INTRODUCTION

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APOPTOSIS IS AN IMPORTANT MECHANISM for eliminating cells without perturbing surrounding cells and tissues and is observed in many biological phenomena, such as involution of the thymus, turnover of enteric crypt epithelial cells, and remodeling of embryonic tissues (25). Recent studies suggested that cells undergo apoptosis as a result of altered expression of some proto-oncogenes and tumor suppresser genes that are controlled by hormones and/or cytokines (5, 12). However, little information is available on factors that trigger the cellular mechanism leading to apoptosis.

The life span of polymorphonuclear leukocytes (PMN) is fairly short compared with those of other leukocytes (20). Even under physiological conditions, cultured PMN spontaneously undergo apoptosis in the absence of any stimuli, and apoptotic cells are phagocytosed by macrophages (16-19). The life cycle of PMN is regulated by many factors, including granulocyte-macrophage colony-stimulating factor (GM-CSF) (4, 6), nerve growth factor (10), interleukin-1 (5), interleukin-2 (14), and granulocyte colony-stimulating factor (G-CSF) (1, 6).

Although large numbers of PMN migrate through the mucosal layers of the intestinal and respiratory tracts and appear in their luminal compartments, the biochemical properties of the infiltrated cells are not known. The circulating PMN (CPMN) have been known to be primed by various ligands, such as lipopolysaccharide (LPS), interleukin-1, and TNF- (7, 22, 23). We previously reported that the CPMN undergo priming during the migration into the oral cavity and spontaneously release reactive oxygen species, including superoxide, hypochloride, and nitric oxide (26). Although oral PMN (OPMN) are further activated by various ligands, the fate of these activated cells is not known. The present work reports the fate and biochemical properties of OPMN.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Chemicals. Superoxide dismutase (SOD), catalase, thiourea, and N-acetyl-cysteine were purchased from Sigma Chemical (St. Louis, MO). All other chemicals used were of analytical grade and obtained from Wako Pure Chemical (Osaka, Japan). Inhibitors of caspase-3 (N-acetyl-Asp- Glu-Val-Asp-aldehyde; Ac-DEVD-CHO) and caspase-1 (Ac-YVDK-CHO) were purchased from Peptide Institute (Osaka, Japan).

Preparation of PMN. OPMN were obtained from healthy human subjects as described previously (15). Briefly, 1 h after brushing the teeth without toothpaste, the oral cavity was thoroughly washed with 15 ml of Krebs-Ringer-phosphate buffer solution (KRP) for 15 periods of 30 s each. The combined solution (150-200 ml) was centrifuged at 250 g for 5 min. The precipitated cells were resuspended in 10 ml of KRP and filtered through a nylon filter (300 mesh) to separate OPMN from oral epithelial cells and cell debris. OPMN-enriched filtrate was centrifuged at 250 g for 5 min, and the precipitated cells were resuspended in 5 ml of KRP. OPMN thus prepared were overlayered on 3 ml of Polymorphprep (Nycomed Pharma, Oslo, Norway) and centrifuged at 450 g for 30 min at 20°C. The cells collected from the interface between KRP and Polymorphprep were washed with KRP, suspended in KRP (1 × 10⁸ cells/ml), and kept on ice until use for the experiments. CPMN were isolated from the fresh blood of healthy volunteers as described for the isolation of OPMN. Rat peritoneal PMNs (RPPMN) were obtained from the rat 16 h after intraperitoneal injection of 2% nutrose as described previously (22). Rat circulating PMNs (RCPMN) were obtained from fresh blood as described previously (22). Cell viability was tested by the trypan blue dye exclusion method.

Culture of PMN. Freshly isolated PMN were cultured in a RPMI1640 medium (10⁶/ml) supplemented with 10% FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin. After varying times of incubation, aliquots of cells were removed, washed once in phosphate-buffered saline (PBS), and analyzed for viability and apoptosis.

Light and electron microscopic observation. At varying times after incubation, cells were centrifuged and the precipitated cells were fixed in 0.1 M sodium cacodylate buffer (pH 7.4) containing 2.5% glutaraldehyde. The PMN thus treated were postfixed in 1% osmium tetroxide, stained en bloc with 2% uranyl acetate, dehydrated with graded ethanol and propylene monoxide, and embedded in resin (Epon 812). Serial sections of each specimen were cut on a diamond knife, mounted on formvar film-coated single-slot grids, and then stained with uranyl acetate and lead citrate solutions.

Analysis of DNA fragmentation. DNA was extracted from the washed OPMN. Briefly, 10⁶ cells were lysed in 0.5 ml of ice-cold 10 mM Tris · HCl buffer, pH 8.0, containing 20 mM EDTA and 0.25% Triton X-100, and suspended for 10 s. The lysate was centrifuged at 15,000 g and 4°C for 10 min. The supernatant fraction was incubated with RNase (20 µg/ml) at 37°C for 1 h, and then, 0.1 volume of 10% sodium dodecyl sulfate (SDS) was added to the mixture, which was then incubated at 55°C for 30 min. DNA was extracted from the supernatant with an equal volume of phenol-chloroform-isoamyl alcohol (25/24/1) and precipitated with 1 volume of 2-propanol in the presence of 0.3 M sodium acetate, pH 5.2. The precipitate was collected by centrifugation at 15,000 g and 4°C for 10 min, rinsed with 70% ethanol, dried, and suspended in 10 mM Tris · HCl buffer, pH 8.0, containing 1 mM EDTA. The DNA samples were electrophoresed on 1.7% agarose gel in 40 mM Tris-acetate buffer, pH 8.0, containing 1 mM EDTA and 0.2 µg/ml ethidium bromide. DNA was visualized by a FluoroImager SI (Molecular Dynamics, Sunnyvale, CA).

Lactate dehydrogenase activity in culture medium. Lactate dehydrogenase in the supernatant of the culture medium was measured using a colorimetric kit (Wako, Osaka).

Assay of cellular glutathione level. Cellular glutathione was determined by the glutathione recycling method using glutathione reductase as described (24).

Assay of caspase activity. Cells (1 × 10⁶) were lysed in 50 µl of lysis buffer (50 mM Tris · HCl, pH 7.4, 0.5% Nonidet P-40, 0.5 mM EDTA, and 150 mM NaCl) at 4°C for 30 min. The lysates were then centrifuged at 20,000 g for 10 min. Caspase activity in the supernatant was determined in 20 mM HEPES buffer, pH 7.5, containing 0.1 M NaCl and 5 mM DTT at 37°C using 10 µM Ac-DEVD-MCA. The fluorescence of released 7-amino-4-methyl-coumarin (AMC) was measured using a fluorospectrophotometer (Molecular Device Gemini). The wavelengths for the excitation and emission were 355 and 460 nm, respectively. One unit of the enzyme is defined as the amount of the enzyme represented for the release of 1 nmol AMC/h.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Change in viability of PMN. Figure 1 shows changes in the viability of cultured OPMN, CPMN, RPPMN, and RCPMN as determined by the trypan blue dye exclusion test. The viability of OPMN and RCPMN markedly decreased within 24 h. In contrast, more than 85% of CPMN and 75% of RPPMN remained intact at 24 h of culture, after which their viability decreased slowly. The activity of lactate dehydrogenase in the culture medium increased more rapidly with OPMN and RCPMN than with CPMN and RPPMN.

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Viability of polymorphonuclear leukocytes (PMN). Freshly isolated oral PMN from human (OPMN), circulating PMN from human (CPMN), circulating PMN from rat (RCPMN), and peritoneal PMN from rat (RPPMN) were suspended in RPMI1640 medium (1 × 106/ml) supplemented with 10% FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin. After varying times of incubation, aliquots were removed and centrifuged at 250 g for 10 min. A: cells were washed once in phosphate-buffered saline (PBS) and assayed for viability using trypan blue dye exclusion method. , CPMN; , OPMN; , RCPMN; , RPPMN; B: supernatants were diluted in PBS, and lactate dehydrogenase (LDH) activity was measured using a colorimetric kit (Wako). , CPMN; , OPMN; , RCPMN; , RPPMN (n = 10).

Morphological changes of OPMN. During culture under physiological conditions, OPMN gradually showed morphological changes characteristic of those of apoptosis (Fig. 2). After isolating from oral cavity, phagocytosis of bacteria was invariably seen (Fig. 2A). After more than 4 h, the nuclei were characterized by continuous condensation of chromatin abutted on the nuclear envelopes (Fig. 2, B-D). Numerous vacuoles were seen throughout the cytoplasm (Fig. 2, C and D). After 12 h of culture, most of the OPMN underwent apoptosis.

View larger version (154K): [in this window] [in a new window]   Fig. 2.   Morphological features of OPMN. Electron micrographs: A: OPMN at 0 h; B: OPMN at 4 h; C: OPMN at 8 h; D: OPMN at 12 h.

DNA fragmentation in OPMN. To elucidate the mechanism for cell death of OPMN, possible involvement of DNA fragmentation was studied. Upon agarose gel electrophoresis, DNA samples isolated from cultured OPMN revealed a marked fragmentation, particularly after 4 h of culture (Fig. 3). In contrast, DNA fragmentation of CPMN became apparent only after 12 h of culture.

View larger version (40K): [in this window] [in a new window]   Fig. 3.   Time-dependent DNA fragmentation of PMN. A: experimental conditions were the same as in Fig. 1. PMN were cultured for 0, 2, 4, 8, and 12 h before the extraction of DNA. Extracted DNA was subjected to 2% agarose gel electrophoresis and visualized by ethidium bromide. DNA from 1 × 106 PMN was applied to each lane. Mr, Molecular marker. B: percentage of apoptotic cells. Apoptotic cells were counted under light microscopy. Open bars, OPMN; filled bars, CPMN.

Changes in cellular glutathione. To determine the reduced glutathione (GSH) status in PMN, glutathione levels in OPMN, CPMN, RPPMN, and RCPMN were determined during culture (Fig. 4). Total glutathione levels (GSH + 2GSSG) in OPMN and RCPMN markedly decreased during 12 h of culture, whereas those in CPMN and RPPMN remained unchanged during the period of observation.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Level of intracellular glutathione. Intracellular glutathione levels were determined by the glutathione recycling method (24). A: * P  0.01, compared with OPMN (0 h) and CPMN (0 h); # P  0.01, compared with OPMN (0 h) and OPMN (12 h); n = 10. B: ## P  0.01, compared with RCPMN (0 h) and RCPMN (12 h); n = 10.

Effect of antioxidants on OPMN. GSH and related thiols play important roles in the survival of various cells. To elucidate the possible involvement of oxidative stress in the mechanism of cell death, the effect of GSH and related thiols on the apoptosis of OPMN was examined. Activation of caspase-3 and apoptosis of OPMN were strongly inhibited by the presence of a low concentration of L-cysteine (Fig. 5). Similar results were also observed with GSH, although its protective effect was lower than that of L-cysteine. L-Cystine scarcely showed such a protective effect. We previously reported that OPMN spontaneously generated reactive oxygen species, including superoxide, hypochloride, and hydroxyl radicals (26). To clarify the role of active oxygen species in OPMN apoptosis, we examined the inhibitory effects of their scavengers on the viability and caspase-3 activity of OPMN. The presence of either SOD, catalase, or thiourea in the medium failed to inhibit the activation of caspase-3 and the occurrence of OPMN apoptosis. These results suggested that changes in the thiol status in and around OPMN might play critical roles in the suppression of their apoptosis.

View larger version (26K): [in this window] [in a new window]   Fig. 5.   Effect of antioxidants on OPMN apoptosis. OPMN (1 × 106 cells/ml) were cultured for 12 h in the presence of various antioxidants. The viability of PMN was determined by the dye-exclusion test (A, C). Caspase-3 activity in OPMN was measured by cleavage of the specific fluorogenic substrate DEVD-AMC (B, D). A: , superoxide dismutase (SOD); , catalase; , SOD + catalase; , thiourea. B: lane 1, untreated cells; lane 2, +10 unit/ml SOD; lane 3, +10 unit/ml catalase; lane 4, 10 unit/ml SOD + 10 unit/ml catalase; lane 5, +1 mM thiourea. C: , L-cysteine; , D-cysteine; , L-cystine; , reduced glutathione (GSH); ×, N-acetyl-cysteine (NAC). D: lane 1, untreated cells; lane 2, +100 µM L-cysteine; lane 3, +100 µM D-cysteine; lane 4, +10 mM NAC; lane 5, +100 µM L-cysteine; lane 6, +1 mM GSH. * P  0.01; n = 10.

Effect of various inhibitors on cell death. To elucidate the possible involvement of protein kinases and caspase in the apoptosis of OPMN, the effect of specific inhibitors was tested. In the presence of herbimycin A in the medium, the activation of caspase-3 and the occurrence of OPMN apoptosis were inhibited (Fig. 6). In contrast, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) did not inhibit the occurrence of apoptosis.

View larger version (16K): [in this window] [in a new window]   Fig. 6.   Effect of protein kinase inhibitors on OPMN apoptosis. OPMN (1 × 106 cells/ml) were cultured for 12 h in the presence of various kinase inhibitors. A: viability of OPMN was determined by the dye-exclusion test. , herbimycin; , 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7). B: caspase-3 activity in OPMN was measured by cleavage of the specific fluorogenic substrate DEVD-AMC. Lane 1, untreated cells; lane 2, +5 µM herbimycin; lane 3, +10 µM H-7. * P  0.01; n = 10.

View larger version (10K): [in this window] [in a new window]   Fig. 7.   Effect of caspase inhibitors on OPMN. OPMN (1 × 106 cells/ml) were cultured for 12 h in the presence of various caspase inhibitors. Viability of OPMN was determined by the dye-exclusion test. Lane 1, untreated cells; lane 2, +10 µM Ac-YVDK-CHO; lane 3, +10 µM Ac-DEVD-CHO. * P  0.01, n = 10.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The present work demonstrates that cultured OPMN rapidly and spontaneously undergo apoptosis, whereas apoptosis of CPMN occurs fairly slowly. Recent studies revealed that apoptosis of PMN was partially inhibited by a variety of cytokines (5, 12). Of the cytokines influencing apoptosis in human PMN, G-CSF has been known to exhibit a potent antiapoptotic activity. Because OPMN spontaneously generated reactive oxygen species, oxidative stress elicited by these metabolites might trigger the reaction leading to cell death. Consistent with this notion is the present finding that GSH levels in OPMN were significantly lower than those in CPMN and decreased markedly during the short time of culture. Takei et al. (21) reported that, after activation of either phorbol 12-myristate 13-acetate or opsonized zymosane, human CPMN rapidly underwent apoptosis by some mechanism that was inhibited by thiourea, a hydroxyl radical scavenger.

Because activation of caspase-3 and the occurrence of OPMN apoptosis were inhibited by fairly low concentrations of either GSH or L-cysteine, oxidative stress and/or change in thiol status in and around these cells might be important for triggering the events leading to apoptosis. OPMN spontaneously generated reactive oxygen species, including superoxide, hypochloride, and hydroxyl radicals (26). To elucidate the role of their reactive species in the mechanism of OPMN apoptosis, we examined the effect of various scavengers. Because SOD, catalase, and thiourea failed to inhibit OPMN apoptosis, factors other than extracellular superoxide, hydrogen peroxide, and hydroxyl radicals might be responsible for the induction of cell death. Although L-cystine is taken up by various cells and reduced to L-cysteine inside cells (2, 3), it failed to inhibit the apoptosis of OPMN. Furthermore, impermeant D-cysteine (~0.1 mM) was as effective as L-cysteine in inhibiting the apoptosis of OPMN. Therefore, extracellular thiols might play important roles in the inhibition of OPMN apoptosis. To gain further insight into the molecular mechanism of OPMN apoptosis, dynamic aspects of extracellular thiols in the oral cavity and in OPMN should be studied further.

When the circulation PMN infiltrate into the peritoneal cavity, they undergo irreversible aging. However, the present work shows that the peritoneal PMN survive longer than PMN obtained from rat blood. Furthermore, GSH in peritoneal PMN was maintained at higher levels than that in PMN from rat blood samples. Thus GSH levels in PMN may not always decrease during aging.

OPMN are always exposed to saliva that contains high concentrations of peroxidase, SCN, and H₂O₂. In addition, the parotid gland possesses the ability to concentrate SCN. HOSCN exists in equilibrium with its conjugate base hypothiocyanite anion (OSCN). Both HOSCN and OSCN are potent oxidants, thereby cellularly oxidizing GSH (8). It is not surprising that the GSH levels in OPMN were lower than those in CPMN.

We previously reported that tyrosine kinase inhibitors, such as herbimycin A, but not protein kinase C inhibitors such as H-7, inhibited the generation of reactive oxygen species by OPMN (13). Tyrosine kinase inhibitor, but not the protein kinase C inhibitor, inhibited the cell death of OPMN. Thus spontaneous generation of reactive oxygen species might play an important role in triggering metabolic events leading to apoptosis.

Bcl-2, a proto-oncogene product, plays a role in inhibiting apoptosis in various cells. Previous studies (9, 11) indicated that the expression of the bcl-2 gene is apparent with early myeloid cells of the bone marrow but not with CPMN. Furthermore, bcl-2 was shown to be below detectable levels and caspase-3 was activated in OPMN (13) . Thus oxidative stress might easily induce apoptosis of OPMN. In fact, inhibitors of caspase-3 inhibited the apoptosis of OPMN. Thus OPMN spontaneously generate reactive oxygen species, thereby increasing oxidative stress, which triggers the metabolic cascade, leading to the activation of caspase-3 in bcl-2-deficient OPMN, thus inducing their apoptosis.