The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

The Journal of Immunology, 2008, 180, 8361 -8368
Copyright © 2008 by The American Association of Immunologists, Inc.
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kojima, F.
Right arrow Articles by Crofford, L. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kojima, F.
Right arrow Articles by Crofford, L. J.

Defective Generation of a Humoral Immune Response Is Associated with a Reduced Incidence and Severity of Collagen-Induced Arthritis in Microsomal Prostaglandin E Synthase-1 Null Mice1

Fumiaki Kojima*, Mohit Kapoor*, Lihua Yang*, Erica L. Fleishaker*, Martin R. Ward{dagger}, Seetha U. Monrad{ddagger}, Ponnappa C. Kottangada*, Charles Q. Pace*, James A. Clark*, Jerold G. Woodward{dagger} and Leslie J. Crofford2,*

* Division of Rheumatology, Department of Internal Medicine and {dagger} Department of Microbiology, Immunology and Molecular Genetics, University of Kentucky, Lexington, KY 40536; and {ddagger} Division of Rheumatology, Department of Internal Medicine, University of Michigan, Ann Arbor, MI 48109


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Microsomal PGE synthase-1 (mPGES-1) is an inducible enzyme that acts downstream of cyclooxygenase and specifically catalyzes the conversion of PGH2 to PGE2. The present study demonstrates the effect of genetic deletion of mPGES-1 on the developing immunologic responses and its impact on the clinical model of bovine collagen-induced arthritis. mPGES-1 null and heterozygous mice exhibited decreased incidence and severity of arthritis compared with wild-type mice in a gene dose-dependent manner. Histopathological examination revealed significant reduction in lining hyperplasia and tissue destruction in mPGES-1 null mice compared with their wild-type littermates. mPGES-1 deficient mice also exhibited attenuation of mechanical nociception in a gene dose-dependent manner. In addition, mPGES-1 null and heterozygous mice showed a marked reduction of serum IgG against type II collagen, including subclasses IgG1, IgG2a, IgG2b, IgG2c, and IgG3, compared with wild-type mice, which correlated with the reduction in observed inflammatory features. These results demonstrate for the first time that deficiency of mPGES-1 inhibits the development of collagen-induced arthritis, at least in part, by blocking the development of a humoral immune response against type II collagen. Pharmacologic inhibition of mPGES-1 may therefore impact both the inflammation and the autoimmunity associated with human diseases such as rheumatoid arthritis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Microsomal PGE synthase-1 (mPGES-1)3 is an inducible enzyme that acts downstream of cyclooxygenase (COX) and specifically catalyzes the conversion of PGH2 to PGE2, most prominently in inflammatory conditions (1, 2). mPGES-1 is an attractive target for drug development, as inhibition would specifically diminish the PGE2 production associated with clinical inflammatory disorders while preserving the production of other PGs. Specific inhibitors of mPGES-1 are not yet widely available; however, knockout mice have been generated that have provided insight into the role of mPGES-1 in eicosanoid biosynthesis in vivo and in vitro (3, 4, 5, 6, 7, 8). Studies using mPGES-1 null mice have demonstrated that this enzyme is a key mediator of inflammation, pain, angiogenesis, fever, bone metabolism, tumorigenesis, atherosclerosis, and reproduction (8, 9, 10, 11, 12, 13, 14, 15, 16, 17).

PGE2 is a major inflammatory mediator in rheumatoid arthritis (RA), and high concentrations of PGE2 are detected in the synovial fluid of patients with RA (18). Our previous studies demonstrate that mPGES-1 is coordinately up-regulated with inducible COX-2 in cultured synovial fibroblasts from RA patients by stimulation with proinflammatory cytokines such as IL-1β and TNF-{alpha} (19, 20). In addition, it has been reported that the expression pattern of mPGES-1 in RA synovium correlates with the degree of disease activity (21, 22).

The collagen-induced arthritis (CIA) model is widely used as a model of RA and is highly dependent on both humoral and cellular immunity (23). TCRβ null mice lacking {alpha}β T cells (24) as well as mice lacking B cells (25) are resistant to CIA; both of these strains have reduced Ab production against type II collagen (CII), indicating the critical role of the CII-specific humoral immune response in the pathophysiology of CIA. CII Abs in RA patients have been shown to recognize pathogenic epitopes on CII similar to those in CIA (26, 27, 28, 29, 30).

mPGES-1 null mice are resistant to chicken CIA, but the mechanisms underlying resistance have not been elucidated (8). The present study demonstrates for the first time that the reduced incidence and severity of CIA in mPGES-1 null mice is associated with significantly reduced levels of CII-specific Abs. These data indicate a significant role for mPGES-1 and PGE2 not only in the inflammatory manifestations of CIA but also in the autoimmune response against CII. Our findings provide novel insights relevant to the therapeutic potential for pharmacologic inhibition of mPGES-1 in chronic autoimmune inflammatory diseases including RA.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

mPGES-1 heterozygous (Het) male and female mice on a DBA1 lac/J background were provided by Pfizer (8). mPGES-1 Het mice were mated to generate mPGES-1 null, Het, and littermate wild-type (WT) mice. Mice were housed in microisolator cages in a pathogen-free barrier facility, and all experiments were performed under the Institutional Animal Care and Use Committee guidelines as set forth by the University of Kentucky, Lexington KY. Genotypes were identified by PCR of tail biopsy DNA extract using two-primer sets for the mPGES-1 null allele (P1, 5'-TGCTACTTCCATTTGTCACGTC-3'; and P2, 5'-ACTCCAAGTACTGAGCCAGCTG-3') and the WT allele (P3, 5'-TCCCAGGTGTTGGGATTTAGAC-3'; and P4, 5'-TAGGTGGCTGTACTGTTTGTTGC-3'). After initial denaturation at 95°C for 15 min, PCR involved 40 cycles of 30 s at 95°C, 30 s at 56°C, and 45 s at 72°C, followed by elongation for 5 min at 72°C. DNA from mPGES-1 WT mice showed one band (412 bp), DNA from mPGES-1 null mice showed one band (720 bp), and DNA from mPGES-1 Het mice showed bands of both 412 and 720 bp (Fig. 1). Our previous study also shows that deletion of the mPGES-1 gene results in impaired mPGES-1 mRNA and protein expression as well as PGE2 production in a mPGES-1 gene dose-dependent manner in embryonic fibroblasts prepared from whole embryos of these mice (4).


Figure 1
View larger version (38K):
[in this window]
[in a new window]

  		FIGURE 1. Genotyping of mPGES-1 WT, Het, and null mice by PCR analysis. The lower band (412 bp) is amplified from the WT alleles and the upper band (720 bp) is from the mPGES-1 null alleles.

 
Immunization and development of CIA

Male and female mice used in this study were 10- to 15-wk old. For immunization, 100 µg of bovine CII (immunization grade; Chondrex) in CFA containing Mycobacterium tuberculosis H37 RA was injected intradermally at the base of the tail under anesthesia with isoflurane on day 0. On day 21 postimmunization, a booster injection of 100 µg of bovine CII in IFA was given. Mice were examined weekly after the first immunization. Arthritis severity was assessed by measuring hind paw thickness with calipers to quantitate edema, measuring the incidence (percentage) of clinical arthritis in front and hind paws, and scoring the degree of clinical arthritis on a scale of 0 (no inflammation) to 3 (severely inflamed) for each paw (the maximum score being 12).

Histological assessment of CIA

At day 65 after the first immunization, mice were euthanized in a CO2 chamber and hind paws were collected. Samples were fixed in 4% paraformaldehyde, decalcified in EDTA, and then embedded in paraffin. Five-micrometer-thick sections were stained with H&E. Histological analysis of arthritis was performed by an observer blinded to the genotypes of the mice. The scores were based on the degree of lining hyperplasia, tissue destruction, and inflammatory cell infiltration (0, within normal limits; 1, minimal; 2, mild; and 3, severe).

Assessment of mechanical nociception

Withdrawal response to mechanical nociceptive stimulation on each hind paw was measured using von Frey filaments (North Coast Medical). Briefly, mice were isolated on mesh floors and the plantar surface of each hind paw was stimulated with von Frey filaments. The force that generated at least three withdrawal responses to five repeated stimuli was recorded. Forces required to buckle the filaments ranged from 0.04 to 4 g.

RT-PCR

RNA from spleen and lymph nodes was extracted with TRIzol reagent according to the manufacturer’s instructions. Reverse transcription was performed using a SuperScript preamplification system (Invitrogen) per the manufacturer’s protocol, with 1 µg of total RNA from the tissue as a template. Subsequent amplifications of the cDNA fragments by PCR with HotStar Taq polymerase (Qiagen) were performed using 0.5 µl of the reverse-transcribed mixture as a template, with specific oligonucleotide primers as follows: mouse mPGES-1, 5'-CACACTGCTGGTCATCAAGA-3' (sense) and 5'-ACACCAAGTCCGCAAGTTC-3' (antisense); mouse COX-2, 5'-GGGCCCTTCCTCCAGTAGCAGA-3' (sense) and 5'-CATCAGACCAGGCACCAGACCAA-3' (antisense); and mouse hypoxanthine phosphoribosyltransferase (HPRT), 5'-GTTGGATACAGGCCAGACTTTGTTG-3' (sense) and 5'-GAGGGTAGGCTGGCCTATAGGCT-3' (antisense). After initial denaturation at 95°C for 15 min, PCR involved amplification cycles of 30 s at 95°C, 30 s at 56°C, and 45 s at 72°C, followed by elongation for 5 min at 72°C. The amplified cDNA fragments were resolved by electrophoresis on a 2% (w/v) agarose gel, stained with ethidium bromide, and analyzed by a Chemidoc Apparatus (Bio-Rad).

Measurement of anti-CII IgG

Blood samples were collected into serum separator tubes (BD Biosciences) by the submandibular bleeding method as previously described (31). Serum was separated by centrifuging at 10000 x g for 90 s. The levels of anti-CII IgG in serum were assessed by ELISA. Ninety-six-well plates were coated with an ELISA grade bovine CII (Chondrex) overnight at 4°C. After blocking with 50 mM TBS (pH 8.0) containing 1% BSA, diluted serum samples (1/50,000 for IgG2a and 2b; 1/20,000 for total IgG at day 42; 1/10,000 for total IgG at day 21 and for IgG1; 1/4,000 for IgG2c; and 1/800 for IgG3) were added and incubated overnight at 4°C. After washing, HRP-conjugated Ab (Bethyl Laboratories) was added for 1 h at room temperature. After further washing, the color was developed with tetramethyl benzidine, terminated by 2M H2SO4, and then measured at 450 nm using a plate reader (Bio-Rad). Pooled serum collected from WT mice with arthritis scores > 8 at day 65 was used for the standard curve. The titer of the pooled standard was defined as 1000 U/ml.

FACS analysis (phenotypic analysis)

Spleen was isolated at 10 and 21 days after the first immunization (without booster injection). Single cell suspensions (1 x 106 cells) were incubated for 5 min with anti-CD16/CD32 (BD Pharmingen) to block Fc{gamma}II/III receptor-mediated nonspecific Ab binding. Cells were then stained with fluorochrome-conjugated mAbs (BD Pharmingen) for 30 min at 4°C. After washing, analysis was performed with a BD FACSCalibur System (BD Biosciences).

Measurement of total IgG and IgM

The levels of IgG and IgM were assessed by ELISA according to the manufacturer’s protocol (Bethyl Laboratories). Briefly, 96-well plates were coated with goat anti-mouse IgG for 1 h. After blocking with 50 mM TBS (pH 8.0) containing 1% BSA for 30 min, plates were incubated with diluted serum samples (1/8000) for 1 h at room temperature. After washing, HRP-conjugated Ab was added for 1 h at room temperature. After further washing, the color was developed with tetramethyl benzidine, terminated by 2M H2SO4, and then measured at 450 nm using a plate reader (Bio-Rad). The titer of the pooled standard was defined as 1000 U/ml.

Assessment of PGE2, total IgG, and IgM production in cultured splenocytes

Splenocytes isolated from mPGES-1 WT and null mice (3.75 x 105 cells/0.3 ml) were cultured in 48-well plates for 4 days with complete RPMI 1640 in the absence or presence of 0.001–10 µg/ml LPS (Escherichia coli 0111:B4, Sigma-Aldrich). The concentration of PGE2 in culture medium was measured by ELISA (Cayman Chemicals). The levels of total IgG and IgM in culture medium were assessed by ELISA with standard samples (Bethyl Laboratories).

Proliferative responses of splenocytes

Proliferation of splenocytes was assessed by a BrdU cell proliferation assay kit (Roche Diagnostics) according to the manufacturer’s protocol. Briefly, spleen was isolated at 10 and 21 days after the first immunization (without booster injection). Single cell suspensions were prepared in complete RPMI 1640 supplemented with 10% FBS, 50 mM 2-ME, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. Splenocytes (2.5 x 105 cells/0.1 ml) were cultured in flat-bottom 96-well plates with or without 100 µg/ml CII (T cell proliferation grade; Chondrex) for 48 h, and then further incubated with BrdU for 18 h. Proliferative activity was estimated from the nuclear incorporation of BrdU as measured by ELISA.

Statistical analysis

Data are expressed as the means ± SEM. Statistical analysis was performed with the Sigmastat 3.5 software (Systat Software). The comparison of more than two groups was analyzed with one-way ANOVA followed by Turkey’s multiple comparison test. For comparison of two groups, Student’s t test was performed after testing for normal distribution. The correlation between two quantitative variables was analyzed by Spearman’s rank test. p < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Clinical course of CIA in mPGES-1 genetic deletion

mPGES-1 null, Het, and WT mice on a DBA1 lac/J background were immunized with bovine CII in CFA on day 0 and arthritis was stimulated by providing a booster injection with CII in IFA at day 21 after the first immunization. As shown in Fig. 2A, the number of days to arthritis onset in mPGES-1 null and Het mice was not significantly different from that of their WT littermates. The incidence of CIA was significantly reduced in mPGES-1 null mice compared with WT mice, with only 30–40% of the paws in mPGES-1 null mice exhibiting arthritis by the final time point compared with 70–80% in WT mice. The arthritis incidence in Het mice was intermediate between that in mPGES-1 null mice and WT littermates, indicating a mPGES-1 gene dose-dependent effect in modulating CIA. The mean arthritis score was also markedly decreased in mPGES-1 null and Het mice compared with WT mice in a gene dose-dependent manner, similar to the pattern observed for arthritis incidence (Fig. 2B). Paw edema as evaluated by paw thickness was significantly reduced in mPGES-1-deficient mice (Fig. 2C). No significant changes in body weight were observed among mPGES-1 WT, Het, and null mice during the course of the study, although there was a trend toward lower body weight for WT mice (Fig. 2D).


Figure 2
View larger version (37K):
[in this window]
[in a new window]

  		FIGURE 2. Effects of mPGES-1 gene deletion on CIA response. Time course of incidence of arthritis (A), arthritis scores (B), paw edema (C), and body weight (D) of mPGES-1 WT (n = 21), Het (n = 27), and null (n = 22) mice in CIA at the indicated days after the first immunization. Data for incidence, arthritis scores, and body weight are expressed as the mean ± SEM from each animal. Data for paw edema are expressed as the mean ± SEM from each hind paw. Single, double, and triple characters indicate significance at p < 0.05, p < 0.01, and p < 0.001, respectively. *, WT vs null; $, WT vs Het; #, Het vs null.

 
Histological features of arthritis in mPGES-1 null mice

Severity of arthritis was further assessed by histopathological evaluation. As shown in Fig. 3A, the joints of mPGES-1 WT mice showed characteristic features of CIA with hyperplasia of synovium, enhanced tissue destruction, and increased infiltration of inflammatory cells (left column). Compared with WT mice, mPGES-1 null mice exhibited less synovial proliferation and tissue destruction (right column). When these histological features were graded by an observer blinded to the genotypes of the sections, the scores for mPGES-1 null mice were reduced compared those for WT littermates (Fig. 3B). The scores in Het mice were intermediate between mPGES-1 null mice and WT littermates, indicating a mPGES-1 gene dose-dependent effect in developing arthritis.


Figure 3
View larger version (96K):
[in this window]
[in a new window]

  		FIGURE 3. Histological analysis of arthritis severity in mPGES-1 null mice. A, Hind paws of mPGES-1 WT, Het, and null mice were collected at day 65 after the first immunization and sections were stained by H&E. Results are representative examples in mPGES-1 WT (left column), Het (middle column), and null (right column) mice with CIA. Top and middle rows show x4 (original magnification) views of footpads and ankle joints, respectively; bottom row shows x10 (original magnification) views of the ankle joints. Arrowheads and stars in the bottom row show enhanced cartilage destruction and hyperplasia of synovium, respectively, in WT mice compared with Het and null mice. B, Histological scores in mPGES-1 WT (open bar, n = 12), Het (gray bar, n = 17), and null (filled bar, n = 9) mice. Sections were blindly examined for lining hyperplasia, tissue destruction, and inflammatory cell infiltration. Data are expressed as the mean ± SEM. *, p < 0.05 indicates significance compared with WT mice.

 
Reduction of mechanical nociception in mPGES-1 deficiency

mPGES-1-deficient mice have previously been reported to have differential pain responses in acetic acid writhing, thermal nociception, and neuropathic pain models (8, 9, 10). To examine the effect of mPGES-1 deficiency on mechanical nociception during the development of CIA, withdrawal latency was measured using von Frey filaments. Inflammation results in mechanical hyperalgesia, which can be measured by withdrawal responses to progressively smaller diameter fragments that generate progressively lower mechanical force to the footpad. As shown in Fig. 4A, all three genotypes showed a progressive decrease in the force required to elicit a withdrawal response of the inflamed hind paw, which paralleled the time course of development and the severity of arthritis shown in Fig. 2. Compared with WT mice, mPGES-1 null mice showed significantly less change from baseline throughout the monitoring period. The withdrawal response in Het mice was intermediate between that of mPGES-1 null and WT mice, indicating a mPGES-1 gene dose-dependent contribution to mechanical nociception in arthritis.


Figure 4
View larger version (26K):
[in this window]
[in a new window]

  		FIGURE 4. Inhibitory effect of mPGES-1 gene deletion on mechanical nociception. A, Time course of mechanical nociception in mPGES-1 WT, Het, and null mice with CIA. Values show the change in mechanical force required to elicit a withdrawal response in hind paws of mPGES-1 WT (n = 15), Het (n = 19), and null (n = 16) mice with CIA at the indicated days after the first immunization. Data are expressed as the mean ± SEM from each hind paw. Single, double, and triple characters indicate significance at p < 0.05, p < 0.01, and p < 0.001, respectively. *, WT vs null; $, WT vs Het. B, Correlation between mechanical nociception and arthritis score in mPGES-1 WT (n = 15) and null (n = 16) mice with CIA at day 49. The mechanical force required to elicit a withdrawal response is plotted against the arthritis score of individual paws at day 49. The correlation was analyzed by Spearman’s rank test and correlation coefficient (r) and p value are indicated.

 
When the withdrawal force was plotted against the arthritis score, there was a significant inverse correlation (Fig. 4B), suggesting that the decreased nociception exhibited by mPGES-1-deficient mice was due at least in part to overall lower arthritis scores.

Induction of mRNA expression for mPGES-1 and COX-2 in spleen and lymph nodes of CII/CFA-immunized mice

To determine the pattern of COX-2 and mPGES-1 induction in immune tissues, the mRNA expression profile of COX-2 and mPGES-1 in WT spleen and lymph nodes was determined by RT-PCR (Fig. 5). This revealed a significant increase in mRNA expression of both enzymes in spleen (Fig. 5A) and lymph nodes (Fig. 5B) after CII/CFA immunization. mPGES-1 mRNA expression showed a gradual increase, with maximum levels on day 21 postimmunization. In contrast, COX-2 mRNA levels rapidly increased by day 10 after immunization and then decreased by day 21. These data clearly support a role for these enzymes in the developing immune response.


Figure 5
View larger version (31K):
[in this window]
[in a new window]

  		FIGURE 5. Induction of mPGES-1 and COX-2 in spleen and lymph nodes of CII/CFA-immunized mice. mRNA expression of mPGES-1 and COX-2 in WT spleen (A) and lymph nodes (B) at days 0 (control nonimmunized mice), 10, and 21 after immunization was determined by RT-PCR. Results are representative examples from 3–6 mice at each time point (top panel). Levels of mRNA expression were normalized against hypoxanthine phosphoribosyltransferase (HPRT); data are shown as the fold induction relative to day 0 expression (assigned the value "1") and are the mean ± SEM from 3–6 mice at each time point. *, p < 0.05, indicates statistical significance compared with day 0.

 
mPGES-1 null mice show reduced levels of anti-collagen Abs

High levels of anti-CII Abs have been shown to be essential for disease development in the CIA model (32, 33). To determine the impact of mPGES-1 deficiency on the CII-specific immune response, serum levels of anti-CII IgG on days 21 (before booster injection) and 42 (21 days after booster injection) were determined. As shown in Fig. 6A, levels of CII-specific IgG were significantly decreased on both days 21 and 42 in mPGES-1 in a gene-dose dependent manner. In addition, day 42 levels of CII-specific IgG subclasses IgG1, IgG2a, IgG2b, IgG2c, and IgG3 were significantly lower in mPGES-1 null mice compared with WT mice (Fig. 6B). The levels of anti-CII IgG subclasses in Het mice were intermediate between those of mPGES-1 null mice and WT littermates, again indicating a gene dose-dependent effect of mPGES-1.


Figure 6
View larger version (28K):
[in this window]
[in a new window]

  		FIGURE 6. Reduction of CII-specific IgG levels in mPGES-1 deficiency. A, Levels of CII-specific total IgG in serum. Serum from mPGES-1 WT (open bar, n = 21), Het (gray bar, n = 27), and null (filled bar, n = 22) mice with CIA was collected on days 21 and 42, and titers of CII specific total IgG were measured by ELISA. B, Levels of CII-specific IgG subclasses in serum. Titers of CII-specific IgG in serum of mPGES-1 WT (open bar, n = 21), Het (gray bar, n = 27), and null (filled bar, n = 22) mice at day 42 were measured by ELISA. Data are expressed as the mean ± SEM in arbitrary units for CII-specific IgG, IgG1, IgG2a, IgG2b, IgG2c, and IgG3. *, Single, double and triple characters indicate significance at p < 0.05, p < 0.01, and p < 0.001, respectively.

 
To determine why we observed reduced Ab production postimmunization in mPGES-1-deficient mice, we ascertained whether null mice displayed altered immune cell populations. We analyzed splenocytes isolated from mPGES-1 null and WT mice after CII immunization by FACS for specific cell surface markers (CD4, CD8, CD25, CD19, CD11c, and Mac-1). The population of cells stained by each marker did not change significantly in the absence of mPGES-1 (data not shown), although there was a trend toward a lower population of Mac-1 positive cells in mPGES-1 null mice (WT, 10.76 ± 1.79% (n = 4); null, 6.18 ± 0.33% (n = 3); p = 0.085). This suggests that the altered serum Ab profile was not due to a different splenic population of immune cells in the absence of mPGES-1.

Reduction of anti-CII Abs in mPGES-1 null mice correlates with the resistance against arthritis

We next analyzed the correlation between serum CII-specific Ab level reductions in mPGES-1 deficiency with the reduction of arthritis observed in this study (Table I). Because mice had not developed visible signs of arthritis by day 21, we used the arthritis index at day 42 to correlate with serum Ab production on day 21 postimmunization. IgG levels on both days 21 and 42 postimmunization correlated to both the incidence and the severity of arthritis on day 42. Levels of CII-specific IgG subclasses also significantly correlated with the incidence and severity of arthritis. We conclude that there is a high likelihood that the reduction of CIA in mPGES-1 null mice is due to the diminished ability of these mice to produce Abs against CII.


View this table:
[in this window]
[in a new window]

  		Table I. Correlation of serum CII specific IgG and subclass levels to incidence and severity of arthritisa

 
Proliferative response of splenocytes in mPGES-1 null mice

To analyze Ag-specific T cell responses in mPGES-1 deficiency, we examined the proliferative activity of splenocytes in response to CII stimulation in vitro. Splenocytes were isolated at days 10 and 21 after immunization with CII in CFA and then stimulated with CII. As shown in Fig. 7, proliferative responses were increased by CII stimulation in both mPGES-1 null and WT cells, with no significant differences observed. This demonstrates that Ag-specific T cell proliferative responses in CIA are not altered in the absence of mPGES-1 and are therefore unlikely to contribute to the altered clinical presentation and immune profile seen in mPGES-1 null mice.


Figure 7
View larger version (17K):
[in this window]
[in a new window]

  		FIGURE 7. CII-induced proliferation of splenocytes with mPGES-1 deficiency. Splenocytes were isolated from mPGES-1, WT, and null mice at days 10 and 21 postimmunization with CII in CFA. Cells were stimulated with CII (100 µg/ml) for 48 h and then further incubated with BrdU for 18 h. Proliferative activity was estimated from the nuclear incorporation of BrdU as measured by ELISA. Data for mPGES-1 deficient and stimulated WT cells are expressed relative to the level of incorporation in unstimulated WT splenocytes (assigned the value "1"), and are the mean ± SEM from three mPGES-1 WT, and null mice at each time point. cont, Control.

 
Effect of mPGES-1 gene deletion on total IgM and IgG production in vivo and in vitro

We next determined the effect of mPGES-1 deficiency on polyclonal Ab production by measuring total IgG and IgM levels. As shown in Fig. 8A, the levels of both total IgG and IgM in serum were significantly reduced in mPGES-1 null mice compared with WT mice postimmunization. The results indicate that the effect of mPGES-1 deletion may not be limited to the CII-specific autoimmune response but also may be due to a reduced nonspecific immune response to CFA.


Figure 8
View larger version (32K):
[in this window]
[in a new window]

  		FIGURE 8. Effect of mPGES-1 gene deletion on total IgG and IgM production in vivo and in vitro. A, Serum from mPGES-1 WT (n = 6), Het (n = 8), and null (n = 6) mice with CIA were collected at the indicated days after the first immunization, and levels of IgG and IgM were measured by ELISA. Data are expressed as the mean ± SEM. Single and double characters indicate significance at p < 0.05 and p < 0.01, respectively. *, WT vs null; $, WT vs Het; #, Het vs null. B, Splenocytes isolated from mPGES-1 WT and null mice were cultured with or without LPS (0.001–10 µg/ml) for 4 days in vitro. Levels of PGE2 and total IgG and IgM in culture medium were measured by ELISA. Data are expressed as the mean ± SEM from 3–5 mPGES-1 WT and null mice. ***, p < 0.001 indicates significance.

 
To determine whether there is a specific defect in the capacity of B cells deficient in mPGES-1 to produce Ab, production of total IgG and IgM was examined in vitro in cultured splenocytes after stimulation with LPS, which is a well known polyclonal activator of B cells (Fig. 8B). Levels of PGE2 in culture supernatant were significantly increased by LPS in WT mice; this increase was completely abolished in mPGES-1 null mice. In this in vitro setting, however, levels of total IgM and IgG were increased by LPS in a concentration-dependent manner in both mPGES-1 null and WT cells, and no significant difference was observed. These results indicate that mPGES-1 is essential for PGE2 biosynthesis in splenocytes. However, the diminished nonspecific and CII-specific Ab response seen in mPGES-1 deficient mice is not due to an intrinsic defect in the Ig production capacity of null B-cells or to the absence of inducible PGE2.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The present study in mPGES-1 null, Het, and WT mice demonstrates an important role for mPGES-1 in the pathogenesis of autoimmune arthritis induced by bovine CII. Genetic deletion of mPGES-1 results in a reduction in inflammatory responses, including arthritis parameters and mechanical nociception, in a gene dose-dependent manner. Further, mPGES-1 deficiency results in a marked reduction in the levels of CII-specific IgG, which correlates with the reduction in arthritis incidence and severity.

We demonstrate that mPGES-1 deficiency results in a reduction in the severity of arthritis with decreased synovial hyperplasia, tissue destruction, and infiltration of inflammatory cells. We also show a reduction in pain perception after mechanical stimulation. Previous studies have shown that COX-2-deficient mice also display a significant reduction in both clinical and histological changes in CIA induced by bovine CII (34). Furthermore, administration of a COX-2 inhibitor, but not a COX-1 inhibitor, reduces paw inflammation (35). A recent study using mice deficient in PGE2 receptor subtypes (EP1–4) as well as selective EP antagonists supports the essential role of PGE2 in CIA induced by bovine CII, mediating its effects via EP2/EP4 signaling (36). Studies using the CII Ab-induced arthritis (CAIA) model, which does not require intrinsic CII Ab production, have also demonstrated the role of mPGES-1 in inflammatory arthritis; in this model, mPGES-1-associated PGE2 exerts its effects via the EP4 receptor (10, 37). These studies indicate that PGE2 plays a role in immune complex arthritis predominantly via EP4 signaling.

A previous study used the identical strain of mPGES-1 null mice as in this study but used chicken CII as the inciting Ag; it reported almost complete elimination of inflammation and histopathological changes in CIA with the absence of mPGES-1 (8). This complete protection from arthritis is in contrast to our current findings, which showed only an ~50% reduction of arthritis in mPGES-1 null mice compared with WT mice. Previous reports have documented differences in clinical CIA related to the source of CII (38). Therefore, the disparate results obtained from the two studies could be explained by the fact that those authors performed their studies using chicken CII, in contrast to our experiments performed with bovine CII. The present study is also the first to report the intermediate CIA phenotype in mice heterozygous for mPGES-1, important in evaluating it as a potential target for drug development.

The difference in time course profiles of mPGES-1 and COX-2 mRNA expression in immune tissues, including spleen and lymph nodes, is one of the important finding in this study. Our previous studies using synovial fibroblasts isolated from patients with RA demonstrated that COX-2 shows a rapid increase of expression after IL-1β stimulation, whereas expression of mPGES-1 increases more gradually when compared with that of COX-2 in both mRNA and protein levels (19, 20). These studies also suggested that increased levels of PGE2 production depend on slowly expressed mPGES-1 in these cells. A similar expression pattern as that seen in RA synovial fibroblasts was also observed in mouse embryonic fibroblasts after IL-1β stimulation (4). The different profile of mPGES-1 and COX-2 expression implies that regulation of these enzymes occurs via different pathways. COX-2 is a classic immediate-early gene (39). In contrast, mPGES-1 expression in response to various stimuli is regulated by the transcription factor Egr-1 (early growth response factor-1) in mouse (40) and human (41). Egr-1 was reported to bind to the proximal GC box in the mPGES-1 gene promoter. The differing transcriptional regulation of mPGES-1 and COX-2 may help to explain the different time course of the expression of these enzymes in spleen and lymph nodes in this study.

The CIA model is strongly dependent on the immunologic events associated with CII-specific autoimmunity. Because the immunomodulatory (as opposed to inflammatory) role of mPGES-1 is unclear, we examined the effects of mPGES-1 genetic deletion on immunologic events in CIA. The present study demonstrates a significant reduction in in vivo levels of CII-specific IgG subclasses (total IgG, IgG1, IgG2a, IgG2b, IgG2c, and IgG3) with mPGES-1 deletion. The reduction of CII-specific Ab production correlates with the reduction in incidence and severity of arthritis. A previous study using mPGES-1 null mice with chicken CII CIA reported that "the absence of mPGES-1 expression in mice does not cause any gross immunological abnormalities in vivo" and that there was no significant difference in the circulating serum levels of anti-CII IgG2a, in contrast to our current observation with bovine CII (8). However, the changes observed in the immunological responses of mPGES-1 null mice in this study are consistent with previous observations in COX-2 null mice using bovine CII to induce arthritis (34). COX-2 null mice demonstrated a marked reduction in the production of anti-CII Abs, implicating a coupled COX-2/mPGES-1/PGE2 pathway in the development of an Ab response to bovine CII.

PGE2 has been reported to modulate immunologic events including dendritic cell maturation, macrophage activation, and B cell and T cell function (42, 43, 44, 45, 46, 47). PGE2 is known to promote class switching to IgG1 in the presence of IL-4, indicating evidence for mechanisms by which PGE2 regulates the immune system and advances a Th2-type response (48). A recent study has demonstrated that activated human B lymphocytes express COX-2 and that nonsteroidal anti-inflammatory drugs (NSAIDs), including selective COX-2 inhibitors, attenuate Ab production in vitro (49). In the present study, we observed no significant effect of mPGES-1 on the levels of Ab production in splenocytes stimulated with LPS in vitro, although mPGES-1 is essential for endogenous PGE2 production in these cells. Similarly, in the present study we observed no significant effect of mPGES-1 deficiency on the proliferation of splenocytes in response to CII in an in vitro model to analyze the T cell proliferative response against CII.

In a separate report, we observed that mPGES-1 is required for PGE2 production in murine dendritic cells. mPGES-1 null dendritic cells exhibit normal maturation and migration, but the level of IL-12 production after LPS stimulation is significantly reduced (S. U. Monrad, F. Kojima, M. Kapoor, E. L. Kuan, S. Sarkar, G. J. Randolf, and L. J. Crofford, submitted for publication). A previous study found that PGE2 produced by mouse splenic macrophages is associated with a COX-2-dependent shift from Th1- to Th2-type cytokines by heat-killed Mycobacterium bovis bacillus Calmette-Guerin (BCG; a vaccination strain for M. tuberculosis), which has been widely used to establish animal models of autoimmune disease including adjuvant arthritis, as well as by CFA, which contains heat-killed M. tuberculosis (50). Furthermore, a recent study demonstrated that administration of a stable PGE analog, misoprostol, exacerbated CIA and was associated with increased levels of IL-17 (51). These findings suggest that an altered cytokine profile related to absent PGE2 might play a role in aberrant development of the CII-specific humoral immune response in CIA in mPGES-1 null mice. Further assessment of the effect of mPGES-1 deficiency and reduced PGE2 on the tissue environment in which APCs encounter Ag and interact with T cells and B ells may provide further understanding of mechanisms of altered humoral immune response observed in the in vivo setting.

The present study provides important information regarding the therapeutic potential for pharmacologic inhibition of mPGES-1 in autoimmune inflammatory diseases, including RA. Taken together with previous studies, a mPGES-1 inhibitor is likely to have anti-inflammatory and analgesic effects in these disorders. However, because the effect of mPGES-1 on Ab production was not limited to the CII-specific response, the findings reported herein may have important implications for a wider range of immunologic processes including autoimmunity, the response to vaccination, and infections.


   Acknowledgment
 
We thank Dr. Donald A. Cohen (University of Kentucky) for valuable advice and support on FACS analysis.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health/ National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant R01 AR049010, National Institutes of Health Grant EY14060, and a travel award from the Japanese Society of Clinical Pharmacology and Therapeutics. Back

2 Address correspondence and reprint requests to Dr. Leslie J. Crofford, Department of Internal Medicine, Rheumatology Division, Room J-509, Kentucky Clinic, University of Kentucky, Lexington, KY 40536. E-mail address: ljcrof2{at}email.uky.edu Back

3 Abbreviations used in this paper: mPGES, microsomal PGE synthase; CIA, collagen-induced arthritis; CII, type II collagen; COX, cyclooxygenase; Het, heterozygous; RA, rheumatoid arthritis. Back

Received for publication December 4, 2007. Accepted for publication April 7, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Jakobsson, P. J., S. Thoren, R. Morgenstern, B. Samuelsson. 1999. Identification of human prostaglandin E synthase: a microsomal, glutathione-dependent, inducible enzyme, constituting a potential novel drug target. Proc. Natl. Acad. Sci. USA 96: 7220-7225. [Abstract/Free Full Text]
  2. Murakami, M., H. Naraba, T. Tanioka, N. Semmyo, Y. Nakatani, F. Kojima, T. Ikeda, M. Fueki, A. Ueno, S. Oh, I. Kudo. 2000. Regulation of prostaglandin E2 biosynthesis by inducible membrane-associated prostaglandin E2 synthase that acts in concert with cyclooxygenase-2. J. Biol. Chem. 275: 32783-32792. [Abstract/Free Full Text]
  3. Uematsu, S., M. Matsumoto, K. Takeda, S. Akira. 2002. Lipopolysaccharide-dependent prostaglandin E2 production is regulated by the glutathione-dependent prostaglandin E2 synthase gene induced by the Toll-like receptor 4/MyD88/NF-IL6 pathway. J. Immunol. 168: 5811-5816. [Abstract/Free Full Text]
  4. Kapoor, M., F. Kojima, M. Qian, L. Yang, L. J. Crofford. 2006. Shunting of prostanoid biosynthesis in microsomal prostaglandin E synthase-1 null embryo fibroblasts: regulatory effects on inducible nitric oxide synthase expression and nitrite synthesis. FASEB J. 20: 2387-2389. [Abstract/Free Full Text]
  5. Kapoor, M., F. Kojima, M. Qian, L. Yang, L. J. Crofford. 2007. Microsomal prostaglandin E synthase-1 deficiency is associated with elevated peroxisome proliferator-activated receptor {gamma}: regulation by prostaglandin E2 via the phosphatidylinositol 3-kinase and Akt pathway. J. Biol. Chem. 282: 5356-5366. [Abstract/Free Full Text]
  6. Trebino, C. E., J. D. Eskra, T. S. Wachtmann, J. R. Perez, T. J. Carty, L. P. Audoly. 2005. Redirection of eicosanoid metabolism in mPGES-1-deficient macrophages. J. Biol. Chem. 280: 16579-16585. [Abstract/Free Full Text]
  7. Boulet, L., M. Ouellet, K. P. Bateman, D. Ethier, M. D. Percival, D. Riendeau, J. A. Mancini, N. Methot. 2004. Deletion of microsomal prostaglandin E2 (PGE2) synthase-1 reduces inducible and basal PGE2 production and alters the gastric prostanoid profile. J. Biol. Chem. 279: 23229-23237. [Abstract/Free Full Text]
  8. Trebino, C. E., J. L. Stock, C. P. Gibbons, B. M. Naiman, T. S. Wachtmann, J. P. Umland, K. Pandher, J. M. Lapointe, S. Saha, M. L. Roach, et al 2003. Impaired inflammatory and pain responses in mice lacking an inducible prostaglandin E synthase. Proc. Natl. Acad. Sci. USA 100: 9044-9049. [Abstract/Free Full Text]
  9. Mabuchi, T., H. Kojima, T. Abe, K. Takagi, M. Sakurai, Y. Ohmiya, S. Uematsu, S. Akira, K. Watanabe, S. Ito. 2004. Membrane-associated prostaglandin E synthase-1 is required for neuropathic pain. NeuroReport 15: 1395-1398. [Medline]
  10. Kamei, D., K. Yamakawa, Y. Takegoshi, M. Mikami-Nakanishi, Y. Nakatani, S. Oh-Ishi, H. Yasui, Y. Azuma, N. Hirasawa, K. Ohuchi, et al 2004. Reduced pain hypersensitivity and inflammation in mice lacking microsomal prostaglandin E synthase-1. J. Biol. Chem. 279: 33684-33695. [Abstract/Free Full Text]
  11. Ikeda-Matsuo, Y., A. Ota, T. Fukada, S. Uematsu, S. Akira, Y. Sasaki. 2006. Microsomal prostaglandin E synthase-1 is a critical factor of stroke-reperfusion injury. Proc. Natl. Acad. Sci. USA 103: 11790-11795. [Abstract/Free Full Text]
  12. Kamei, D., M. Murakami, Y. Nakatani, Y. Ishikawa, T. Ishii, I. Kudo. 2003. Potential role of microsomal prostaglandin E synthase-1 in tumorigenesis. J. Biol. Chem. 278: 19396-19405. [Abstract/Free Full Text]
  13. Engblom, D., S. Saha, L. Engstrom, M. Westman, L. P. Audoly, P. J. Jakobsson, A. Blomqvist. 2003. Microsomal prostaglandin E synthase-1 is the central switch during immune-induced pyresis. Nat. Neurosci. 6: 1137-1138. [Medline]
  14. Saha, S., L. Engstrom, L. Mackerlova, P. J. Jakobsson, A. Blomqvist. 2005. Impaired febrile responses to immune challenge in mice deficient in microsomal prostaglandin E synthase-1. Am. J. Physiol. 288: R1100-R1107.
  15. Wang, M., A. M. Zukas, Y. Hui, E. Ricciotti, E. Pure, G. A. FitzGerald. 2006. Deletion of microsomal prostaglandin E synthase-1 augments prostacyclin and retards atherogenesis. Proc. Natl. Acad. Sci. USA 103: 14507-14512. [Abstract/Free Full Text]
  16. Kubota, K., T. Kubota, D. Kamei, M. Murakami, I. Kudo, T. Aso, I. Morita. 2005. Change in prostaglandin E synthases (PGESs) in microsomal PGES-1 knockout mice in a preterm delivery model. J. Endocrinol. 187: 339-345. [Abstract/Free Full Text]
  17. Inada, M., C. Matsumoto, S. Uematsu, S. Akira, C. Miyaura. 2006. Membrane-bound prostaglandin E synthase-1-mediated prostaglandin E2 production by osteoblast plays a critical role in lipopolysaccharide-induced bone loss associated with inflammation. J. Immunol. 177: 1879-1885. [Abstract/Free Full Text]
  18. Egg, D., R. Gunther, M. Herold, F. Kerschbaumer. 1980. Prostaglandins E2 and F2 {alpha} concentrations in the synovial fluid in rheumatoid and traumatic knee joint diseases. Z. Rheumatol. 39: 170-175. [Medline]
  19. Stichtenoth, D. O., S. Thoren, H. Bian, M. Peters-Golden, P. J. Jakobsson, L. J. Crofford. 2001. Microsomal prostaglandin E synthase is regulated by proinflammatory cytokines and glucocorticoids in primary rheumatoid synovial cells. J. Immunol. 167: 469-474. [Abstract/Free Full Text]
  20. Kojima, F., H. Naraba, Y. Sasaki, R. Okamoto, T. Koshino, S. Kawai. 2002. Coexpression of microsomal prostaglandin E synthase with cyclooxygenase-2 in human rheumatoid synovial cells. J. Rheumatol. 29: 1836-1842. [Abstract/Free Full Text]
  21. Murakami, M., K. Nakashima, D. Kamei, S. Masuda, Y. Ishikawa, T. Ishii, Y. Ohmiya, K. Watanabe, I. Kudo. 2003. Cellular prostaglandin E2 production by membrane-bound prostaglandin E synthase-2 via both cyclooxygenases-1 and -2. J. Biol. Chem. 278: 37937-37947. [Abstract/Free Full Text]
  22. Westman, M., M. Korotkova, E. af Klint, A. Stark, L. P. Audoly, L. Klareskog, A. K. Ulfgren, P. J. Jakobsson. 2004. Expression of microsomal prostaglandin E synthase 1 in rheumatoid arthritis synovium. Arthritis Rheum. 50: 1774-1780. [Medline]
  23. Seki, N., Y. Sudo, T. Yoshioka, S. Sugihara, T. Fujitsu, S. Sakuma, T. Ogawa, T. Hamaoka, H. Senoh, H. Fujiwara. 1988. Type II collagen-induced murine arthritis. I. Induction and perpetuation of arthritis require synergy between humoral and cell-mediated immunity. J. Immunol. 140: 1477-1484. [Abstract]
  24. Corthay, A., A. Johansson, M. Vestberg, R. Holmdahl. 1999. Collagen-induced arthritis development requires {alpha} β T cells but not {gamma} {delta} T cells: studies with T cell-deficient (TCR mutant) mice. Int. Immunol. 11: 1065-1073. [Abstract/Free Full Text]
  25. Svensson, L., J. Jirholt, R. Holmdahl, L. Jansson. 1998. B cell-deficient mice do not develop type II collagen-induced arthritis (CIA). Clin. Exp. Immunol. 111: 521-526. [Medline]
  26. Clague, R. B., L. J. Moore. 1984. IgG and IgM antibody to native type II collagen in rheumatoid arthritis serum and synovial fluid: evidence for the presence of collagen-anticollagen immune complexes in synovial fluid. Arthritis Rheum. 27: 1370-1377. [Medline]
  27. Tarkowski, A., L. Klareskog, H. Carlsten, P. Herberts, W. J. Koopman. 1989. Secretion of antibodies to types I and II collagen by synovial tissue cells in patients with rheumatoid arthritis. Arthritis Rheum. 32: 1087-1092. [Medline]
  28. Cook, A. D., M. J. Rowley, I. R. Mackay, A. Gough, P. Emery. 1996. Antibodies to type II collagen in early rheumatoid arthritis: correlation with disease progression. Arthritis Rheum. 39: 1720-1727. [Medline]
  29. Cook, A. D., A. Stockman, C. A. Brand, B. D. Tait, I. R. Mackay, K. D. Muirden, C. C. Bernard, M. J. Rowley. 1999. Antibodies to type II collagen and HLA disease susceptibility markers in rheumatoid arthritis. Arthritis Rheum. 42: 2569-2576. [Medline]
  30. Cook, A. D., R. Gray, J. Ramshaw, I. R. Mackay, M. J. Rowley. 2004. Antibodies against the CB10 fragment of type II collagen in rheumatoid arthritis. Arthritis Res. Ther. 6: R477-R483. [Medline]
  31. Golde, W. T., P. Gollobin, L. L. Rodriguez. 2005. A rapid, simple, and humane method for submandibular bleeding of mice using a lancet. Lab. Anim. 34: 39-43.
  32. Watson, W. C., A. S. Townes. 1985. Genetic susceptibility to murine collagen II autoimmune arthritis: proposed relationship to the IgG2 autoantibody subclass response, complement C5, major histocompatibility complex (MHC) and non-MHC loci. J. Exp. Med. 162: 1878-1891. [Abstract/Free Full Text]
  33. Williams, P. J., R. H. Jones, T. W. Rademacher. 1998. Correlation between IgG anti-type II collagen levels and arthritic severity in murine arthritis. Autoimmunity 27: 201-207. [Medline]
  34. Myers, L. K., A. H. Kang, A. E. Postlethwaite, E. F. Rosloniec, S. G. Morham, B. V. Shlopov, S. Goorha, L. R. Ballou. 2000. The genetic ablation of cyclooxygenase 2 prevents the development of autoimmune arthritis. Arthritis Rheum. 43: 2687-2693. [Medline]
  35. Ochi, T., Y. Ohkubo, S. Mutoh. 2003. Role of cyclooxygenase-2, but not cyclooxygenase-1, on type II collagen-induced arthritis in DBA/1J mice. Biochem. Pharmacol. 66: 1055-1060. [Medline]
  36. Honda, T., E. Segi-Nishida, Y. Miyachi, S. Narumiya. 2006. Prostacyclin-IP signaling and prostaglandin E2-EP2/EP4 signaling both mediate joint inflammation in mouse collagen-induced arthritis. J. Exp. Med. 203: 325-335. [Abstract/Free Full Text]
  37. McCoy, J. M., J. R. Wicks, L. P. Audoly. 2002. The role of prostaglandin E2 receptors in the pathogenesis of rheumatoid arthritis. J. Clin. Invest. 110: 651-658. [Medline]
  38. van Vollenhoven, R. F., C. Nagler-Anderson, A. Soriano, G. W. Siskind, G. J. Thorbecke. 1988. Tolerance induction by a poorly arthritogenic collagen II can prevent collagen-induced arthritis. Cell. Immunol. 115: 146-155. [Medline]
  39. Crofford, L. J., B. Tan, C. J. McCarthy, T. Hla. 1997. Involvement of nuclear factor {kappa}B in the regulation of cyclooxygenase-2 expression by interleukin-1 in rheumatoid synoviocytes. Arthritis Rheum. 40: 226-236. [Medline]
  40. Naraba, H., C. Yokoyama, N. Tago, M. Murakami, I. Kudo, M. Fueki, S. Oh-Ishi, T. Tanabe. 2002. Transcriptional regulation of the membrane-associated prostaglandin E2 synthase gene: essential role of the transcription factor Egr-1. J. Biol. Chem. 277: 28601-28608. [Abstract/Free Full Text]
  41. Subbaramaiah, K., K. Yoshimatsu, E. Scherl, K. M. Das, K. D. Glazier, D. Golijanin, R. A. Soslow, T. Tanabe, H. Naraba, A. J. Dannenberg. 2004. Microsomal prostaglandin E synthase-1 is overexpressed in inflammatory bowel disease: evidence for involvement of the transcription factor Egr-1. J. Biol. Chem. 279: 12647-12658. [Abstract/Free Full Text]
  42. Roper, R. L., D. H. Conrad, D. M. Brown, G. L. Warner, R. P. Phipps. 1990. Prostaglandin E2 promotes IL-4-induced IgE and IgG1 synthesis. J. Immunol. 145: 2644-2651. [Abstract]
  43. Garrone, P., L. Galibert, F. Rousset, S. M. Fu, J. Banchereau. 1994. Regulatory effects of prostaglandin E2 on the growth and differentiation of human B lymphocytes activated through their CD40 antigen. J. Immunol. 152: 4282-4290. [Abstract]
  44. Stein, S. H., R. P. Phipps. 1992. Anti-class II antibodies potentiate IgG2a production by lipopolysaccharide-stimulated B lymphocytes treated with prostaglandin E2 and IFN-{gamma}. J. Immunol. 148: 3943-3949. [Abstract]
  45. Stein, S. H., R. P. Phipps. 1991. Antigen-specific IGG2A production in response to prostaglandin E2, immune complexes, and IFN-{gamma}. J. Immunol. 147: 2500-2506. [Abstract/Free Full Text]
  46. Phipps, R. P., S. H. Stein, R. L. Roper. 1991. A new view of prostaglandin E regulation of the immune response. Immunol. Today 12: 349-352. [Medline]
  47. Kabashima, K., D. Sakata, M. Nagamachi, Y. Miyachi, K. Inaba, S. Narumiya. 2003. Prostaglandin E2-EP4 signaling initiates skin immune responses by promoting migration and maturation of Langerhans cells. Nat. Med. 9: 744-749. [Medline]
  48. Roper, R. L., B. Graf, R. P. Phipps. 2002. Prostaglandin E2 and cAMP promote B lymphocyte class switching to IgG1. Immunol. Lett. 84: 191-198. [Medline]
  49. Ryan, E. P., S. J. Pollock, T. I. Murant, S. H. Bernstein, R. E. Felgar, R. P. Phipps. 2005. Activated human B lymphocytes express cyclooxygenase-2 and cyclooxygenase inhibitors attenuate antibody production. J. Immunol. 174: 2619-2626. [Abstract/Free Full Text]
  50. Shibata, Y., R. A. Henriksen, I. Honda, R. M. Nakamura, Q. N. Myrvik. 2005. Splenic PGE2-releasing macrophages regulate Th1 and Th2 immune responses in mice treated with heat-killed BCG. J. Leukocyte Biol. 78: 1281-1290. [Abstract/Free Full Text]
  51. Sheibanie, A. F., T. Khayrullina, F. F. Safadi, D. Ganea. 2007. Prostaglandin E2 exacerbates collagen-induced arthritis in mice through the inflammatory interleukin-23/interleukin-17 axis. Arthritis Rheum. 56: 2608-2619. [Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kojima, F.
Right arrow Articles by Crofford, L. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kojima, F.
Right arrow Articles by Crofford, L. J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS