Published online before print October 4, 2005
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* Department of Biomedical Sciences, Florida Atlantic University, Boca Raton;
Department of Physiology, Brody School of Medicine at East Carolina University, Greenville, North Carolina;
Japan BCG Laboratory, Tokyo; and
404 Palmeto Drive, Caswell Beach, 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
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(IFN-) production with reduced IL-4 and IL-10 production in recall responses. This treatment also resulted in decreased total serum IgE levels. Treatment of C57Bl/6 mice with HK-BCG (0.5 mg dose) also induced a mixture of Th1 and Th2 responses, although IFN- production was markedly increased, and IL-4 was decreased compared with Balb/c mice. Thus, our results indicate that by 14 days following treatment of mice with high doses of HK-BCG, splenic PGE2-MØ formation is associated with a PGHS-2-dependent shift from Th1-to-Th2 immune responses.
Key Words: PGHS-2 (Cox-2) • cPGES • purified protein derivative • splenic macrophages • PGE2 • Th1-to-Th2 shift
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Complete Freund’s adjuvant [CFA; heat-killed (HK)-M. tuberculosis] and HK-BCG in mineral oil have been widely used to establish animal models of autoimmune diseases, such as experimental autoimmune encephalomyelitis, neuritis, uveitis, thyroiditis, orchitis, and adjuvant arthritis [4 , 5 ]. The adjuvants enhance Th1-mediated macrophage (MØ) activation and Th2-mediated antibody formation [6 ]. Pathogenic roles of Th1/Th2 responses appear to be varied among autoimmune disease models [5 ], which may be related to the range of HK-mycobacteria concentrations (0.05–0.5 mg) used in these animal models [5 , 7 ].
Much attention has been directed to the role of mycobacterial heat shock protein 65 (HSP65), an immunodominant antigen. It has been proposed that a host Th1 response to HSP65 plays a protective role in mycobacterial infections [8
]. However, Th1-to-Th2 shifts of immune responses against mycobacteria result in the formation of antibodies against HSP65, which does not have a decisive, protective role against infection [2
]. Furthermore, as antibodies or T cells specific for bacterial HSP65 potentially cross-react with host HSPs, these immune responses may have pathogenic roles in autoimmune diseases [9
]. Th1 cytokines, including interferon- (IFN-), promote or counteract pathogenesis, depending on the autoimmune disease [9
]. Therefore, Th1-to-Th2 shifts of immune responses may be associated with the pathogenesis of various autoimmune diseases as well as chronic intracellular infections [10–12].
In previous studies, we have reported that normal, splenic MØ stimulated ex vivo do not produce significant levels of prostaglandin E2 (PGE2), distinct from MØ originating from other tissues [13 ]. However, high doses of HK-BCG or related products such as HK-Propionibacterium acnes (Corynebacterium parvum) in vivo induce stable and sustained formation of PGE2-releasing MØ (PGE2-MØ) in the spleen [6 , 14 15 16 ]. The mechanism for formation of these MØ is still unclear. Previous studies [15 , 16 ] indicate that the formation of splenic PGE2-MØ is dependent on radiosensitive bone marrow cells, which may supply precursors of splenic PGE2-MØ. Alternatively, an inflammatory cytokine "milieu" may up-modulate PGE2 biosynthesis directly by splenic MØ [17 18 19 ].
The spleen is the lymphoid tissue where PGE2-MØ and immune lymphocytes interact in chronic inflammatory diseases. PGE2 inhibits the production of Th1 cytokines, such as interleukin (IL)-2, IL-12, and IFN- [20
]. 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 [20
, 21
]. Therefore, we have examined the hypothesis that splenic PGE2-MØ development in vivo following treatment with high doses of HK-BCG is time-, dose-, and PGG/H synthase (PGHS)-2-dependent and promotes a Th1-to-Th2 shift in specific immune responses. Because of described differences in immune responses between Balb/c and C57Bl/6 mice [18
, 20
], we have included results obtained with both strains.
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Preparations of HK-BCG
As described previously [6
], cultured M. bovis BCG Tokyo 172 strain was washed, autoclaved, and lyophilized. The powder of HK-BCG was suspended in saline and dispersed by brief (10 s) sonication immediately prior to injection. These HK-BCG preparations contained undetectable levels of endotoxin (<0.03 EU/ml), as determined by the Limulus amebocyte lysate assay (Sigma Chemical Co., St. Louis, MO) [6
]. HK-BCG was also suspended in mineral oil (M-5904, Sigma Chemical Co.) in some experiments.
Treatment of mice with HK-BCG
Groups of mice received 0.01, 0.1, 0.5, or 1 mg HK-BCG, intraperitoneally (i.p.), on Day 0. Controls received 0.2 ml saline. In some experiments, mice receiving BCG and controls were further treated i.p with 10 mg/kg NS-398 or nimesulide (Cayman Chemical, Ann Arbor, MI) daily, starting on Day 1. Control groups of mice received 0.5% ethanol in saline (0.2 ml/dose). Unless indicated, spleens and sera were harvested on Day 14. To compare the effects of HK-BCG suspended in saline or mineral oil, additional groups of mice were given HK-BCG in mineral oil i.p. Controls received 0.2 ml mineral oil.
Cytokine production in recall response of spleen cell cultures
Spleens from each group of mice were isolated, pooled, minced with scissors, digested with 50 U/ml collagenase D (C2139, Sigma Chemical Co.) in RPMI 1640 plus 10% fetal bovine serum (FBS), 37°C, for 60 min, and filtered through a 100-µm mesh. Single-cell suspensions were prepared by washing digested cells with RPMI 1640 containing 100 µg/ml DNase (DN-25, Sigma Chemical Co.). After washing with serum-free RPMI 1640, cell suspensions were applied to the top of a discontinuous Percoll gradient (35/60%). Following centrifugation, 800 g, 30 min, 22°C, cells at the Percoll interface were collected. Spleen cells were suspended in RPMI 1640 plus 10% FBS at 4 x 106 cells/ml and incubated with endotoxin-free mycobacterial HSP65 (Stressgen, Victoria, BC, Canada) or purified protein derivative (PPD; Japan BCG Laboratory, Tokyo) at 1 or 5 µg/ml, respectively, for 4 days. In some experiments, 10–6 M nimesulide was added to the cultures. After incubation, culture supernatants were collected, and IL-4, IL-10, and IFN- levels were measured by the respective enzyme-linked immunosorbent assay (ELISA; PharMingen, San Diego, CA).
PGE2-MØ
Plastic adherent splenic MØ were isolated from spleen-cell suspensions prepared above [13
]. Splenic MØ (2x106/ml) were cultured in serum-free RPMI-1640 medium with 10–6 M calcium ionophore A23187 (Sigma Chemical Co.), 1 µg/ml arachidonic acid (AA; Cayman Chemical), or 1 µg/ml bacterial endotoxin [lipopolysaccharide (LPS), Sigma Chemical Co.] for 2 h. In some experiments, splenic cells (2x106/ml) were cultured in RPMI-1640 medium plus 2% FBS with 5 µg/ml PPD or 1 µg/ml HSP65 for 2 days in the presence of the PGHS inhibitors nimesulide, indomethacin, or NS-398, all at 1 µM. PGE2 levels in the culture supernatants were measured by competitive ELISA (Cayman Chemical).
Magnetic separation of F4/80-positive cells
Red cell-free spleen cells (108 cells) were stained with 5 µg/ml monoclonal antibody (mAb) F4/80 recognizing spleen MØ (Accurate Chemical and Scientific Corp., Westbury, NY), followed by addition of 200 µl magnetic microbead-conjugated goat anti-rat immunoglobulin G (IgG; 130-048-501, Miltenyi Biotec, Auburn, CA). F4/80- positive and -negative cells were isolated according to the company’s instructions. The content of F4/80 cells, determined cytometrically, in positive and negative preparations, was 90% and less than 2%, respectively (data not shown).
PGE synthase (PGES) assay
PGES activity in cell lysates was measured as conversion of PGH2 to PGE2 [22
]. Adherent, splenic MØ in 400 µl 10 mM Tris, pH 8.0, were disrupted by sonication using a Branson sonifier (10 s, three times at 1-min intervals). After centrifugation of the sonicates at 1700xg for 10 min, 4°C, the supernatants were used as the source of enzyme activity. An aliquot of each lysate (10 µg protein) was incubated with 0.5 µg PGH2 (Cayman Chemical) for 30 s at 24°C in 0.1 ml 0.1 M Tris, pH 8.0, containing 1 mM reduced L-glutathione (Sigma Chemical Co.) and 5 µg indomethacin. After terminating the reaction by addition of 100 mM FeCl2, PGE2 in the supernatants was quantified by competitive ELISA (Cayman Chemical). Protein concentrations were determined by bicinchoninic acid (BCA) assay (Pierce, Rockford, IL) using bovine serum albumin as standard.
Total serum IgE, antigen-specific IgG1 and IgG2a
Total serum IgE was determined by ELISA using purified mouse IgE 
isotype as the standard rat anti-mouse IgE mAb (clone R35-72) as capture antibody and biotinylated rat mAb detecting IgE (clone R25-92), as detection antibody (all reagents from PharMingen) [6
]. Levels of PPD-specific IgG1/IgG2a and HSP65-specific IgG1 and IgG2a were measured by ELISA with 96-well plates coated overnight at 4°C with 0.5 µg PPD or 0.1 µg HSP65 per well in 100 µl 0.05 M sodium carbonate buffer, pH 9.6 [6
]. Biotinylated rat mAb, detecting IgG1 and IgG2a, were clones A85-1 and R19-15, respectively (PharMingen).
Western blotting
Splenic MØ were prepared as described above, harvested, and washed three times with cold saline. Washed cells were resuspended in lysis buffer {50 mM Tris, pH 7.5, 150 mM NaCl, 1:500 Sigma protease inhibitor cocktail (P8340, Sigma Chemical Co.), 1% Nonidet P-40, and 1% sodium deoxycholate [13
]}. Debris was eliminated by centrifugation (5 min, 1000xg). Protein concentration in the lysate was measured using BCA as described above. Equal amounts of protein were loaded onto sodium dodecyl sulfate-polyacrylamide minigels and separated by electrophoresis (200 V for 45 min). Proteins were then transferred to a polyvinylidene difluoride (Sigma Chemical Co.) membrane, which was blocked with 5% nonfat dry milk and incubated with antibody [anti-PGHS-1, 1:1000; anti-PGHS-2, 1:4000; anticytosolic PGES (anti-cPGES), 1:1000; antimicrosomal PGES-1 (anti-mPGES-1), 1:1000, all from Cayman Chemical] in 5% nonfat dry milk overnight at 4°C. Following incubation with peroxidase-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA), proteins were detected by enhanced chemiluminescence (Amersham, Piscataway, NJ) following the manufacturer’s instructions [13
].
Statistics
Data were analyzed by one-way ANOVA. For cell culture studies, tissues isolated from at least four mice were pooled unless indicated; cells were cultured in at least triplicate in each group. P < 0.05 was considered statistically significant.
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Figure 1. HK-BCG induces splenic PGE2-MØ formation. Groups of mice (four/group) received i.p. HK-BCG suspended in saline or mineral oil on Day 0. (A) Balb/c and C57Bl/6 mice were treated, respectively, with 1 mg and 0.5 mg HK-BCG in saline. Spleens were harvested on indicated day including Day 0 (untreated). Splenic MØ were isolated, pooled in each group, and incubated for 2 h in serum-free RPMI-1640 medium (106 MØ/ml) in the presence of 1 µg/ml AA, 1 µM A23187, 1 µg/ml LPS, or media alone (Medium). (B) Splenic MØ were isolated from mice 14 days after receiving HK-BCG suspended in saline or mineral oil at the indicated doses. PGE2 release was elicited in vitro by 1 µM A23187 for 2 h. (C) Spleen cells were isolated from mice 14 days after receiving 1 mg HK-BCG in saline and incubated in RPMI 1640 plus 2% FBS (2x106 cells/ml) in the presence of mycobacterial antigens (5 µg/ml PPD or 1 µg/ml HSP65). In some groups, spleen cells were treated further with 1 µM NS-398, 1 µM nimesulide (Nime), 1 µM indomethacin (Indo), or saline (control) for 48 h. (D) Spleen cells were isolated from Balb/c mice, which received 1 mg HK-BCG in mineral oil. Recall responses and PGE2 production were determined with methods similar to those in C. PGE2 was measured by ELISA. Mean ± SD, n = 3. Each result represents a group of four mice from three experiments.
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To determine whether recall responses in vitro elicit PGE2 release, spleen cells isolated from mice treated with HK-BCG in saline were stimulated with the mycobacterial antigens, PPD or HSP65. As shown in Figure 1C , these antigens induced spleen cells to release PGE2 in both strains of mice, dependent on the HK-BCG dose and suggesting that interaction between antigen-specific lymphocytes and splenic MØ triggers PGE2 biosynthesis. Furthermore, in Balb/c mice treated with 1 mg HK-BCG in saline or mineral oil, PGE2 biosynthesis was inhibited by NS-398, nimesulide, or indomethacin, consistent with mediation of PGE2 synthesis by PGHS-2 in splenic PGE2-MØ (Fig. 1D) .
Our results clearly indicate that HK-BCG, in a dose-dependent manner, induces splenic PGE2-MØ formation within 7–14 days. Inhibition by the PGHS-2 selective inhibitors nimesulide and NS398 implies a dependence on PGHS-2 for PGE2 synthesis. There is no difference in the magnitude of PGE2-MØ formation in response to HK-BCG suspended in saline or in mineral oil. Therefore, HK-BCG suspended in saline was used to further characterize PGE2-MØ and determine whether these cells contribute to the Th1-to-Th2 shift of immune responses.
Protein detection of PGHS-1, PGHS-2, mPGES-1, and cPGES in splenic PGE2-MØ
PGE2-MØ metabolize endogenous AA to PGE2 through the rate-limiting enzymes PGHS and PGES. Two major isoforms of PGHS convert AA to PGH2: PGHS-1, a constitutive form, and PGHS-2, an inducible form. PGH2 is subsequently converted to PGE2 by cPGES and mPGES-1 [22
, 23
]. Murakami et al. [22
] reported that mPGES-1 is a terminal enzyme of PGHS-2-mediated PGE2 synthesis, and PGHS-2 and mPGES-1 are induced in various cells, including peritoneal MØ, by proinflammatory stimuli. Normal, splenic MØ expressed PGHS-1, mPGES-1, and cPGES but not PGHS-2 (Fig. 2
). This profile was unchanged in splenic MØ isolated from Balb/c mice 14 days after receiving 0.01 mg HK-BCG in saline (data not shown). When Balb/c mice were treated with 1 mg HK-BCG, PGHS-2 was detected on Days 7 and 14 (Fig. 2)
. The levels of PGHS-1, mPGES-1, and cPGES remained similar to those in normal, splenic MØ (Fig. 2)
. Therefore, increased PGHS-2 levels, but not mPGES-1 levels, were associated with increases in PGE2 release by splenic PGE2-MØ. Figure 2B
shows that PGHS-2 was highly enriched in F4/80-positive MØ on Day 14.
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Figure 2. PGHS-1, PGHS-2, cPGES, and mPGES-1 levels in HK-BCG-treated mice. (A) Balb/c mice received 1 mg HK-BCG in saline, i.p., on Day 0, and Days 0 (untreated), 3, 7, and 14 spleens were harvested. Lysates of splenic M
Ø isolated from each group of animals were analyzed by Western blotting. (B) F4/80-positive and -negative cells were isolated from Day 14 spleen cells as indicated in Materials and Methods. PGHS-2 expression in each fraction was determined by Western blotting. Each lane was loaded with 5 µg total protein. Results are representative of three separate experiments.
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Figure 3. PGES activities in splenic M
Ø isolated from BCG-treated mice. Groups of Balb/c mice (four/group) received 0 (0.2 ml saline), 0.01, or 1 mg HK-BCG i.p. in saline on Day 0; Day 14 spleens were harvested. Splenic M
Ø were isolated, pooled in each group, and sonicated. PGES activities in cell lysates were measured as described in Materials and Methods. PGE2 was measured by ELISA. Mean ± SD, n = 3. In the absence of exogenous PGH2, endogenous PGE2 levels were  0.87 ng/10 µg protein.
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Figure 4. Total serum IgE levels and mycobacterial antigen-specific IgG1 and IgG2a levels. Groups of Balb/c mice (five/group) received 0 (0.2 ml saline), 0.01, or 1 mg HK-BCG in saline on Day 0; Day 14 sera were harvested. Total IgE levels in the sera were measured by sandwich ELISA. Levels of PPD-specific IgG1 and IgG2a or HSP65-specific IgG1 and IgG2a in sera were measured as described in Materials and Methods. The sera were diluted 1/100 and 1/25 with saline for assays of antigen-specific IgG1 (open bars) and IgG2a (solid bars), respectively. Values are mean ± SD; n = 5. *, **, and ***, P < 0.05, P < 0.01, and P < 0.005, respectively, compared with the saline control group. Each result represents mice from two experiments.
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Figure 6. PGHS-2 inhibitors (NS-398, nimesulide) in vivo inhibit Th2 cytokine production and decrease serum IgE levels but enhance IFN- production. (A) Groups of Balb/c mice (five/group) received 1 mg HK-BCG i.p. in saline on Day 0. Controls received 0.2 ml saline. BCG-treated mice and controls were treated i.p. daily starting on Day 1 with 10 mg/kg NS-398 or 0.5% ethanol in saline (0.2 ml). Day 14 sera and spleens were harvested. Total IgE in sera was measured by ELISA. Mean ± SD, n = 5. *, P < 0.05, compared with the BCG group. Spleen cells were cultured in the presence of 5 µg/ml PPD (open bars), 1 µg/ml HSP65 (solid bars), or medium alone (shaded bars) for 4 days; levels of IFN-, IL-4, and IL-10 in the culture supernatants were measured by ELISA. Mean ± SD, n = 3. * and **, P < 0.01, and P < 0.005, respectively, compared with the saline control group. (B) Groups of C57Bl/6 mice (four/group) received i.p. 0.5 mg HK-BCG on Day 0. Controls received 0.2 ml saline. Mice were treated i.p. daily starting on Day 1 with 10 mg/kg nimesulide or 0.5% ethanol in saline (0.2 ml). Day 14 sera and spleens were harvested, and spleen cells were cultured in the presence of 5 µg/ml PPD for 4 days. The levels of total IgE in sera and IL-4 and IFN- in the culture supernatants were measured by ELISA. Mean ± SD, n = 3. *, P < 0.05; **, P < 0.01, compared with saline treatment. Each result represents a group of four mice from two experiments.
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(Th1 response) were detected compared with untreated mice (Fig. 5
). These spleen cells also produced IL-10 (Th2 response, Fig. 5
) but only minimal levels of IL-4 (data not shown). In contrast, spleen cells from animals receiving 1 mg HK-BCG produced less IFN- (Fig. 5)
but increased amounts of IL-10 (Fig. 5)
and IL-4 (Fig. 6
). No IL-5 was detected (data not shown). It is interesting that HSP65 (1 µg/ml) stimulated IFN- production but not IL-4 or IL-10 production in recall responses (Fig. 5)
. When spleen cell cultures were treated with 1 µM nimesulide, a PGHS-2 inhibitor, IFN- production in recall responses was enhanced significantly. These results suggest that endogenous PGE2 inhibits IFN- production, consistent with a shift from Th1-to-Th2 response in animals treated with 1 mg HK-BCG. However, the in vitro treatment with nimesulide only slightly reduced IL-10 production (Fig. 5A)
and did not alter PPD-stimulated IL-4 production (data not shown), indicating that under these conditions, there is limited effect of PGE2 on these Th2 cytokine responses.
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Figure 5. Th1 and Th2 cytokine production in recall responses. (A) Groups of Balb/c mice (five/group) received 0 mg (0.2 ml saline), 0.01 mg, or 1 mg HK-BCG i.p. on Day 0; Day 14 spleen cells were isolated and stimulated in vitro with 5 µg/ml PPD or 1 µg/ml HSP65 for 4 days. In some cultures, 1 µM nimesulide was added. The levels of IFN- and IL-10 in the culture supernatants were measured by ELISA, as described in Materials and Methods. Values are mean ± SD from triplicate cultures. The data shown are for a group of five mice and are representative of two independent experiments. *, **, and ***, P < 0.05, P < 0.01, and P < 0.005, respectively, compared with the saline control group. (B) Groups of C57Bl/6 mice (four/group) received 0 (0.2 ml saline), 0.01, and 0.5 mg HK-BCG i.p. on Day 0; Days 14 and 21 spleens were isolated, and spleen cells were cultured in the presence of 5 µg/ml PPD for 4 days. The levels of IFN- and IL-4 in the supernatants were measured by ELISA. Mean ± SD, n = 3. * and **, P < 0.05, and P < 0.01, respectively, compared with the saline control group.
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and IL-4 produced were much higher and lower, respectively, than those seen with spleen cells from Balb/c mice (Figs. 5
and 6)
. These responses were essentially unchanged when spleen cells were isolated 21 days following treatment with HK-BCG, as was also shown for PGE2 production (Figs. 1
and 6)
. When spleen cells were depleted of CD4+ cells by anti-CD4 antibody (clone GK1.5) plus guinea pig serum, the remaining cells did not express detectable levels of IFN-, IL-10, or IL-4 in recall responses (data not shown), indicating that production of these cytokines was CD4+ cell-dependent.
PGE2 is known to inhibit production of IL-12, a Th1 cytokine inducing IFN- production [27
]. However, in our studies, IL-12 was undetectable (<12 pg/ml) in the groups where IFN- levels were above baseline (data not shown). Despite the undetectable levels of IL-12 in the presence of nimesulide, where PGE2 production is inhibited, IFN- levels are increased (Fig. 5)
. In our previous report [28
], 1–10 µm chitin particles induced splenic MØ to produce IL-12, which was inversely dependent on the PGE2 concentration. For example, when splenic MØ from normal C57Bl/6 and from high-dose HK-BCG groups were stimulated in vitro with 1–10 µm chitin particles at 100 µg/ml for 24 h, IL-12 levels were detected as 75 and <12 pg/ml/106 MØ, respectively. These results are consistent with earlier observations [20
, 27
, 28
] that inhibition of IL-12 production by endogenous PGE2 contributes to the reduction of Th1 responses.
In vivo effects of PGHS-2 inhibition in BCG-treated mice
To assess the effects of PGHS-2 activity on immune responses, Balb/c mice, which received 1 mg HK-BCG, were treated in vivo with NS-398. Recall responses of spleen cell cultures with PPD or HSP65 were determined 14 days after BCG treatment. Figure 6 shows that spleen cells isolated from animals treated with NS-398 in vivo and stimulated in vitro with PPD produced more IFN- but less IL-4 or IL-10 than those not treated with the PGHS-2 inhibitor. IL-4 and IL-10 were not detected in HSP65-stimulated recall responses, with or without NS-398. Production of HSP65-stimulated IFN- was enhanced significantly by the treatment with NS-398 (Fig. 6)
. Finally, in vivo inhibition of PGHS-2 reduced total serum IgE levels (Fig. 6)
.
The pattern of response for total serum IgE in C57Bl/6 mice treated in vivo with NS-398 was similar to that for Balb/c mice. The IL-4 produced in PPD recall responses with cells from C57Bl/6 mice also followed a similar pattern to Balb/c mice, but the levels were reduced approximately tenfold (Fig. 6)
. Only relatively low levels (<100 pg/ml) of IL-10 were seen in C57Bl/6 mice (data not shown). The IFN- responses again followed a similar pattern to Balb/c mice, but the levels were increased markedly (Fig. 6)
. The results contrast with the recall responses for cells treated in vitro with a PGHS-2 inhibitor, described above. Following in vivo administration of a PGHS-2 inhibitor, the Th1-to-Th2 shift of splenic lymphocyte responses to mycobacterial antigens and serum IgE production develops in a PGHS-2-dependent manner.
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Unlike MØ isolated from peritoneum, bone marrow, or blood, normal, splenic MØ exhibit only a relatively low level of PGE2-release (<1 ng PGE2/106 MØ) [13
]. When activated in vitro by bacterial endotoxin and IFN-, splenic MØ produce a maximum of <2 ng/ml PGE2 [13
]. Additional factors must contribute to the enhanced PGE2 release by splenic PGE2-MØ isolated from mice 7–21 days after HK-BCG treatment. In mice depleted of bone marrow by 89Sr, we previously found that splenic PGE2-MØ are not formed following administration of HK-C. parvum, although these mice show increases in total splenic MØ and myeloid precursors [15
, 16
]. It is likely, therefore, that PGE2-MØ precursors are generated in the radiosensitive bone marrow following HK-BCG treatment, then migrate, and localize in the spleen [16
]. This process takes at least 5–7 days after administration of HK-BCG. Splenic PGE2-MØ are located strategically to interact with lymphocytes and induce the shift of Th1-to-Th2 responses during mycobacterial infections. Once PGE2-MØ are established, they persist for long periods [15
], therefore prolonging the effect of PGE2 on immune regulation. It should also be noted that splenic PGE2-MØ formation is independent of circulating monocytes [15
, 16
]. Th1/Th2 cells that develop in the spleen eventually migrate to inflammatory sites [1
].
Our results (Figs. 2 and 3) show that PGHS-2 is increased with increased PGE2-MØ formation and is associated specifically with F4/80+ cells. PGES activity and the mPGES-1 protein level are not increased in response to BCG treatment. In contrast, normal peritoneal MØ express high levels of mPGES-1 and PGHS-2 and release a large amount of PGE2 [22 , 24 , 29 ] in responding to LPS in vitro. Therefore, the regulatory mechanisms PGHS-2 and PGE2 synthesis in splenic PGE2-MØ are distinct from peritoneal MØ. Although PGHS-2 may be necessary for the increased PGE2 following BCG treatment, we have not demonstrated that this enzyme is sufficient or the rate-limiting factor. Secretory phospholipase A2 type V is also known to be involved in prostaglandin production in splenic MØ, mast cells, and mesangial cells [19 , 30 ], which makes this enzyme a possible candidate for regulation following BCG treatment. In addition, we have not investigated a possible role for the constitutively expressed PGES mPGES-2 [31 ]. Further analysis will be required to identify the required enzymes and rate-limiting factor(s) for PGE2 synthesis in splenic PGE2-MØ.
Balb/c mice, compared with C57Bl/6 mice, frequently show the induction of significant Th2 responses after infection or immunization, such as with BCG [32
] and Leishmania major [21
, 33
]. Normal, splenic MØ isolated from Balb/c mice and treated in vitro with LPS produce more PGE2 than those from C57Bl/6 mice [18
] and have a greater sensitivity to the Th1-suppressive effect of PGE2 [18
, 19
]. However, our studies indicate that C57Bl/6 mice express splenic PGE2-MØ and a mixed Th1 and Th2 lymphocyte response against mycobacteria in responding to high-dose HK-BCG with a pattern similar to that for Balb/c mice. The remarkable differences between these two strains are the increased amount of IFN- and decreased amount of IL-4 produced.
Infections caused by Chlamydia pneumoniae and Helicobacter pylori as well as Mycobacterium sp have been implicated as co-risk factors in atherosclerosis [34 , 35 ]. Seroepidemiological studies [36 ] indicate that patients with atherosclerosis express high antibody titers against mycobacterial HSP65, a prokaryotic HSP of the 60-kDa family (HSP60/65), indicating a Th2-mediated, humoral response against this antigen. Prokaryotic HSP60/65 are >97% homologous, whereas prokaryotic and human/mouse HSP60/65 have >70% amino acid sequence homology. By attacking stressed arteries that express endogenous HSP60, anti-HSP65 may contribute to atherosclerosis through antigenic mimicry [36 ]. Microbial HSP60/65 serves as an immunodominant antigen in protection from and in the pathogenesis of infectious diseases [34 , 36 37 38 ]. The development of Th1 cells against HSP65 mediates resistance to M. tuberculosis, which constitutes a major vaccine strategy against tuberculosis [8 ]. However, in recurrent and chronic infections, anti-HSP65, which does not have a decisive, protective role against mycobacterial infections, may promote the further progression of atherosclerotic plaques [36 , 39 ]. Similarly, Th1-to-Th2 shifts of the immune response may be associated with the pathogenesis of various chronic, immunologically mediated diseases.
Live BCG induces not only host resistance to M. tuberculosis but also tumoricidal activity and has been used widely for treatment of superficial bladder cancer [40 , 41 ]. Live BCG also induces a nonspecific MØ microbicidal activity against Listeria monocytogenes [42 , 43 ] and Toxoplasma gondii [44 ]. It has been reported that a low dose (0.01 mg) of HK-BCG suspended in saline induced a Th1 lymphocyte response against mycobacteria, but the effects were not sufficient to protect against mycobacterial infections, whereas immunization with HK-mycobacteria in mineral oil (CFA) was protective [45 ]. Although mineral oil provides additional adjuvant activity, our study indicates that mineral oil itself has no effect on the formation of splenic PGE2-MØ.
In conclusion, previously, we found that development of splenic PGE2-MØ is dependent on radiosensitive bone marrow cells and that the PGE2-MØ are probably derived directly from the bone marrow [6 , 15 , 16 ]. Here, we have continued our investigation of the unique splenic PGE2-MØ population, which plays a novel, PGHS-2-dependent, immunoregulatory role in the Th1-to-Th2 shift of immune responses against mycobacterial antigens including HSP65. It is possible that PGE2 as an antiapoptotic agent also promotes antigen-presenting activity of dendritic cells [46 ], whose maturation is induced directly by BCG, and may skew the immune response toward a Th2-like profile [47 ]. PGE2 is known to increase regulatory T cell differentiation and function, which could contribute to the immune responses of BCG-treated mice [48 , 49 ]. Finally, it remains to be elucidated whether similar mechanisms are involved in the pathogenesis of Leishmania infection, Trichophyton dermatophytosis, syphilitic infection, or human immunodeficiency virus infection progressing to AIDS [21 , 50 51 52 53 ], where Th1-to-Th2 shifts of immune responses and splenic PGE2-MØ formation are frequently seen. Th1-to-Th2 shifts are also observed in neonatal, aged, tumor-bearing, diabetic, and hypercholesterolemic animals [14 , 54 55 56 57 58 ].
Received June 15, 2005; revised July 20, 2005; accepted August 16, 2005.
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and B cell stimulatory factor-1 reciprocally regulate Ig isotype production Science 236,944-947
(cPLA2 ) and secretory phospholipase A2 (sPLA2) in hydrogen peroxide-induced arachidonic acid release in murine mesangial cells: sPLA2 regulates cPLA2 activity that is responsible for arachidonic acid release J. Biol. Chem. 278,24153-24163This article has been cited by other articles:
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