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Originally published online as doi:10.1189/jlb.0504311 on January 18, 2005

Published online before print January 18, 2005
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(Journal of Leukocyte Biology. 2005;77:544-551.)
© 2005 by Society for Leukocyte Biology

Differential effects of IL-10 on prostaglandin H synthase-2 expression and prostaglandin E2 biosynthesis between spleen and bone marrow macrophages

Yoshimi Shibata*,1, Akihito Nishiyama*, Hiroyoshi Ohata*, Jon Gabbard*, Quentin N. Myrvik{dagger} and Ruth Ann Henriksen{ddagger}

* Department of Biomedical Sciences, Florida Atlantic University, Boca Raton;
{ddagger} Department of Physiology, Brody School of Medicine at East Carolina University, Greenville, North Carolina; and
{dagger} Myrvik Enterprises, Southport, North Carolina

1 Correspondence: Department of Biomedical Sciences, Florida Atlantic University, 777 Glades Rd., P.O. Box 3091, Boca Raton, FL 33431-0991. E-mail: yshibata{at}fau.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
Different populations of mononuclear phagocytes (MØ) show considerable diversity of cellular function including prostaglandin E2 (PGE2) biosynthesis. Certain bacterial components enhance PGE2 biosynthesis differentially in selected populations of MØ. Interleukin (IL)-10 is proposed to inhibit modulation of PGE2 biosynthesis by down-regulating prostaglandin G/H synthase-2 (PGHS-2) expression. To assess whether IL-10 regulates PGE2 biosynthesis and PGHS-2 expression, splenic and bone marrow MØ were isolated from IL-10-deficient (IL-10–/–), C57Bl/6 [wild-type (WT) control], and Balb/c (comparison control) mice and were treated with lipopolysaccharide (LPS) and/or interferon-{gamma} (IFN-{gamma}) as a model of bacterial inflammation. LPS-induced PGHS-2 expression was similar for splenic MØ isolated from the three strains of mice. However, PGE2 released by LPS-treated splenic MØ was significantly higher in IL-10–/– and Balb/c than in WT cells. In the presence of LPS and IFN-{gamma}, PGHS-2 expression and PGE2 release by IL-10–/– and Balb/c splenic MØ were enhanced compared with stimulation with LPS alone or IFN-{gamma} alone. However, there was no significant increase in PGE2 release from WT splenic MØ treated with LPS plus IFN-{gamma} despite increased PGHS-2 expression. In sharp contrast, PGHS-2 expression and PGE2 release by bone marrow MØ were greatly enhanced in IL-10–/– cells compared with control cells. Our results indicate that IL-10 regulation of MØ PGE2 biosynthesis and PGHS-2 expression is compartment-dependent and that PGE2 production is not linked directly to PGHS-2 levels. Furthermore, our findings emphasize strain-specific differences between C57Bl/6 and Balb/c mice, and Balb/c appears more similar to the IL-10–/– than to the C57Bl/6 with respect to prostanoid production.

Key Words: PGE2 • PGHS-2 • splenic macrophages • marrow macrophages • LPS


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
Mononuclear phagocytes (MØ) are a major source of prostaglandin E2 (PGE2), an arachidonic acid (AA) metabolite that regulates the immune response, hematopoiesis, inflammation, tissue injury and repair, and bone resorption. The regulation of these events may be closely related to the regulation of PGE2 release by MØ [1 ]. The effective in vivo expression of these responses, furthermore, may depend on the presence of an adequate number of MØ with appropriate functions in specialized locations [2 , 3 ]. We are interested in the lymphoid tissue of spleens where PGE2-releasing MØ (PGE2-MØ) and immune lymphocytes interact in chronic inflammatory diseases including mycobacterial infections. PGE2 inhibits the production of T helper cell type 1 (Th1) cytokines, such as interleukin (IL)-2, IL-12, and interferon-{gamma} (IFN-{gamma}) [4 ]. In contrast, PGE2, depending on stimulatory conditions, has no effect or enhances production of Th2 cytokines, such as IL-4, IL-5, and IL-10 [4 , 5 ]. Therefore, increases in splenic PGE2-MØ may underlie the Th1-to-Th2 shift of immune responses, which are major pathogenic events in chronic inflammatory diseases. More studies are needed to elucidate the mechanisms of splenic PGE2 production and splenic PGE2-MØ formation.

Normal splenic MØ, unlike peritoneal and bone marrow MØ or monocytes, produce relatively low levels of PGE2 (<5 ng PGE2/5x106 MØ/ml) [6 ]. However, previous studies [7 ] have shown that in vivo responses to chronic inflammatory conditions, including mycobacterial infection, are manifested by the emergence of splenic MØ with the capacity to form large amounts of PGE2 (>50 ng/ml). The mechanism for splenic PGE2-MØ formation appears to be complex. Our studies have shown that their formation is dependent on radiosensitive bone marrow cells, which may supply precursors of splenic PGE2-MØ [2 ]. It is likely that the precursors migrate and localize to the spleen, where mature forms of PGE2-MØ are established [6 ]. Alternatively, an inflammatory cytokine "milieu" may directly up-modulate PGE2 biosynthesis by splenic MØ [8 , 9 ].

PGE2-MØ metabolize endogenous AA to PGE2 through cyclooxygenase [prostaglandin H synthase (PGHS), EC 1.4.99.1], rate-limiting enzymes for prostaglandin, and thromboxane production. Two major isoforms of PGHS exist: PGHS-1, a constitutive form, and PGHS-2, an inducible form, which is rapidly up-regulated in response to lipopolysaccharide (LPS) and proinflammatory cytokines [10 11 12 ]. The formation of splenic PGE2-MØ is expected, therefore, to be dependent on the level and activity of PGHS-2 induced [8 ].

Cytokines, which are present in pro- and anti-inflammatory conditions, modulate PGE2 synthesis by MØ cell lines [13 ], MØ freshly isolated from the peritonea, and lungs or those derived from monocytes [8 , 11 , 12 , 14 ]. Tumor necrosis factor {alpha} (TNF-{alpha}) [15 ], IL-1{alpha} [15 , 16 ], and IFN-{gamma} [17 , 18 ] have been demonstrated to induce PGHS-2 expression, whereas IL-4 [19 ], IL-13 [20 , 21 ], IL-10 [22 ], and transforming growth factor-ß [23 ] can inhibit PGHS-2 induction. The question addressed in these studies is whether the level of PGHS-2 expression under selected inflammatory conditions is dependent on IL-10 and directly correlated with PGE2 biosynthesis in splenic MØ.

IL-10 is a MØ deactivator, blocking LPS-induced synthesis of TNF-{alpha}, IL-1ß, IL-6, IL-8, IL-12, and granulocyte macrophage-colony stimulating factor by human monocytes [24 ], mouse peritoneal MØ [25 ], and mouse splenic MØ [26 ]. It has been reported that IL-10 can inhibit PGHS-2 induction in vitro in human monocytes and neutrophils [19 , 22 ]. Recently, Berg et al. [8 ] demonstrated that LPS induces normal murine spleen MØ to express PGHS-2 and synthesize PGE2, which are down-regulated by endogenous IL-10. Our results with IL-10–/– MØ show that in the presence of LPS, IL-10 down-regulates PGE2 synthesis without an equivalent effect on PGHS-2 expression.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
Animals
Healthy 8- to 12-week-old IL-10–/– female mice on C57Bl/6 background were obtained from Jackson Laboratories (Bar Harbor, ME). Wild-type (WT) C57Bl/6 and Balb/c female mice were obtained from Jackson Laboratories. All mice were maintained in microisolator cages under specific, pathogen-free conditions in the animal care facility at East Carolina University (Greenville, NC) and Florida Atlantic University (Boca Raton).

Reagents
LPS from Escherichia coli (serotype 0111:B4, phenol extraction, L-2630) and mouse recombinant IFN-{gamma} (I-4777) were obtained from Sigma Chemical Co. (St. Louis, MO) and reconstituted in pyrogen-free saline. Recombinant murine IL-10 was obtained from PharMingen (San Diego, CA). A23187 (Sigma Chemical Co.) was dissolved in dimethyl sulfoxide at 1 mg/ml. AA (Sigma Chemical Co.) was dissolved in 100% ethanol at 1 mg/ml. Rabbit polyclonal anti-murine PGHS-1 and rabbit polyclonal anti-PGHS-2 were obtained from Cayman Chemicals (Ann Arbor, MI).

MØ preparations from spleen and bone marrow
Spleens in each group of mice (at least five mice per group) were isolated and pooled. Excised spleens were minced with scissors, digested with 50 U/ml collagenase D (C-2139, Sigma Chemical Co.) in RPMI 1640 plus 10% fetal bovine serum (FBS) at 37°C for 60 min, and filtered through a 100-µm mesh. After washing digested cells with RPMI 1640 in the presence of 100 µg/ml DNase (DN-25, Sigma Chemical Co.), cells were suspended in RPMI 1640 plus 10% FBS. Bone marrow cells were isolated by flushing the marrow cavities of the femurs with ice-cold RPMI 1640 and gently refluxing the expelled cell plug with a Pasteur pipette to form a single-cell suspension. To enrich MØ fraction [2 , 6 ], spleen cell and bone marrow cell suspensions were layered over a discontinuous Percoll gradient (35/60%, Sigma Chemical Co.). Following centrifugation (800 g for 30 min at 22°C), cells in the layer between 35% and 60% Percoll were collected, washed, and suspended in RPMI 1640 plus 10% FBS. These cells were plated at 3–5 x 106 cells/ml per 35 mm culture dish (Falcon, Oxnard, CA) and incubated at 37°C in 5% CO2 in air. After 2 h incubation, the cells were washed with Ca2+- and Mg2+-free 0.15 M phosphate-buffered saline for removal of the nonadherent cells and held on ice for 30 min. The adherent cells were harvested by scraping and were washed twice with serum-free RPMI 1640. Viability was >90% (trypan blue exclusion). Adherent spleen cells were >85%, and adherent marrow cells were >92% MØ, estimated by phagocytosis of immunoglobulin G (IgG)-opsonized sheep red cells [2 ] and/or cytometrically following staining with antimembrane-activated complex 1 [27 ].

Cell culture protocols
Splenic MØ and bone marrow MØ were cultured at 5 x 106 cells/ml in RPMI 1640 supplemented with 5% FBS, penicillin (100 U/ml), streptomycin (100 U/ml), and amphotericin B (2.5 µg/ml) in 12 x 75 mm culture tubes (0.5 ml/tube, Costar, Corning, NY). Cells were incubated in medium alone or medium supplemented with LPS (10 µg/ml), IFN-{gamma} (10 ng/ml), or LPS plus IFN-{gamma} mixture (both at same final concentrations). Where indicated, exogenous IL-10, at 0.1, 1, or 10 ng/ml, was added to the culture. After 24 h, the cells were washed with cold saline and treated with lysis buffer as described below. To elicit PGE2 release, the cultured cells were washed, suspended in serum-free RPMI 1640, and incubated with agonists (A23187, AA, or LPS) at 37°C. Supernatants from triplicate cultures were harvested after 2 h and stored at –70°C before analysis for PGE2.

Quantification of PGE2 and thromboxane B2 (TxB2)
PGE2 and TxB2 levels in tissue-culture supernatants were determined using enzyme immunoassay kits (Cayman Chemicals) according to the manufacturer’s instructions.

Western blotting
Splenic and bone marrow MØ were cultured as described above, harvested, and washed three times with cold saline. Washed cells were suspended in lysis buffer [50 mM Tris (pH 7.5), 150 mM NaCl, Sigma protease inhibitor cocktail (1:500, P8340, Sigma Chemical Co.), 1 % Nonidet P-40, and 1% sodium deoxycholate]. Debris was eliminated by centrifugation (5 min, 1000 g). Protein concentration was measured using a commercial reagent based on bicinchoninic acid staining (Pierce, Rockford, IL) using bovine serum albumin as standard. Equal amounts of cellular protein were loaded onto sodium dodecyl sulfate-polyacrylamide gel and separated by electrophoresis (200 V for 45 min). Proteins were transferred to polyvinylidene difluoride membrane, and the membrane was blocked with 5% nonfat dry milk. The membrane was then incubated with antibody (anti-PGHS-1, 1:1000; anti-PGHS-2, 1:4000) overnight at 4°C. Following incubation with peroxidase-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch, West Glove, PA), proteins were detected by chemiluminescence (Amersham, Piscataway, NJ) following the manufacturer’s instructions.

Statistics
Data were analyzed by one-way ANOVA. For cell culture studies, tissues isolated from at least five mice were pooled unless indicated; these cells were cultured in at least triplicate in each group. P value of less than 0.05 is considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 CONCLUSION
 REFERENCES
 
PGE2 release by splenic MØ isolated from IL-10–/–, C57Bl/6 (WT), and Balb/c mice
Normal murine splenic MØ release PGE2 at minimum levels compared with bone marrow MØ [3 , 6 ]. To determine whether endogenous IL-10 regulates PGE2 released by splenic MØ in response to the proinflammatory stimuli LPS and/or IFN-{gamma}, splenic MØ were isolated from C57Bl/6 (WT) and IL-10–/– mice. Recently, Kuroda and Yamashita [9 ] found differential release of PGE2 by splenic MØ isolated from WT and Balb/c mice when stimulated by LPS in vitro. We, therefore, used splenic MØ from Balb/c mice as comparison controls. The MØ were primed with LPS (10 µg/ml), IFN-{gamma} (10 ng/ml), LPS combined with IFN-{gamma} (at same final concentrations), or medium alone for 24 h, followed by the elicitation of PGE2 release with 1 µM calcium ionophore A23187 for 2 h. As shown in Figure 1 , PGE2 release was slightly but significantly enhanced in Balb/c and IL-10–/– mice compared with WT when splenic MØ were primed with LPS. This enhancement was eliminated when 1 µM NS-398 was added to the LPS-primed cultures (data not shown). Although IFN-{gamma} did not prime PGE2 biosynthesis by itself, it showed a synergistic effect in the presence of LPS. Priming effects of LPS and IFN-{gamma} were not observed in splenic MØ from WT mice (Fig. 1) .



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  		Figure 1. The effects of endogenous IL-10 on PGE2 biosynthesis in splenic and bone marrow MØ (5x106 MØ/ml), which were isolated from IL-10–/–, WT (C57Bl/6), and Balb/c mice, and primed with LPS (10 µg/ml), IFN-{gamma} (10 ng/ml), LPS mixed with IFN-{gamma} (at same final concentrations), or medium alone for 24 h. These MØ were elicited by 1 µM A23187 for 2 h, and PGE2 levels in supernatants were measured by enzyme-linked immunosorbent assay (ELISA). Mean ± SD, n = 3. *, P < 0.05; **, P < 0.01; #, P < 0.001, compared with those of WT (C57Bl/6) mice. Results are representative of two separate experiments. KO, Knockout.

 
PGE2 release by bone marrow MØ isolated from IL-10–/–, C57Bl/6 (WT), and Balb/c mice
When elicited with 1 µM A23187, bone marrow MØ from Balb/c, WT, or IL-10–/– mice released tenfold more PGE2 than normal splenic MØ (Fig. 1) . For the control cells, Balb/c, or WT, PGE2 release was not increased when bone marrow MØ were primed with LPS or IFN-{gamma} alone, although LPS plus IFN-{gamma} significantly enhanced PGE2 release in Balb/c mice. In sharp contrast, bone marrow MØ from IL-10–/– mice showed tenfold increases in PGE2 release in response to LPS alone, without further enhancement by the mixture of IFN-{gamma} and LPS (Fig. 1) . Elicitation with 1 µg/ml AA also showed similar profiles of PGE2 release but at lower levels than those elicited by A23187 (Table 1 ). However, the elicitation by 1 µg/ml LPS did not significantly enhance PGE2 production compared with that by buffer alone in any of the primed groups (Table 1) . This may be a consequence of the experimental conditions, which resulted in the cells becoming refractory to the lower concentration of LPS used for elicitation after the long period of priming at higher LPS.


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  		Table 1. PGE2 Release by Primed Bone Marrow M{cjs0625} That Were Treated with A23187, AA, or LPS

 
Thromboxane release by MØ
To determine whether the effects observed for PGE2 production were general for all prostanoids, TxB2 levels were also determined in MØ supernatants. For splenic MØ pretreated with LPS and/or IFN-{gamma}, there was no increase in TxB2 release following pretreatment with LPS and/or IFN-{gamma} compared with medium alone (Fig. 2 ). Furthermore, as shown in Figure 2 , bone marrow MØ from C57Bl/6 and Balb/c mice released TxB2 at significantly higher levels compared with those from splenic MØ, similarly to what was observed for PGE2 (Fig. 1) . However, priming with LPS or LPS combined with IFN-{gamma} did not result in increases in TxB2 release but tended to suppress thromboxane production (Fig. 2) . Our results further demonstrate that endogenous IL-10 has no significant effect on thromboxane production by splenic or bone marrow MØ (Fig. 2) .



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  		Figure 2. The effects of endogenous IL-10 on TxB2 release by splenic and bone marrow MØ (5x106 MØ/ml), which were isolated from IL-10–/– and Balb/c mice, and primed with LPS (10 µg/ml), IFN-{gamma} (10 ng/ml), LPS mixed with IFN-{gamma} (at same final concentrations), or medium alone for 24 h. These MØ were elicited by 1 µM A23187 for 2 h, and TxB2 levels in the supernatants were measured by ELISA. Mean ± SD, n = 3. *, P < 0.05; **, P < 0.01, compared with those of medium-treated cells. Results are representative of two separate experiments.

 
Effect of endogenous IL-10 on protein expression of PGHS-1 and PGHS-2
To determine whether endogenous IL-10 alters the level of protein expression of PGHS-1 or PGHS-2, Western blot analyses for PGHS-1 and PGHS-2 were performed. Untreated splenic MØ isolated from Balb/c, WT, and IL-10–/– mice showed PGHS-1 expression, and the levels were relatively stable following treatment with IFN-{gamma} and/or LPS (Fig. 3 ). Conversely, PGHS-2 protein in untreated splenic MØ was undetectable. Stimulation of splenic MØ with LPS resulted in marked increases in PGHS-2 protein at comparable levels among the three strains of mice. The mixture of LPS and IFN-{gamma} further enhanced PGHS-2 levels in all strains, although the increase was greater in Balb/c than in WT and IL-10–/– (Fig. 3) .



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  		Figure 3. PGHS-1 and PGHS-2 levels in splenic MØ that were isolated from IL-10–/–, WT, and Balb/c mice and primed with LPS and/or IFN-{gamma} in vitro. Each lane was loaded with 5 µg total protein. Results are representative of three separate experiments.

 
As for spleen MØ, PGHS-2 was not detected in untreated bone marrow MØ. Bone marrow MØ from Balb/c and WT mice, also showed increased PGHS-2 levels in response to LPS (Fig. 4 ). These levels were further enhanced when LPS was combined with IFN-{gamma} (Fig. 4) . In IL-10–/– mice, PGHS-2 levels induced by LPS or LPS plus IFN-{gamma} were further enhanced compared with WT or Balb/c mice (Fig. 4) . Bone marrow MØ PGHS-1 expression was unchanged among the treatment groups as well as among the three mouse strains (Fig. 4) .



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  		Figure 4. PGHS-1 and PGHS-2 levels in bone marrow MØ that were isolated from IL-10–/–, WT, and Balb/c mice and primed with LPS and/or IFN-{gamma} in vitro. Each lane was loaded with 5 µg total protein. Results are representative of three separate experiments.

 
Effects of exogenous IL-10 on PGHS-2 expression and PGE2 synthesis
To determine whether the effects observed in IL-10–/– cells could be reversed by addition of exogenous IL-10, splenic and bone marrow MØ were isolated from WT mice and primed with LPS plus IFN-{gamma}. In the absence of added IL-10, the culture supernatants of splenic and bone marrow MØ contained IL-10 at 121 and 267 pg/ml/5 x 106 MØ, respectively. Neither splenic nor bone marrow MØ isolated from IL-10–/– and primed with LPS plus IFN-{gamma} produced IL-10 (<25 pg/ml). Exogenous IL-10, at 0.1, 1, or 10 ng/ml, was added to the IL-10–/– MØ cultures. In splenic MØ, 10 ng/ml, but not 0.1 or 1 ng/ml IL-10, inhibited PGHS-2 protein expression and PGE2 synthesis. In sharp contrast, for bone marrow MØ from IL-10–/– mice, IL-10 at these concentrations inhibited PGE2 synthesis and PGHS-2 expression in a dose-dependent manner (see Fig. 6 ). As shown above (Fig. 1) , when WT splenic or marrow MØ were primed with LPS plus IFN-{gamma}, PGE2 synthesis was not increased. Exogenous IL-10 did not inhibit PGE2 synthesis by WT splenic or marrow MØ (Figs. 5 and 6 ), although IL-10 at 10 ng/ml inhibited PGHS-2 expression in these MØ. Finally, exogenous IL-10, like endogenous IL-10, did not inhibit PGHS-1 protein expression by splenic and marrow MØ (Figs. 3 4 5 6) . Taken together, our results indicate that the inhibitory effects of IL-10 on PGE2 synthesis in inflammation are significant in splenic and marrow MØ, but the magnitude of the IL-10 effect is greater in bone marrow MØ than in splenic MØ.



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  		Figure 6. The effects of exogenous IL-10 on PGHS-2 expression and PGE2 synthesis in bone marrow MØ (5x106 MØ/ml), which were isolated from IL-10–/– and WT mice, and used. All other procedures were identical to those in Figure 5 . Mean ± SD, n = 3–4. *, P < 0.05; #, P < 0.001; ##, P < 0.001, compared with those of LPS + IFN-{gamma} alone.

 


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  		Figure 5. The effects of exogenous IL-10 on PGHS-2 expression and PGE2 synthesis in splenic MØ (5x106 MØ/ml), which were isolated from IL-10–/– and WT mice, and primed with LPS (10 µg/ml) mixed with IFN-{gamma} (10 ng/ml) in the presence of 0 (medium), 0.01, 1, or 10 ng/ml murine IL-10 for 24 h. PGHS-1 and PGHS-2 levels in these MØ were determined by Western blotting. Results are representative of two separate experiments. The primed MØ were elicited by 1 µM A23187 for 2 h, and PGE2 levels in the supernatants were measured by ELISA. Mean ± SD, n = 4. #, P < 0.001, compared with those of LPS + IFN-{gamma} alone. Similar results were obtained in a separate experiment.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
CONCLUSION
 REFERENCES
 
Regulation of PGE2 production
MØ are a major source of PGE2, a key mediator regulating inflammatory responses [28 29 30 31 32 33 ]. Heterogeneity of PGE2 biosynthesis among MØ populations has been well documented [3 , 34 ]. Normal splenic MØ express relatively low levels of PGE2 (<5 ng PGE2/5x106 MØ/ml). However, 7–14 days after C57Bl/6 and CBA/J mice are challenged with mycobacteria or related components, splenic MØ show a tenfold increase in PGE2 release (>50 ng/ml) [2 , 3 , 6 ]. Exact mechanisms for splenic PGE2-MØ formation are still unknown. The present in vitro studies examined PGE2 biosynthesis in MØ treated with LPS and/or IFN-{gamma}, proinflammatory agents used to simulate conditions present in bacterial inflammation. We have investigated the role of IL-10 in regulating in vitro PGE2 production by splenic and bone marrow MØ with cells derived from IL-10–/– and two control strains of mice. As the IL-10–/– mice are on a C57Bl/6 (WT) background, this species is the most relevant control for determining the effects of IL-10. When splenic MØ were primed with LPS, there was a significant increase in PGE2 in the IL-10–/– cells as well as in Balb/c cells. When LPS-primed IL-10–/– splenic MØ are compared with WT cells, the results indicate that endogenous IL-10 causes significant suppression of PGE2 production to approximately the level seen in unprimed cells (Fig. 1) . We found that IL-10 also contributes to the down-regulation of PGE2 biosynthesis by LPS-primed bone marrow MØ. In this case, the difference in PGE2 production between WT and Balb/c was less pronounced than for the corresponding splenic cells. It is interesting that PGE2 synthesis is much greater in bone marrow MØ than in splenic MØ as is the magnitude of the IL-10 effect (Fig. 1) . Our in vitro studies indicate that IL-10 does not regulate thromboxane production (Fig. 2) . The results for Balb/c and WT mice also indicate a genetically determined difference in the murine inflammatory response, consistent with an earlier report [35 ].

LPS priming of MØ enhancement of PGHS-2 is independent of IL-10
We further analyzed the effects of IL-10 on PGHS-2 expression in relation to PGE2 biosynthesis in splenic and marrow MØ. Our studies demonstrate that LPS induces PGHS-2 protein expression by IL-10–/– splenic MØ with a profile similar to that seen in splenic MØ from WT or Balb/c mice, suggesting that this response is not highly dependent on IL-10. Neither the inflammatory mediators nor IL-10 appear to exert any regulatory effect on PGHS-1 protein expression, similar to previous reports indicating that PGHS-1 expression is generally constitutive rather than inducible [8 ]. Previous studies of PGE2 production by rat alveolar MØ in response to LPS, inflammatory cytokines, and/or mitogens concluded that PGE2 production is significantly associated with an increase in PGHS-2 protein levels [12 ]. Examination of our data (Figs. 1 3 and 4) indicates that in IL-10–/– mice, there appears to be a dependence of PGE2 production on PGHS-2 levels as previously reported. This is particularly evident in bone marrow MØ, where the magnitude of PGE2 production is greatest. However for WT cells, the levels of PGE2 synthesis do not correspond with PGHS-2 levels. Increased PGE2 production does not appear to occur in the absence of increased expression of PGHS-2, although increased PGHS-2 expression does not necessarily lead to increased PGE2 production. Therefore, factors other than PGHS-2 expression must contribute to the IL-10-dependent regulation of PGE2 biosynthesis.

In our previous studies [2 , 3 , 6 , 36 ] of C57Bl/6 and CBA/J mice immunized with bacillus Calmette-Guerin (BCG) or Cryptosporidium parvum, establishment of high PGE2-releasing splenic MØ required 7–14 days (>50 ng PGE2/5x106 MØ). These MØ expressed PGHS-1 and PGHS-2, although the contribution of endogenous IL-10 to PGHS-2-mediated PGE2 synthesis by the MØ was unclear. It is interesting that splenic MØ isolated 1 day after immunization showed relatively high PGHS-2 expression in an IL-10-independent manner (Y. Shibata, unpublished observation). The expression of PGHS-2 was not observed on day 2 after BCG immunization. Unlike splenic MØ isolated 7–14 days after immunization, however, high PGHS-2 levels on day 1 were not associated with high PGE2 synthesis, as splenic MØ isolated from days 1–3 after BCG immunization showed no increase in PGE2 release (<5 ng/ml) [2 , 6 ]. This in vivo observation suggests that there is also a temporal component to the development of PGE2 production.

Contribution of IFN-{gamma}
Our results indicate that in the presence of LPS, PGE2 production in IL-10–/– and Balb/c splenic MØ is enhanced by addition of IFN-{gamma}. In WT mice, there was no significant increase in PGE2 release in the presence of LPS or IFN-{gamma}, singly or combined (Fig. 1) , despite the increase in PGHS-2 expression in the presence of LPS (Fig. 3) . IFN-{gamma} in synergy with LPS is reported to induce PGHS-2 expression in peritoneal MØ [37 ] through the activation of nuclear factor (NF)-{kappa}B [38 ]. Our results with splenic and bone marrow MØ also suggest a synergistic effect of IFN-{gamma} on PGHS-2 expression (Figs. 3 and 4) .

Effect of exogenous IL-10 on PGHS-2 induction and PGE2 synthesis
The results (Figs. 5 and 6) indicate that exogenous IL-10 inhibits PGE2 synthesis and PGHS-2 induction in IL-10–/– splenic and bone marrow MØ, confirming a regulatory role for IL-10. However, for splenic MØ, where levels of PGE2 synthesis are quite low, this inhibition appears to require relatively high doses of exogenous IL-10. Along with the quantitative difference in PGE2 production, this difference in IL-10 sensitivity could also indicate a difference in regulatory mechanisms for cells of splenic and marrow origins. It may also be that exogenous IL-10 added to the media of the splenic MØ does not functionally or temporally reproduce IL-10 levels developed in vivo.

Taken together, the results suggest that another effector, such as PGE synthase or possibly a specific phospholipase A2, is involved in the regulation of PGE2 biosynthesis and that regulation of this effector is directly or indirectly dependent on IL-10. Alternatively, the effects of IL-10 on PGE2 production may be through regulation of PGHS-2 activity. One cannot rule out the contribution of PGHS-3, a PGHS-1 variant that was recently identified [39 ].

Role of IL-10
There are multiple possible mechanisms for IL-10 regulation of PGHS-2 synthesis [8 ]. It is established that LPS, IL-1ß, and TNF-{alpha} enhance PGHS-2 mRNA stability [40 ], whereas exogenous IL-10 in vitro accelerates the degradation of PGHS-2 mRNA in human monocytes [22 ], consistent with our findings for addition of exogenous IL-10-to-IL-10–/– cells (Figs. 5 and 6) . In addition, previous studies [41 42 43 44 45 ] suggest that LPS-induced PGHS-2 expression and PGE2 synthesis are associated with p38 mitogen-activated protein kinase (MAPK) activation in human monocytes and neutrophils. IL-10 appears to down-regulate p38 MAPK activation [45 46 47 ], which is an upstream kinase regulating NF-{kappa}B activation in the neutrophil [48 ], suggesting that p38 MAPK might play a role in transcriptional and post-transcriptional regulation of the PGHS-2 gene.

Regardless of how endogenous IL-10 regulates PGE2 synthesis in the absence of IL-10, splenic MØ activated by LPS and IFN-{gamma} produce a maximum of less than 8 ng/ml PGE2/5 x 106 MØ. Additional factors must contribute to the enhanced PGE2 release by splenic PGE2-MØ isolated from mice 7–14 days after BCG immunization [6 , 36 ]. These results are consistent with our previous hypothesis that splenic PGE2-MØ are derived from radiosensitive bone marrow cells [2 , 6 ]. It remains to be elucidated whether priming of bone marrow MØ in vitro with LPS or LPS plus IFN-{gamma} mimics the formation of splenic PGE2-MØ in vivo and whether IL-10 down-regulates PGHS-2-mediated PGE2 biosynthesis in splenic PGE2-MØ isolated from BCG-immunized mice.


   CONCLUSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
REFERENCES
 
These in vitro studies comparing IL-10–/– and WT cells have shown that IL-10 contributes to the down-regulation of PGE2 biosynthesis in splenic and marrow MØ previously primed with LPS. Increased PGE2 synthesis is associated with increased PGHS-2 expression, but additional factors contribute to regulation of prostanoid production.


   ACKNOWLEDGEMENTS
 
This work was supported in part by National Institutes of Health grant HL71711 and Department of Defense grant DAMD17-03-1-0004 (Y. S.). The authors thank Emma Hardison for her excellent technical support.

Received May 27, 2004; revised December 16, 2004; accepted December 20, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
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