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Am J Physiol Cell Physiol 293: C184-C190, 2007. First published March 21, 2007; doi:10.1152/ajpcell.00346.2006
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PROTEIN AND VESICLE TRAFFICKING, CYTOSKELETON

Differential subcellular localization of COX-2 in macrophages phagocytosing heat-killed Mycobacterium bovis BCG

Makiko Yamashita,1 Shoutaro Tsuji,1 Akihito Nishiyama,1 Quentin N. Myrvik, Ruth Ann Henriksen,2 and Yoshimi Shibata1

1Department of Biomedical Sciences, Florida Atlantic University, Boca Raton, Florida; and 2Department of Physiology, Brody School of Medicine at East Carolina University, Greenville, North Carolina

Submitted 21 June 2006 ; accepted in final form 27 January 2007


   ABSTRACT
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Cyclooxygenase-2 (COX-2)-mediated prostaglandin E2 (PGE2) biosynthesis by macrophages downregulates microbicidal activities in innate and acquired immune responses against intracellular bacteria. Previous studies in mice showed that intraperitoneal administration of heat-killed Mycobacterium bovis bacillus Calmette-Guérin (HK-BCG) resulted in induction of splenic PGE2-releasing macrophages in 7–14 days. In contrast, HK-BCG induced catalytically inactive COX-2 at relatively high levels in the macrophages within 1 day. In the present study, we found that COX-2 was localized subcellularly in the nuclear envelope (NE) 7 and 14 days after HK-BCG treatment, whereas COX-2 was dissociated from the NE 1 day after treatment. At 1 day after treatment, the majority of COX-2-positive macrophages had phagocytosed HK-BCG. In contrast, no intracellular HK-BCG was detected 7 and 14 days after treatment in COX-2-positive macrophages, where COX-2 was associated with the NE. However, when macrophages phagocytosed HK-BCG in vitro, all COX-2 was associated with the NE. Thus the administration of HK-BCG induces the biphasic COX-2 expression of an NE-dissociated catalytically inactive or an NE-associated catalytically active form in splenic macrophages. The catalytically inactive COX-2-positive macrophages develop microbicidal activities effectively, since they lack PGE2 biosynthesis.

nuclear envelope; autoimmune disease; prostaglandin E2

CYCLOOXYGENASE-2 (COX-2, prostaglandin G/H synthase-2) is induced in local macrophages in response to exogenous and endogenous inflammatory agents and is rate limiting for macrophage prostaglandin E2 (PGE2) biosynthesis. PGE2 released by macrophages regulates various immune responses in autocrine and paracrine fashions. For example, PGE2 inhibits IL-12 production by macrophages (36), inducible nitric oxide (NO) synthase/NO production (12), and NADPH oxidase with release of superoxide anion (14). In contrast, PGE2 promotes IL-10 production by macrophages and Th2 cells (29, 32), dendritic cell antigen presentation (37), regulatory T cell differentiation and function (1), and CXCR4/stromal cell-derived factor-1-mediated hematopoietic stem cell migration (11). Regulation of these events, therefore, may depend on the regulation of PGE2 release by COX-2-positive macrophages (6). The effective in vivo expression of these responses, furthermore, may depend on the presence of an adequate number of COX-2-positive macrophages in specific locations (25, 29, 31). Although the regulation of PGE2 biosynthesis by local tissue macrophages in vivo has been reported, the regulatory mechanisms remain unclear and controversial (8, 13, 25, 35).

In the spleen, PGE2-releasing macrophages may interact closely with lymphocytes to induce a Th1-to-Th2 shift of immune responses (29) in chronic inflammatory diseases, which include mycobacterial infections (22), Leishmania infection (3), syphilitic infection (9), human immunodeficiency virus infection progressing to acquired immunodeficiency syndrome (23), and animal models of autoimmune diseases that are established with Freund's complete adjuvant [heat-killed (HK) Mycobacterium tuberculosis in mineral oil] (2). Recently, we found (28, 29) that various strains of mice (Balb/c, C57BL/6, and IL-10–/–) develop splenic COX-2-positive F4/80-positive macrophages not only 5–21 days, but also 1 day (occasionally 2–3 more days), after intraperitoneal administration of HK Mycobacterium bovis bacillus Calmette-Guérin (HK-BCG). At 5–21 days after treatment, 7- to 10-fold more PGE2 is released by splenic macrophages from HK-BCG-treated than from untreated mice. In sharp contrast, at 1 day after treatment, COX-2 is catalytically inactive, and there is no increase in PGE2 (28). However, in macrophages freshly isolated from normal spleen and peritoneum and treated in vitro with HK-BCG, we found expression of catalytically active COX-2 within 1 day. Thus, at 1 day after HK-BCG treatment in vivo, but not in vitro, splenic macrophages appear to uniquely express catalytically inactive COX-2.

Catalytically active COX-2 is an integral membrane protein that lacks a transmembrane domain and associates with only one face of the membrane bilayer through a monotopic membrane binding domain (34). The active enzyme is localized in the nuclear envelope (NE) and the endoplasmic reticulum (17, 20, 34). PGE2 is effectively synthesized from arachidonic acid (AA) by the combined actions of cytosolic phospholipase A2 (cPLA2), COX-2, and microsomal PGE synthase (mPGES), which are localized in the perinuclear membrane (18, 19). In this study, we have determined that COX-2 activity in splenic macrophages isolated from HK-BCG-treated mice is associated with specific subcellular localization.


   MATERIALS AND METHODS
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RESULTS AND DISCUSSION
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Animals. Nonpregnant female C57BL/6 mice (8–14 wk old; Harlan Laboratory, Indianapolis, IN) were maintained in barrier-filtered cages and fed Purina laboratory chow and tap water ad libitum. Experimental protocols were approved by the Institutional Animal Care and Use Committee of Florida Atlantic University.

Intraperitoneal administration of HK-BCG. As described previously (28), cultured bacteria of the M. bovis BCG Tokyo 172 strain were washed, autoclaved, and lyophilized. The 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 amoebocyte lysate assay (Sigma Aldrich, St. Louis, MO) (28). Groups of mice (3/group) received 1 mg of HK-BCG (5 x 108 bacilli/mg) intraperitoneally on day 0. Control mice received 0.1 ml of saline. Spleens were harvested 1, 7, and 14 days after treatment.

Splenic macrophage preparation. Mice were anesthetized by injection of ketamine (50 mg/kg ip) and xylazine (5 mg/kg ip). Spleens from each group of mice were isolated and minced with scissors. Spleen cells were suspended in RPMI 1640 + 10% FBS at 37°C for 1 h and filtered through a 100-µm mesh. For enrichment of the macrophage fraction (24, 31), spleen cell suspensions were plated at 2 x 107 cells/100-mm culture dish (Falcon, Oxnard, CA) and incubated at 37°C in 5% CO2 in air. After 2 h of incubation, cells were washed with Ca2+- and Mg2+-free PBS for removal of nonadherent cells. Culture dishes were placed on ice for 30 min before a cell scraper (Corning, Corning, NY) was used to harvest the adherent cells, which were washed twice with serum-free RPMI 1640. Viability determined by trypan blue exclusion was >90%. Adherent spleen cells were ~70% macrophages, as estimated by phagocytosis of IgG-opsonized sheep red blood cells and/or cytometrically after the cells were stained with anti-F4/80 (24, 26, 31).

Treatment of splenic macrophages with HK-BCG or LPS in vitro. Adherent splenic macrophages obtained from normal mice (see above) were plated at 106 cells/well of 12-well culture plates (Falcon) and incubated at 37°C in 5% CO2 in air. Cells were cultured with HK-BCG (100 µg/ml) or LPS (1 µg/ml) for an additional 24 h.

Subcellular localization of COX-2 by confocal microscopic analysis. Splenic and peritoneal macrophages prepared as described above were fixed in 4% paraformaldehyde in PBS for 30 min. The fixed cells were permeabilized with PBS containing 0.1% Triton X-100 for 5 min and incubated for 3 h in blocking buffer consisting of PBS with 10% FBS at room temperature. Antibodies were diluted in the same blocking buffer. Cells were incubated with anti-COX-2 antibody (1:500 dilution; Cayman Chemical, Ann Arbor, MI) overnight at 4°C. Subsequently, cells were washed three times with PBS and incubated with FITC-conjugated donkey anti-rabbit IgG (1:500 dilution; Jackson ImmunoReseach, West Grove, PA) for 1 h at room temperature. For detection of the nucleus and HK-BCG, propidium iodine (PI) was mixed at 10 µg/ml with the second antibody solution. After they were washed three times, the cells were examined with a laser scanning confocal microscope (Radiance 2100, Bio-Rad). The images were processed with Adobe Photoshop software. For confirmation of the immunologic specificity of the COX-2 antibody, a blocking peptide (250 µg/ml) of COOH-terminal amino acids 570–598 of COX-2 (DPQPTKTATINASASHSRLDDINPTVLIK; Cayman Chemical) was included in some experiments.

Subcellular fractionation. The method for subcellular fractionation was modified from that published previously (34). Splenic macrophages prepared as described 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. Removal of cell membranes was verified by microscopic examination. Cell debris was removed by low-speed centrifugation (700 g) for 10 min, and nuclei were collected by further centrifugation of the supernatants at 10,000 g for 10 min. Membrane and cytosolic fractions were prepared by ultracentrifugation of the resulting supernatants at 100,000 g for 90 min. Nuclei and membrane fractions were resuspended in 0.1 M Tris·HCl (pH 7.5). Protein concentrations were measured with a bicinchoninic acid assay (Pierce, Rockford, IL) and BSA as standard.

Western blot analysis. Equal amounts of protein were loaded onto SDS-polyacrylamide minigels and separated by electrophoresis (200 V for 45 min). Proteins were then transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA), which was blocked with 10% nonfat dry milk and incubated with anti-COX-2 antibodies (Cayman Chemical) in 5% nonfat dry milk overnight at 4°C. After incubation with peroxidase-conjugated donkey anti-rabbit IgG (1:20,000 dilution; Jackson ImmunoResearch), proteins were detected by chemiluminescence (ECL plus, Amersham, Piscataway, NJ) following the manufacturer's instructions. Specificity of the COX-2 antibody was confirmed with the COX-2 blocking peptide (see above).

COX activity assay. COX activity in isolated cellular fractions was measured with a COX assay kit (Cayman Chemical) following the manufacturer's instructions, with AA as a substrate and N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) as a co-substrate. 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.

Mouse anti-BCG antiserum. Mice were immunized intraperitoneally with 1 mg of HK-BCG, and serum was isolated 14 days later. For the detection of intracellular HK-BCG by confocal analysis, anti-BCG serum was diluted 1:100 with blocking buffer consisting of PBS with 10% FBS. The primary antibody was detected by FITC-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch).

Statistics. Differences between mean values were analyzed by Student's t-test with Statcel software. P < 0.05 is considered statistically significant.


   RESULTS AND DISCUSSION
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 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
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Background. Previously, we demonstrated the biphasic COX-2 protein expression by splenic F4/80-positive macrophages after HK-BCG administration and the discordance between COX-2 expression and PGE2 production 1 day after treatment (28). Treatment of mice with 1 mg of HK-BCG was chosen to achieve an inflammatory response comparable to that associated with mycobacterial infections or Freund's complete adjuvant, as used in models of autoimmune disease. Splenic macrophages isolated from untreated mice release minimal levels of PGE2 (30). Splenic macrophages obtained from mice 7 or 14 days after treatment with 1 mg of HK-BCG and stimulated in vitro with calcium ionophore A-23187 or AA (a COX substrate) released more than eight times more PGE2 than untreated controls (28). We have shown that PGE2 biosynthesis by splenic macrophages 14 days after treatment is inhibited by NS-398, nimesulide, or indomethacin, indicating dependence on COX-2 (29). In contrast, 1 and 3 days after HK-BCG treatment, splenic macrophages showed little increase in PGE2 release (28).

COX and PGE synthases (PGES) are key enzymes for PGE2 biosynthesis. We therefore investigated expression of these enzymes in splenic macrophages by Western blot analysis (28). Splenic macrophages isolated from untreated mice expressed COX-1, cytosolic PGES (cPGES), and mPGES, but not COX-2. COX-2 was detected 1, 7, and 14 days, but not 3 days, after treatment with 1 mg of HK-BCG. COX-1, mPGES-1, and cPGES levels 1, 7, and 14 days after treatment with HK-BCG were similar to those in untreated splenic macrophages. However, 1 day after HK-BCG treatment, the increased COX-2 expression did not result in enhanced PGE2 biosynthesis. Cell-free PGE2 biosynthesis assays confirmed that COX and PGES were catalytically inactive and active, respectively, 1 day after treatment (28). Thus catalytically inactive and active COX-2 were associated with the PGE2 synthesis activities of macrophages 1 and 7 (or 14) days after treatment, respectively.

Different localization of COX-2 expressed by splenic macrophages 1, 7, and 14 days after HK-BCG treatment. To further understand the changes in COX-2 expression and activity, we examined the subcellular localization of COX-2 in splenic macrophages by confocal microscopy. Staining of HK-BCG with PI and anti-BCG serum is shown in Fig. 1A. At 1 day after treatment, COX-2 was dissociated from the PI-stained NE (Fig. 1B, Table 1). PI-stained HK-BCG microbes were also detected 1 day after treatment (Fig. 1B). In contrast, significant numbers of macrophages showed COX-2 localized in the NE and more diffusely distributed in the cytoplasm 7 and 14 days after treatment (Fig. 1B, Table 1). COX-2 localization in these macrophages is similar to localization of catalytically active COX-2 expressed by serum-stimulated NIH/3T3 cells (17). However, not all macrophage COX-2 was associated with the NE 7 and 14 days after treatment; in some cells, COX-2 was dissociated from the NE, as at 1 day after treatment. Table 1 summarizes macrophage cell counts based on NE-associated and NE-dissociated COX-2 distributions. Most COX-2-positive splenic macrophages (99.8%) expressed NE-dissociated COX-2 1 day after treatment, whereas COX-2 was only 54 and 53% dissociated from the NE 7 and 14 days after treatment, respectively, in COX-2-positive macrophages (Table 1). Significant numbers of macrophages expressing NE-dissociated COX-2 also contained intracellular PI-stained HK-BCG microbes (Fig. 1B, Table 1). We found no detectable intracellular BCG in macrophages expressing NE-associated COX-2, suggesting phagocytosis-dependent NE-dissociated COX-2 expression.


Figure 1
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  		Fig. 1. Subcellular localization of COX-2 expressed by splenic macrophages after treatment with heat-killed Mycobacterium bovis bacillus Calmette-Guérin (HK-BCG). A: adherent splenic macrophages from animals treated with HK-BCG were stained with propidium iodide (PI) mixed with normal mouse serum (red) or HK-BCG-immunized mouse serum (green), and HK-BCG microbes were detected by confocal microscopy. B: C57BL/6 female mice (3/group) were treated with HK-BCG (1 mg ip) on day 0, and splenic macrophages were prepared. Cells were examined by confocal microscopy after immunofluorescence staining with anti-COX-2 (green) and PI (red) for the nucleus. COX-2-positive staining was completely blocked by a COX-2-specific blocking peptide (not shown).

 

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  		Table 1. Subcellular localization of COX-2 in splenic macrophages isolated from HK-BCG-treated mice

 
In vitro treatment of macrophages with HK-BCG resulted in NE-associated COX-2. To determine whether HK-BCG induces NE-dissociated COX-2 expression in vitro, splenic macrophages were isolated from normal mice and treated in vitro with HK-BCG (100 µg/ml) or LPS (1 µg/ml) as a positive control for 24 h. Only NE-associated COX-2 expression was detected in HK-BCG- or LPS-treated splenic macrophages (Fig. 2). Treatment with A-23187 for 2 h elicited release of significant amounts of PGE2 from these COX-2-positive macrophages (data not shown). Intracellular HK-BCG was also detected by PI staining (Fig. 2). Our results indicate that NE-dissociated COX-2 expression is induced by HK-BCG in vivo (Fig. 1B), but not in vitro, in splenic macrophages. Peritoneal macrophages isolated 1 day after intraperitoneal administration of HK-BCG also expressed NE-dissociated catalytically inactive COX-2 but not NE-associated catalytically active COX-2 (unpublished observations). Thus our results clearly indicate that the phagocytosis of HK-BCG by macrophages in vivo is related to NE-dissociated COX-2 expression in splenic and, probably, also peritoneal macrophages.


Figure 2
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  		Fig. 2. In vitro treatment of macrophages with HK-BCG resulted in COX-2 expression associated with the nuclear envelope (NE). Normal splenic macrophages were treated with HK-BCG (100 µg/ml) or LPS (1 µg/ml) for 24 h. Cells were examined by confocal microscopy after immunofluorescence staining with anti-COX-2 (green) and PI (red) for the nucleus. Results are representative of 3 separate experiments.

 
Differential expression of COX-2 in subcellular fractions. To further confirm that COX-2 dissociated from the NE is catalytically inactive, splenic macrophages were homogenized and subjected to differential centrifugation 1 and 7 days after treatment. More COX-2 protein was detected in the nuclear than in the membrane fraction 7 days after treatment (Fig. 3A). The profile of COX-2 distribution is similar to that determined for murine macrophages stimulated with LPS (21). Significantly more COX activity was detected in the nuclear fraction than in the membrane and cytosolic fractions (Fig. 3B). In contrast, COX-2 was predominantly detected in the membrane, but not in the nuclear, fraction 1 day after treatment. However, 1 day after treatment, COX activity was only minimal in all the subcellular fractions. Taken together, these results suggest that, 1 day after HK-BCG treatment, COX-2 that is dissociated from the NE and localized in the membrane fraction is catalytically inactive.


Figure 3
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  		Fig. 3. Distribution and activity of COX in subcellular fractions. Splenic macrophages were homogenized and separated into nuclear (N), microsomal membrane (M), and cytosolic (C) fractions by differential centrifugation. A: protein (20 µg) from each fraction was used for detection of COX-2 by Western blot. Immunoreactivity of the COX-2 antibody was blocked by a COX-2-specific peptide (data not shown). B: COX activity in each fraction was measured using the COX assay kit (Cayman Chemical) following the manufacturer's instructions, and specific enzyme activities were calculated. Differences were analyzed by Student's t-test with Statcel software. Values are means ± SE (n = 3). **Significantly different (P < 0.01) from C on day 7 and N, M, and C on day 1.

 
Ontogenic heterogeneity of splenic macrophages between 1 day and 7 or 14 days after treatment. The differential COX-2 activities of splenic macrophages 1 day and 7 or 14 days after treatment (28) appear to be related to their ontogenic heterogeneity. Although COX-2-positive macrophages treated for 1 day and 7 or 14 days expressed F4/80 (28, 29), those treated for 1 day, which seem to be resident in the spleen, directly phagocytose HK-BCG. Our previous studies demonstrated that PGE2-releasing macrophages are derived from radiosensitive bone marrow 7 and 14 days after treatment (24, 28, 31). It is likely that, at the early stages (1–2 days) after HK-BCG treatment, macrophage precursors are induced to migrate from the bone marrow and localize in the spleen, where mature forms of PGE2-releasing macrophages are established within 5–21 days (24, 31). Our results indicate that these bone marrow-derived macrophages express catalytically active COX-2 (PGE2-releasing macrophages), apparently without the phagocytosis of HK-BCG. At 2–3 days after in vivo HK-BCG treatment, COX-2 expression in splenic macrophages is significantly reduced by an undetermined mechanism. We have not determined whether macrophages expressing NE-dissociated COX-2 at 7 and 14 days after treatment include resident macrophages and the recently bone marrow-derived macrophages.

Cytoplasmic COX-2 in splenic macrophages 1 day after treatment. Phagocytosis of HK-BCG in vivo, but not in vitro, appears to be essential for development of NE-dissociated catalytically inactive COX-2 (Figs. 1B and 2, Table 1). In their analysis of COX mutants, Spencer et al. (34) reported that the proteins, which lacked enzyme activity, were distributed in the microsomal membrane fraction. They suggested that these mutant proteins were mostly present as unfolded aggregates that precipitated with membrane fractions. Although Spencer et al. also suggested that the membrane binding domains of COX are important in maintaining the catalytic activity, a precise explanation for the association of cellular localization with enzyme activity or inactivity is unknown. Recently, D'Avila et al. (5) reported that intrapleural administration of live BCG induces lipid-laden pleural macrophages in a Toll-like receptor type 2-dependent, but phagocytosis-independent, manner. After in vivo or in vitro treatment with live BCG, macrophages expressed COX-2 localized at the lipid bodies within 24 h and mediated a large amount of PGE2 synthesis (5). In our study, lipid bodies were not specifically identified. However, we did find that COX-2 was not preferentially co-localized with intracellular HK-BCG (Fig. 1B) or lysosome-associated membrane protein 1-positive late phagosomes (data not shown). Liou et al. (16) found that COX-2 was present in cytosolic vesicle-like structures in PMA-stimulated bovine aortic endothelial cells in vitro and that PGI2 synthesis by these cells was not enhanced compared with that by unstimulated cells. In PMA- and IL-1beta-treated fibroblasts, catalytically active COX-2 was found in the plasma membrane co-localized with caveolin (15). Thus regulation of COX-2 activity associated with its subcellular localization appears to be complex, dependent on cell types and specific activating agents.

Additionally, the adequate coupling of COX-2 with PLA2s providing substrate or the terminal PGES is important for PGE2 synthesis (18, 19, 33). We found previously that PGES activity is intact in macrophages 1 day after treatment with HK-BCG (28). Therefore, mPGES and COX-2 may be in different subcellular compartments 1 day after treatment. In support of this conclusion, we have detected mPGES localized in the NE 1 and 14 days after treatment (data not shown).

Immunologic roles of catalytically inactive COX-2-positive macrophages. Mycobacteria, including M. bovis BCG, are powerful Th1 adjuvants and are used to induce autoimmune diseases in animal models. In response to these components, macrophages become bactericidal, with increases in NADPH oxidase/superoxide anion release (10), inducible NO synthase/NO production (4), and IFN-{gamma}/IL-12/TNF-{alpha} synthesis (27). These components concomitantly induce COX-2 expression and PGE2 biosynthesis. PGE2 downregulates Th1 responses and microbicidal activities (7, 12, 29). It is therefore reasonable to speculate that catalytically inactive COX-2-positive macrophages enhance Th1 responses and development of bactericidal activities more effectively than macrophages with catalytically active COX-2.

It has been well established that bacterial immunomodulators [HK-BCG or HK-Corynebacterium parvum (Propionibacterium acnes)] given intraperitoneally attenuate the release of PGE2 and other eicosanoids by peritoneal macrophages compared with untreated animals (8, 13, 25). Our present and previous findings (28) indicate that the differential subcellular localization of COX-2 in splenic macrophages after intraperitoneal administration of HK-BCG results in different capacities for PGE2 biosynthesis. This is the first report demonstrating that NE-dissociated COX-2, which lacks catalytic activity, is induced in local macrophages phagocytosing HK-BCG. Interestingly, NE-dissociated COX-2 expression is not seen in macrophages phagocytosing HK-BCG in vitro. The reason for this difference is not clear. Thus our study indicates that the generally accepted concept that, in the presence of bacterial components, resident COX-2-negative macrophages are converted to COX-2-positive macrophages with release of relatively large amounts of PGE2 may need further investigation, particularly with respect to the effects of in vivo phagocytosis of intracellular bacteria by macrophages.


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This work was supported by National Heart, Lung, and Blood Institute Grant RO1 HL-71711, US Department of Defense Grant 17-03-1-0004, the Charles E. Schmidt Biomedical Foundation (Y. Shibata), and Florida Atlantic University.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Y. Shibata, Dept. of Biomedical Sciences, Florida Atlantic Univ., 777 Glades Rd., PO Box 3091, Boca Raton, FL 33431-0991 (e-mail: yshibata{at}fau.edu)

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


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