirin as part of the therapy.
AUTHOR CONTRIBUTIONS
Dr. Datta had full access to all of the data in the study and
takes responsibility for the integrity of the data and the accuracy of the
data analysis.
Study design. Zhang, Datta.
Acquisition of data. Zhang, Bertucci, Smith, Xu.
Analysis and interpretation of data. Zhang, Bertucci, Xu, Datta.
Manuscript preparation. Zhang, Bertucci, Xu, Datta.
Statistical analysis. Zhang.
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