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The Journal of Immunology, 2007, 179, 7072 -7078
Copyright © 2007 by The American Association of Immunologists, Inc.
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Catalytically Inactive Cyclooxygenase 2 and Absence of Prostaglandin E2 Biosynthesis in Murine Peritoneal Macrophages following In Vivo Phagocytosis of Heat-Killed Mycobacterium bovis Bacillus Calmette-Guérin1

Makiko Yamashita*, Tsutomu Shinohara*, Shoutaro Tsuji*, Quentin N. Myrvik{dagger}, Akihito Nishiyama*, Ruth Ann Henriksen{ddagger} and Yoshimi Shibata2,*

* College of Biomedical Sciences, Florida Atlantic University, Boca Raton, FL 33431; {dagger} Palmeto Dr, Caswell Beach, NC 28461; and {ddagger} Department of Physiology, Brody School of Medicine, East Carolina University, Greenville, NC 27834


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Over 25 years ago, it was observed that peritoneal macrophages (M{phi}) isolated from mice given heat-killed Mycobacterium bovis bacillus Calmette-Guérin (HK-BCG) i.p. did not release PGE2. However, when peritoneal M{phi} from untreated mice are treated with HK-BCG in vitro, cyclooxygenase 2 (COX-2), a rate-limiting enzyme for PGE2 biosynthesis, is expressed and the release of PGE2 is increased. The present study of peritoneal M{phi} obtained from C57BL/6 mice and treated either in vitro or in vivo with HK-BCG was undertaken to further characterize the cellular responses that result in suppression of PGE2 release. The results indicate that M{phi} treated with HK-BCG in vivo express constitutive COX-1 and inducible COX-2 that are catalytically inactive, are localized subcellularly in the cytoplasm, and are not associated with the nuclear envelope (NE). In contrast, M{phi} treated in vitro express catalytically active COX-1 and COX-2 that are localized in the NE and diffusely in the cytoplasm. Thus, for local M{phi} activated in vivo by HK-BCG, the results indicate that COX-1 and COX-2 dissociated from the NE are catalytically inactive, which accounts for the lack of PGE2 production by local M{phi} activated in vivo with HK-BCG. Our studies further indicate that the formation of catalytically inactive COX-2 is associated with in vivo phagocytosis of HK-BCG, and is not dependent on extracellular mediators produced by in vivo HK-BCG treatment. This attenuation of PGE2 production may enhance M{phi}-mediated innate and Th1-acquired immune responses against intracellular infections which are suppressed by PGE2.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Phagocytosis of intracellular bacteria by macrophages (M{phi})3 results in cellular activation with expression of cyclooxygenase-2 (COX-2), a rate-limiting enzyme for PGE2 biosynthesis. PGE2 down-regulates innate and Th1-mediated immune responses induced by bacteria in autocrine and paracrine fashions. For example, PGE2 inhibits inducible NO synthase/NO synthesis, NAPDH oxidase/superoxide anion release, and IL-12/IL-18/TNF-{alpha} synthesis (1, 2, 3). In contrast, PGE2 promotes IL-10 production by M{phi} (4, 5), a Th1-to-Th2 shift of acquired immune responses (4, 5), dendritic cell Ag presentation (6), regulatory T cell differentiation and function (7), bone marrow progenitor cell migration via the CXCR4/stromal cell-derived factor-1 (CXCL12) system (8), IL-23 production (9), and M{phi} production of matrix metalloproteinase 9 (10). Regulation of these events, therefore, may depend on the regulation of PGE2 release by COX-2+ M{phi} (11). The formation of these PGE2-releasing, activated M{phi} (PGE2-M{phi}) appears to be regulated by multiple bacterial and host factors, including the tissue origin of the M{phi} (12, 13, 14).

Humes et al. (15) reported for the first time in 1980 that PGE2 release by peritoneal M{phi} isolated from mice that are given heat-killed Mycobacterium bovis bacillus Calmette-Guérin (HK-BCG) or HK-Corynebacterium parvum (Propionibacterium acnes) i.p. is significantly reduced compared with that released by untreated peritoneal M{phi}. Studies by other groups including our own confirmed this phenomenon (13, 16, 17). Because peritoneal M{phi} elicited by i.p. HK-C. parvum in both monocytopenic and control mice show diminished PGE2 biosynthesis, the phenomenon is not dependent on monocyte-derived M{phi} migration but rather is dependent on the direct interaction between local M{phi} and bacteria (13). A precise explanation for the suppression of PGE2 release has not been established. In sharp contrast to long-held views, recent studies indicate that in vitro these bacteria induce COX-2 expression and PGE2 biosynthesis by various M{phi} preparations including normal peritoneal M{phi}, blood monocytes, and M{phi} cell lines (18, 19, 20, 21, 22, 23, 24, 25, 26). Although the regulation of COX-1 and COX-2 expression has been extensively studied (27), exact mechanisms for regulation of PGE2 biosynthesis by M{phi} activated with HK-BCG in vitro or in vivo are still unclear (12).

PGE2 biosynthesis is initiated by activation of phospholipase A2 (PLA2) to release arachidonic acid (AA), which is metabolized by constitutive COX-1 and inducible COX-2 yielding PGH2, which is converted to PGE2 by cytosolic PGE synthase or microsomal PGES (28, 29). Catalytically active COX-1 and COX-2 are localized in the nuclear envelop (NE) and endoplasmic reticulum (ER) of PGE2-releasing cells (30, 31, 32). More recent studies have suggested that for functional coupling and PGE2 biosynthesis, cytosolic PLA2, COXs, and PGESs appear to be localized in the perinuclear region (28, 33).

Previously, we demonstrated that i.p. administration of HK-BCG results in at least two different forms of COX-2+ splenic M{phi}: M{phi} obtained beginning at 1 day following HK-BCG treatment, which have catalytically inactive COX-2 dissociated from the NE and do not release PGE2, and M{phi} obtained 7 days following treatment which have catalytically active COX-2 localized at the NE and release PGE2. The dissociation of COX-2 from the NE is associated with in vivo phagocytosis of HK-BCG (34). Although capable of phagocytosis, the splenic M{phi} expressing catalytically active COX-2 do not contain intracellular HK-BCG (14, 34). Neither of these COX-2+ M{phi} subsets can be induced in vitro, where only catalytically active COX-2+ M{phi} are seen, and only in the presence of phagocytosed HK-BCG (34). Because formation of splenic and peritoneal PGE2-M{phi} have distinct features (13), we sought to determine whether catalytically inactive COX-2 is induced in peritoneal M{phi} that phagocytose HK-BCG in vivo. We found that endogenous factors involved in phagocytosis of HK-BCG in vivo, but not extracellular signaling molecules produced locally by HK-BCG, are responsible for regulating the subcellular localization COX-2.


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

Nonpregnant female C57BL/6 mice, 8–14 wk old, were obtained from Harlan Breeders. Mice were maintained in barrier-filtered cages and fed Purina laboratory chow and tap water ad libitum. Experimental protocols used in this study were approved by the Institutional Animal Care and Use Committee at Florida Atlantic University.

Intraperitoneal administration of HK-BCG, HK-C. parvum, or LPS

As described previously (35), cultured M. bovis BCG Tokyo 172 strain were washed, autoclaved, and lyophilized. This HK-BCG powder was suspended in pyrogen-free saline and dispersed by brief (10 s) sonication immediately before use. These HK-BCG preparations contained undetectable levels of endotoxin (<0.03 endotoxin units/ml), as determined by the Limulus amebocyte lysate assay (Sigma-Aldrich) (35). Groups of mice (three per group) received 1, 0.1, or 0.01 mg of HK-BCG (5 x 108 bacilli/mg) i.p.. Controls received 0.1 ml of saline. Peritoneal lavage was performed at 6 or 24 h. As comparison controls, peritoneal M{phi} were also obtained from mice given 1 mg of HK-C. parvum (13) or 100 µg of LPS (Escherichia coli:0111:B4, phenol; Sigma-Aldrich) i.p. The schedules were identical with those used for HK-BCG.

Peritoneal M{phi} preparation and treatment with HK-BCG, HK-C. parvum, or LPS in vitro

Peritoneal lavage was performed as previously described (13). Nucleated peritoneal cells were counted in a Coulter counter (model Z1; Beckman Coulter). Differential cell counts were performed on cytospin preparations (Shandon Southern Instruments) stained with Diff-Quik. To enrich plastic-adherent M{phi}, peritoneal cells at 1 x 106 cells/ml suspended in RPMI 1640 plus 5% FBS were incubated in culture dishes (Falcon) for 2 h. Nonadherent cells (lymphocytes) were removed by washing with warmed medium. Adherent cells were cultured with 100 µg/ml HK-BCG, HK-C. parvum, or 1 µg/ml LPS for an additional 6 or 24 h. In some experiments, plasma and cell-free peritoneal lavage fluid isolated from the HK-BCG-treated mice were added to peritoneal M{phi} cultures with HK-BCG.

Cocultures of peritoneal M{phi} treated with HK-BCG in vitro and in vivo

Peritoneal M{phi} were labeled with 1 µM carboxyfluorescein diacetate (CFDA; Molecular Probes) at 37°C for 15 min and washed with RPMI 1640 plus 5% FBS. CFDA-labeled M{phi} (106 cells/ml) were cultured with 100 µg/ml HK-BCG for 6 h and mixed with peritoneal cells at 106 cells/ml isolated from mice in which 1 mg of HK-BCG was given i.p. 6 h before harvest. The mixed cells were cultured for an additional 18 h.

PGE2 assay

For assay of PGE2 release, plastic adherent peritoneal M{phi} (1 x 106 cells/ml) were cultured in serum-free RPMI 1640 with 1 µM calcium ionophore A23187 (Sigma-Aldrich) for 2 h. PGE2 levels in the culture supernatants were measured by competitive ELISA (Cayman Chemical).

Subcellular localization of COX-1 and COX-2 by confocal microscopy

Peritoneal M{phi} prepared as described above were fixed with 4% paraformaldehyde in PBS for 30 min. The fixed cells were permeabilized with 0.1% Triton X-100 in PBS for 5 min and incubated in blocking buffer consisting of PBS with 10% FBS for 3 h at 22°C before incubation with anti-COX-1 or anti-COX-2 Ab (Cayman Chemical), 1:500 in blocking buffer, overnight at 4°C. Subsequently, cells were washed with PBS three times and incubated with FITC-conjugated donkey anti-rabbit IgG (1:500; Jackson ImmunoResearch Laboratories) for 1 h at 22°C. For detection of nuclei and HK-BCG, propidium iodine (PI) was mixed at 10 µg/ml with the secondary Ab solution. After washing three times, cells were examined with a laser scanning confocal microscope (Bio-Rad Radiance 2100). The images were processed with Adobe Photoshop software.

Subcellular fractionation

The method for subcellular fractionation was modified from that published previously (36). Peritoneal M{phi} prepared above were resuspended in 0.1 M Tris-HCl (pH 7.5), disrupted with a Dounce homogenizer, and forced through 26-gauge needles on ice. Disruption of cellular membranes was verified by microscopic examination. Cellular debris was removed by low-speed centrifugation (700 x g for 10 min), and the supernatants were further centrifuged at 10,000 x g for 10 min to collect nuclei. The resulting supernatants were subjected to ultracentrifugation at 100,000 x g for 90 min to isolate microsomal membrane and cytosolic fractions. Nuclear and membrane fractions were resuspended in 0.1 M Tris-HCl (pH 7.5). Protein concentrations were measured with a bicinchoninic acid assay (Pierce) and BSA as standard.

COX activity assay

The peroxidase component of COX in isolated cellular fractions was measured with a COX assay kit (Cayman Chemical) briefly as follows. The activity was determined with AA as a substrate and N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) as cosubstrate. Equal amounts of protein (20 µg) were incubated at 25°C in a reaction mixture consisting of AA, TMPD, and heme in 0.1 M Tris-HCl (pH 7.5). The absorbance change, due to oxidation of TMPD during the initial 5 min, was measured at 590 nm. The specific enzyme activities were calculated and indicated as nanomoles per minute per milligram.

Western blot analysis

Peritoneal M{phi}, prepared as described above, were washed three times with cold saline. Washed cells were resuspended in lysis buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 4 mM EDTA, 0.1% SDS, 1:500 protease inhibitor mixture (P8340; Sigma-Aldrich)), 1% Nonidet P-40, and 1% sodium deoxycholate). Debris was eliminated by centrifugation (10 min, 10,000 x g). Protein concentration in the lysate was measured with a bicinchoninic acid assay (Pierce) and BSA as standard. Equal amounts of protein from each sample were separated by SDS-PAGE. Proteins were then transferred to a polyvinylidene difluoride membrane (Millipore). The membrane was blocked with 10% nonfat dry milk and incubated with Abs (anti-COX-1, 1:2,000; anti-COX-2, 1:4,000 (Cayman Chemical); anti-GAPDH, 1:4,000 (Novus Biologicals) for the detection of GAPDH as constitutively expressed protein control) in 5% nonfat dry milk, overnight at 4°C. Following incubation with peroxidase-conjugated donkey anti-rabbit IgG (1:20,000; Jackson ImmunoResearch Laboratories), proteins were detected by chemiluminescence (ECL plus; Amersham) following the manufacturer’s instructions.

Statistics

Data for PGE2 release were analyzed by one-way ANOVA. For cell culture studies, tissues isolated from at least three mice were pooled unless indicated; these cells were cultured in at least triplicate in each group. Differences between mean values for the COX activity assays were analyzed by Student’s t test with Statcel software. A value of p < 0.05 is considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Difference in PGE2 release from peritoneal M{phi} treated in vivo or in vitro with HK-BCG

Intraperitoneal administration of 1 mg of HK-BCG was chosen to achieve an inflammatory response in mice like that associated with mycobacterial infection. This dose was also used previously to induce splenic PGE2-M{phi} resulting in a Th1-to-Th2 shift of immune response (5). Previous in vitro studies showed that M{phi} phagocytose mycobacteria through TLR2 and that expression of COX-2 and PGE2 biosynthesis are dependent on MAPK and NF-{kappa}B activation (19). Results shown in Fig. 1 indicate a difference in PGE2 production by peritoneal M{phi} dependent on exposure to HK-BCG in vitro or in vivo. Calcium ionophore A23187-elicited production of PGE2 by resident peritoneal M{phi} treated in vitro with HK-BCG is increased at 6 and 24 h, but is unchanged following in vivo treatment (Fig. 1, {blacksquare}). At 6 h, the constitutive production of PGE2 by peritoneal M{phi} treated in vitro with HK-BCG is also increased, but is suppressed for cells treated in vivo (Fig. 1, {square}).


Figure 1
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  		FIGURE 1. Differential PGE2 biosynthesis by M{phi} treated in vitro or in vivo with HK-BCG. For in vivo HK-BCG treatment, groups of C57BL/6 mice received 1 mg of HK-BCG i.p. After 6 or 24 h, peritoneal lavage cells were harvested. For in vitro HK-BCG treatment, normal resident peritoneal M{phi} were incubated with 100 µg/ml HK-BCG for 6 or 24 h, and cells were harvested. C indicates cells exposed to saline for 24 h. To determine PGE2 release, the M{phi} suspension (106/ml) was stimulated with 1 µM A23187 ({blacksquare}) or medium ({square}), for 2 h. PGE2 was assayed by ELISA. Mean ± SD, n = 3. *, p < 0.005 compared with C (saline) in the same group. #, p < 0.005 and ##, p < 0.0005 compared with the corresponding in vitro group, respectively.

 
Distinct subcellular localization of COX in M{phi} activated with HK-BCG in vivo and in vitro

We determined the COX levels in peritoneal M{phi} activated with HK-BCG in vivo and in vitro. As shown in Fig. 2, in vitro treatment of peritoneal M{phi} resulted in increased COX-2 levels, without a change in COX-1. In contrast, for M{phi} from mice treated with 1 mg of HK-BCG in vivo, an increase in COX-2 was accompanied by a decrease in COX-1 (Fig. 2). The results were similar at 6 and 24 h.


Figure 2
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  		FIGURE 2. The detection of COX-1 and COX-2 in M{phi} treated in vitro or in vivo with HK-BCG. Peritoneal M{phi} treated in vivo or in vitro with HK-BCG were prepared as indicated in Fig. 1. COX-1, COX-2, and GAPDH were determined by Western blotting as indicated in Materials and Methods. GAPDH bands show equivalent loading of samples.

 
We further determined the subcellular localization of COX-2 by confocal microscopy. As shown in Fig. 3, M{phi} activated in vitro expressed COX-2 that was localized in the NE. In contrast, M{phi} activated in vivo had a dense form of COX-2 distributed in the cytoplasm, but not localized in the NE (Fig. 3). COX-1 in untreated M{phi} was consistently expressed in the NE and ER, which was not changed for M{phi} treated in vitro. However, when M{phi} were activated in vivo, COX-1 appeared as the dense form dissociated from the NE with a pattern similar to that seen for COX-2 (Fig. 4).


Figure 3
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  		FIGURE 3. Subcellular localization of COX-2 in peritoneal M{phi} after HK-BCG treatment. Peritoneal M{phi} treated in vivo or in vitro with HK-BCG for 24 h were prepared as indicated in Fig. 1. Cells were examined by confocal microscopy following staining with anti-COX-2 (green) and PI (red) for the nucleus. HK-BCG is also stained by PI.

 

Figure 4
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  		FIGURE 4. Subcellular localization of COX-1 in peritoneal M{phi} after HK-BCG treatment. Peritoneal M{phi} treated in vivo or in vitro with HK-BCG for 24 h were prepared as indicated in Fig. 1. Cells were examined by confocal microscopy following staining with anti-COX-1 (green) and PI (red) for the nucleus. HK-BCG is also stained by PI.

 
Differential COX distribution in M{phi} subcellular fractions

To further confirm that COX-2 dissociated from the NE is catalytically inactive, peritoneal M{phi} treated in vivo with 1 mg of HK-BCG were homogenized and subjected to differential centrifugation. For M{phi} activated in vitro, relatively more COX protein and activity were detected in the nuclear and membrane fractions than in the cytosolic fraction (Fig. 5). The profiles of COX-1 and COX-2 distribution are similar to previous reports using various PGE2-releasing cells (36, 37). In contrast, COX protein isolated from M{phi} treated in vivo was predominantly detected in the membrane fraction (Fig. 5A), but the COX activity was not greater than the background level seen in the nuclear and cytosolic fractions (Fig. 5B).


Figure 5
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  		FIGURE 5. Distribution and activity of COX in subcellular fractions. Peritoneal M{phi} treated with HK-BCG in vitro or in vivo for 24 h as indicated in Fig. 1 were homogenized and separated into nuclear (N), membrane (M), and cytosolic (C) fractions by differential centrifugation as detailed in Materials and Methods. A, Protein (20 µg) in each fraction was used for COX-1 and COX-2 detection by Western blotting. B, The COX activity in each fraction was measured by using a COX assay kit (Cayman Chemical) following the manufacturer’s instructions. The specific enzyme activities were calculated and indicated as nanomoles per minute per milligram. Mean ± SD, n = 3. *, p < 0.001 compared with the activity of the corresponding in vitro fraction.

 
Taken together, these results indicate that, following in vivo treatment with 1 mg of HK-BCG, COX-1 and COX-2 are dissociated from the NE and catalytically inactive. It appears that these inactive forms may be associated with aggregation of COX protein. In contrast, peritoneal M{phi} exposed to HK-BCG in vitro expressed both COX protein and activity in the nuclear and membrane fractions.

Kinetics of COX localization and phagocytosis of HK-BCG

To determine the kinetics of the subcellular translocation of COX-1 and expression of COX-2, peritoneal M{phi} were harvested at 2, 6, and 24 h after i.p. administration of 1 mg of HK-BCG. The localization of COX-1 and COX-2 in each sample was analyzed by confocal microscopy. Fig. 6 shows that 97% of untreated peritoneal M{phi} expressed COX-1, whereas COX-1+ M{phi} were reduced to 56, 53, and 47% at 2, 6, and 24 h after treatment with 1 mg of HK-BCG. This reduction corresponds to the reduced COX-1 protein levels determined by Western blot (Fig. 2). The percentage of COX-1 present in the NE-dissociated dense form increased from 52% (30% of total cells) at 2 h until at 24 h nearly all COX-1 (44% of total cells) was in this form. Interestingly, ≥90% of the NE-dissociated (dense form) COX-1+ M{phi} had phagocytosed HK-BCG that was stained by PI (Fig. 6). We previously demonstrated that HK-BCG stained with PI are almost totally costained with anti-BCG Abs (34).


Figure 6
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  		FIGURE 6. Time and HK-BCG dose dependence of COX-1 localization. Groups of C57BL/6 female mice (three per group) received 0.01, 0.1, or 1 mg of HK-BCG i.p. on day 0. Peritoneal lavage cells were harvested at times indicated. Subcellular localization of COX-1 as well as identification of intracellular HK-BCG were performed by confocal microscopy as described under Materials and Methods. Mean ± SE, n = 3. Percentage of COX-1+ M{phi} (Figure 6), percentage of M{phi} with NE-dissociated COX-1 ({square}), percentage of M{phi} with NE-dissociated COX-1 and PI-stained BCG (Figure 6), percentage of M{phi} with NE-associated COX-1 ({blacksquare}), and percentage of M{phi} with NE-associated COX-1 and PI-stained BCG (Figure 6).

 
COX-2, which was present in <1% of untreated cells, was seen in 60% of M{phi} at 2 h with the NE-dissociated dense form present in 41% of these cells (25% of total cells) (Fig. 7). These NE-dissociated (dense form) COX-2+ cells increased to >90% of COX-2+ cells (70% of total cells) at 6 h. As for COX-1, most (95, 95, and 93% at 2, 6, and 24 h, respectively) of M{phi} expressing NE-dissociated COX-2 contained intracellular PI-stained HK-BCG. Figs. 6 and 7 also show that, at 24 h, COX-1 was lost from half of the cells originally containing COX-1, and at the same time 81% of cells now express COX-2. The results further suggest that some cells contain both isoforms of COX.


Figure 7
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  		FIGURE 7. Time and HK-BCG dose dependence of COX-2 localization. Groups of C57BL/6 female mice (three per group) received 0.01, 0.1, or 1 mg of HK-BCG i.p. on day 0. Peritoneal lavage cells were harvested at times indicated. Subcellular localization of COX-2 as well as identification of intracellular HK-BCG were performed by confocal microscopy as described under Materials and Methods. Mean ± SE, n = 3. Percentage of COX-2+ M{phi} (Figure 7), percentage of M{phi} with NE-dissociated COX-2 ({square}), percentage of M{phi} with NE-dissociated COX-2+ and PI-stained BCG (Figure 7), percentage of M{phi} with NE-associated COX-2 ({blacksquare}), and percentage of M{phi} with NE-associated COX-2 and PI-stained BCG (Figure 7).

 
Dose dependence of COX localization

The subcellular translocation of COX-1 and localization of COX-2 were also investigated at lower doses of HK-BCG (0.1 or 0.01 mg). The results shown in Fig. 6 indicate that the fractions of M{phi}-expressing COX-1 and NE-associated COX-1 are both reduced dose dependently at 24 h. Intracellular HK-BCG was detected in over 80% of NE-dissociated (dense form) COX-1+ M{phi} (Fig. 6). Our results clearly indicate that the NE-dissociated (dense form) COX-1 pattern is associated with phagocytosis of HK-BCG.

At 24 h, there were 44 or 35% COX-2+ M{phi} in response to 0.1 or 0.01 mg of HK-BCG, respectively, compared with 81% at 1 mg of HK-BCG (Fig. 7). M{phi} with NE-dissociated (dense form) COX-2 increased dose-dependently to 98% of total COX-2+ M{phi} at 1 mg of HK-BCG. At all doses, nearly all of these NE-dissociated COX-2+ cells contained PI-stained intracellular HK-BCG. Although this was the predominant COX-2+ phenotype in response to 1 mg of HK-BCG at 24 h, there were a few cells observed with NE-associated COX-2 containing HK-BCG (Fig. 7). Thus, in vivo treatment with 1 mg of HK-BCG resulted in a dramatic shift from NE-associated COX-1 to NE-dissociated and catalytically inactive COX-2 at 24 h.

Intraperitoneal administration of HK-C. parvum and LPS induces NE-dissociated and -associated COX-2, respectively, in peritoneal M{phi}

Additional studies following intraperitoneal administration of bacterial endotoxin (LPS) or HK-C. parvum showed that soluble LPS induced catalytically active NE-associated COX-2, and HK-C. parvum induced catalytically inactive, NE-dissociated COX-2 at 24 h (data not shown). These results further support the conclusion that NE-dissociated (dense form) COX-2 expression depends on phagocytosis.

The effects of peritoneal fluid isolated from HK-BCG-treated mice on localization of COX-2 in vitro

Our results suggest that extracellular factors produced by in vivo HK-BCG treatment regulates the localization of COX-2 in peritoneal M{phi}. To test this hypothesis, CFDA-labeled or unlabeled normal peritoneal M{phi} were challenged with HK-BCG and cocultured with peritoneal cells, cell-free peritoneal fluid, or sera isolated from HK-BCG-treated mice. All CFDA-labeled M{phi} expressed NE-associated COX-2 in response to HK-BCG in vitro and the localization was unchanged by coculture with peritoneal cells, peritoneal fluid, or sera from HK-BCG-treated mice (Table I) or from normal mice (data not shown). Thus, it appears that extracellular mediators produced by HK-BCG treatment in vivo do not induce the NE-dissociated form of COX-2 in M{phi} treated in vitro with HK-BCG.


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  		Table I. The effects of peritoneal fluid isolated from HK-BCG-treated mice on the localization of COX-2 in vitro

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
NE-dissociated COX

It is well-established that catalytically active COX-2 is localized in the NE/ER of activated human monocytes and umbilical vein endothelial cells, as well as murine NIH 3T3 cells, and splenic M{phi}, where it mediates PGE2 biosynthesis (30, 31, 32, 34). However, our present and previous studies (34) demonstrate that most splenic and peritoneal M{phi} isolated from mice 24 h after receiving 1 mg of HK-BCG i.p. express catalytically inactive COX-2. This inactive COX-2 appears as a densely stained structure and is completely dissociated from the NE. Constitutively expressed COX-1 is also detected in a dense form dissociated from the NE in these M{phi}. Thus, catalytically inactive forms of COX-1 and COX-2 are present, without PGE2 biosynthesis, in activated splenic (34) and peritoneal M{phi}. However, it is of particular note that the stimulation of normal M{phi} with HK-BCG in vitro results in expression of catalytically active COX-2, which is localized in the NE/ER. Therefore, factors induced in vivo by HK-BCG administration are obligatory for establishing the catalytically inactive form of COX and consequently the regulation of PG production.

Role of phagocytosis

Another provocative finding is that intracellular HK-BCG is observed in nearly all NE-dissociated COX-2+ peritoneal M{phi} isolated from mice 24 h after i.p. administration of HK-BCG (Fig. 7). Thus, phagocytosis of HK-BCG in the local tissues is significantly associated with expression of the NE-dissociated COX-2, although there are a few COX-2+ M{phi} without intracellular HK-BCG and a few COX-2– M{phi} with intracellular HK-BCG in the same samples. These and our earlier studies indicate that the in vivo phagocytosis of HK-BCG by M{phi} in various tissues results in the formation of catalytically inactive COX-2. In preliminary studies in which HK-BCG was administered intranasally, this phenomenon was also demonstrated in alveolar M{phi} (data not shown). However, for mice receiving lower doses of HK-BCG, M{phi} subsets with NE-associated (active) COX-2 and intracellular HK-BCG are present in relatively greater numbers in peritoneal M{phi} (Fig. 7). In contrast, peritoneal M{phi} activated in vitro with HK-BCG show only NE/ER-associated, catalytically active COX-2 with intracellular HK-BCG.

Furthermore, our studies with peritoneal M{phi} in culture (Table I) do not support the hypothesis that extracellular factors produced in response to HK-BCG in the peritoneal cavity or present in sera regulate the subcellular localization of COX isozymes. Thus, it appears that other endogenous factors associated with phagocytosis in vivo but not in vitro are important for the formation of catalytically inactive COX-2. Because the coculture studies presented in Table I were performed for only one set of time intervals, it is possible that, for instance, the early transient expression of a particular cellular mediator is critical for regulating the subcellular location of COX and its activity.

As presented in Fig. 5, the membrane (M) fraction from the M{phi} treated in vivo has less COX activity than the same fraction taken from cells treated in vitro, despite almost equivalent COX protein levels. This suggests that either 1) the COX isozymes in the membrane fractions of the cells treated in vivo have been structurally modified, rendering them less active, or 2) there is a nonstructural (biochemical) basis for this difference. One possibility is that the reduction/oxidation (redox) states of the membrane fractions differ between M{phi} exposed to HK-BCG in vivo and those exposed in vitro. The activities of both COX-1 and COX-2 are modulated by ambient hydroperoxide availability (38). It may be speculated that the sequestered NE-dissociated COX found in peritoneal M{phi} from HK-BCG-treated mice is, at least in part, a consequence of a low "peroxide tone" which limits COX activity.

COX localization in other studies

Other recent studies indicate that COX-2 localizes not only to the NE but also other subcellular sites. D’Avila et al. (39) have reported that intrapleural administration of live BCG induces lipid-laden pleural M{phi} in a TLR2-dependent but phagocytosis-independent manner. In these M{phi}, COX-2 is expressed and localized at lipid bodies within 24 h, and mediates a large amount of PGE2 synthesis. For phorbol ester (PMA)-stimulated bovine aortic endothelial cells, Liou et al. (40) found that COX-2 is present in cytosolic vesicle-like structures, and that PGI2 synthesis by these cells is not enhanced. In PMA- and IL-1β-treated fibroblasts, catalytically active COX-2 is found in the plasma membrane colocalized with caveolin (41). Girotti et al. (42) indicated that catalytically active COX-2 is localized in the phagosomes of peritoneal M{phi} after in vitro phagocytosis of zymosan particles. The localization of cPLA2{alpha} followed by COX-2 to the phagosome correlated with the time course of PGE2 production, suggesting that the phagosome membrane may serve as a site for release of AA and prostanoid production. However, the magnitudes of PGE2 biosynthesis at the phagosome and NE were not reported (42). It is predicted that without colocalization of COX-2, cPLA2{alpha}, and PGES, PGE2 synthesis does not occur and COX-2 is apparently inactive. Thus, regulation of COX-2 activity associated with its subcellular localization appears to be complex, dependent on cell types and specific activating agents.

COX-2 is not associated with phagosome

In our study using M{phi} activated in vivo, the dense form of COX-2 does not appear to be directly associated with intracellular HK-BCG (Fig. 3) or lysosome-associated membrane protein 1-positive late phagosomes (data not shown). Furthermore, activation of M{phi} in vitro also indicated that there is no direct association of COX-2 with intracellular HK-BCG (Fig. 3). Spencer et al. (36) demonstrated in their mutation analysis of COXs that the mutant proteins, which lack membrane binding domains and enzyme activity, are distributed in the microsomal fraction. They suggested that these mutant proteins are mostly present as unfolded aggregates. Although the membrane-binding domains of COX-1 and COX-2 appear to be important for maintaining their catalytic activity, the mechanisms underlying NE dissociation and enzyme inactivation are still unknown.

Role of COX-2 localization

In response to bacterial components, M{phi} become bactericidal with increases in NADPH oxidase/superoxide anion release, inducible NO synthase/NO production, and IL-12/TNF-{alpha} synthesis. PGE2 down-regulates Th1 responses and bactericidal activity toward intracellular organisms. It is therefore reasonable to speculate that catalytically inactive COX-2+ M{phi} enhance the development of bactericidal activities more effectively than M{phi} with catalytically active COX-2. In our studies, at 24 h following treatment with 1 mg of HK-BCG, both COX-1 and COX-2 are NE dissociated and inactive. Compartmentalization of COX might aid the development of bactericidal activity by placing this enzyme in a location where catalysis cannot occur. Whether additional posttranslational modification is involved in this regulation of COX activity is not known.

Conclusion

Our present and previous findings (12, 34) indicate that normal peritoneal and splenic M{phi} treated with HK-BCG in vitro express catalytically active COX-2 and release increased amounts of PGE2 within 24 h. However, administration of HK-BCG activates various tissue M{phi} locally and systemically to express either catalytically active or inactive COX-2, dependent on route of administration, dose, timing, in vivo phagocytosis and the presence of bone marrow-derived PGE2-M{phi} progenitors, which localize and mature at inflammatory sites. Although more studies are needed to elucidate regulatory mechanisms for the diversity of COX-2+ M{phi} formation in vivo, it appears that the distinct COX-2+ M{phi} subsets may play pro- and anti- inflammatory roles.


   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 RO1 HL71711, Department of Defense DAMD 17-03-1-0004, the Charles E. Schmidt Biomedical Foundation (to Y.S.), and Florida Atlantic University. Back

2 Address correspondence and reprint requests to Dr. Yoshimi Shibata, College of Biomedical Sciences, Florida Atlantic University, 777 Glades Road, P.O. Box 3091, Boca Raton, FL 33431-0991. E-mail address: yshibata{at}fau.edu Back

3 Abbreviations used in this paper: M{phi}, macrophage; COX, cyclooxygenase; HK, heat killed; BCG, bacillus Calmette-Guérin; NE, nuclear envelope; ER, endoplasmic reticulum; PGES, PGE synthase; cPLA2, cytosolic phospholipase A2; PI, propidium iodide; AA, arachidonic acid; TMPD, N,N,N',N'-tetramethyl-p-phenylenediamine; CFDA, carboxyfluorescein diacetate. Back

Received for publication April 30, 2007. Accepted for publication September 1, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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