Epidermal growth factor receptor inhibitors enhance susceptibility to Fas-mediated apoptosis in oral squamous cell carcinoma cells.

Molecular inhibition of epidermal growth factor receptor (EGFR) signaling is a promising cancer treatment strategy. We examined whether inhibition of EGFR signaling would affect the susceptibility of oral squamous cell carcinoma (OSCC) cells to Fas-mediated apoptosis. Treatment of OSCC cells with an anti-EGFR monoclonal antibody, C225, and an EGFR tyrosine kinase inhibitor, AG1478, which target the extracellular and intracellular domains of the receptor, respectively, inhibited phosphorylation of EGFR and its downstream effector molecule Akt and amplified the induction of Fas-mediated apoptosis. In OSCC cells treated with EGFR inhibitors, Fas-mediated apoptosis was accompanied by caspase-8 activation but not Bid cleavage. Caspase-3 and -8 inhibitors reduced the effect of EGFR inhibitors on Fas-mediated apoptosis in OSCC cells, but a caspase-9 inhibitor did not. These results indicate that the pro-apoptotic activity of EGFR inhibitors in OSCC cells depends on the extrinsic pathway of the caspase cascade. Although EGFR inhibitors did not affect the expression of Fas, the Fas-associated death domain protein, or procaspase-8 in OSCC cells, the inhibition downregulated cellular FLICE-inhibitory protein (c-FLIP). Moreover, knockdown of c-FLIP in HSC-2 cells with a small interfering RNA strongly enhanced Fas-mediated apoptosis. These results suggest that the EGFR signaling pathway may, in part, regulate Fas-mediated apoptosis in OSCC cells through c-FLIP expression.

Effect of combining epidermal growth factor receptor inhibitors and cisplatin on proliferation and apoptosis of oral squamous cell carcinoma cells.

Epidermal growth factor (EGF) is known to be involved in the proliferation and metastasis of squamous cell carcinoma (SCC), suggesting that the EGF receptor (EGFR) must also contribute to SCC development. In combination with conventional anti-cancer drugs, agents that block EGFR may represent an efficient means of inhibiting proliferation and inducing apoptosis in SCC cells. We investigated the effects of combining an anti-EGFR monoclonal antibody (C225) or an EGFR-selective tyrosine kinase inhibitor (AG1478) with the conventional anti-cancer drug cisplatin on the oral SCC (OSCC) cell lines NA and Ca9-22. We detected constitutive expression of EGFR on the cell membranes of both cell lines. OSCC cell proliferation was inhibited by C225, AG1478 and cisplatin in a dose-dependent manner. The combination of C225 or AG1478 with cisplatin at concentrations

Enhanced susceptibility to tumor necrosis factor-related apoptosis-inducing ligand-mediated apoptosis in oral squamous cell carcinoma cells treated with phosphatidylinositol 3-kinase inhibitors.

In general, oral squamous cell carcinoma (OSCC) cells are relatively resistant to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis during culture in vitro. Here, we studied the role of phosphatidylinositol 3-kinase (PI 3-K)/Akt in survival and apoptosis of these cells. The PI 3-K inhibitors wortmannin and LY294002 markedly suppressed phosphorylation of Akt and accelerated TRAIL-mediated apoptosis in OSCC cells. Addition of TRAIL to PI 3-K inhibitor-treated cells resulted in caspase-8 activation and loss of mitochondrial membrane potential. Furthermore, inhibitors of caspase-3, -8 and -9 reduced the accelerative effect of PI 3-K inhibitors on TRAIL-mediated apoptosis. These results suggest that the pro-apoptotic effect of PI 3-K inhibitors on TRAIL-mediated apoptosis may contribute to both the extrinsic and intrinsic pathways. Although PI 3-K inhibitors did not affect expression of the TRAIL receptors DR4 and DR5, we observed a marked reduction in expression of cellular FLICE-inhibitory protein (c-FLIP), Bcl-2, cellular inhibitor of apoptosis protein-1 (cIAP-1) and X-linked IAP (XIAP), whereas Bax was up-regulated and no significant difference was observed in expression of Bcl-xL, Bak or cIAP-2. Therefore, the PI 3-K/Akt signaling pathway provides partial regulation of TRAIL-mediated apoptosis in OSCC cells via modulation of c-FLIP, Bcl-2, Bax, cIAP-1 and XIAP expression. These results suggest that PI 3-K inhibitors may represent a novel strategy for overcoming resistance to TRAIL-mediated apoptosis in OSCC cells.

Thalidomide suppresses melanoma growth by activating natural killer cells in mice.

Although thalidomide (Thd) is being extensively investigated for its effects on cytokine production and T cell costimulation, it is poorly understood whether it is capable of modulating the activities of natural killer (NK) cells. In this study, Thd effects on NK cell activity were examined with a murine model of melanoma, which is mostly rejected by NK cell-dependent mechanism. Administration of Thd significantly (p<0.01 on Day 21) suppressed the growth of subcutaneous B16F1 melanoma. In Thd-treated mice, marked splenomegaly and augmented splenocyte count were observed. Additionally, the percentage of splenic NK1.1+ cells was elevated to approximately 2.5-fold within 10 days after Thd treatment. The expression of interferon inducible protein (IP)-10, interferon (IFN)-gamma, interleukin (IL)-12 and IL-18 was remarkably upregulated. Production of the cytotoxic molecule perforin was also augmented. These data suggest that Thd strongly activates NK cell activity in mice, possibly resulting in enhanced tumor surveillance defense.

Alveolar bone graft for patients with cleft lip/palate using bone particles and titanium mesh: A quantitative study.

PURPOSE: This study evaluated the bone volume, height, and width that can be obtained in alveolar ridge augmentation using titanium mesh and autogenous bone particles in patients with cleft lip/palate. PATIENTS AND METHODS: Subjects were 15 patients with cleft lip/palate requiring tertiary bone graft for implant therapy. Computed tomography (CT) scans were taken before removing the mesh, from 1 to 21 months after bone grafting. Forty-three reconstructed images corresponding to the positions for implant placement were selected for this study. The percent defect filled with bone (%BONE), defined as the percentage of newly formed bone in the space created by the mesh, was measured for image analyses. In linear analyses, 4 parameters were used: increased bone height (IBH), percent increased bone height (%IBH), increased bone width (IBW), and percent increased bone width (%IBW). Factors influencing the quantitative data and the clinical courses of placed implants were also explored. RESULTS: The average %BONE was 91.1%. IBH averaged 4.4 mm, whereas %IBH averaged 88.5%. IBW averaged 4.6 mm, whereas %IBW averaged 86.4%. Little correlation was present between the quantitative data and patient age, or time interval. A significant correlation was identified between the data for span of the grafted area and %BONE (correlation coefficient value = -0.36). However, the diminishing rate was very low. No implants were lost postoperatively. CONCLUSIONS: Alveolar ridge augmentation with titanium mesh and autogenous bone particles from the anterior iliac crest has very high predictability as a preimplant procedure in patients with cleft lip/palate.

Enhancement of susceptibility to Fas-mediated apoptosis in oral squamous cell carcinoma cells by phosphatidylinositol 3-kinase inhibitor.

In general, oral squamous cell carcinoma (OSCC) cells are relatively resistant to Fas-mediated apoptosis during in vitro culture. Here, we studied the role of survival/apoptosis associated phosphatidylinositol 3-kinase (PI 3-K)/Akt in this process. We found that both PI 3-K inhibitors, wortmannin and LY294002, markedly suppressed the phosphorylation of Akt and accelerated Fas-mediated apoptosis in OSCC cells. It was found that caspase-3 and -8 inhibitors reduced the accelerative effect of PI 3-K inhibitor on Fas-mediated apoptosis in OSCC cells, but not caspase-9 inhibitor. Although PI 3-K inhibitors did not affect the Fas expression of OSCC cells, cellular FLICE-inhibitory protein (c-FLIP) levels were markedly reduced by PI 3-K inhibitor treatment. Moreover, antisense oligonucleotide to c-FLIP confirmed that the down-regulation of c-FLIP enhanced the sensitization to Fas-mediated apoptosis in OSCC cells. These results suggest that PI 3-K/Akt signaling pathway may, in part, regulate Fas-mediated apoptosis in OSCC cells through c-FLIP expression.

Gene expression profiles of oral leukoplakia and carcinoma: genome-wide comparison analysis using oligonucleotide microarray technology.

Although leukoplakia is the most common precancerous lesion of the oral cavity, its molecular biological properties are largely unknown. The aim of this study was to identify the genes responsible for its pathogenesis and malignant transformation using oligonucleotide microarray technology. The expression profiles of 8,800 genes in human oral leukoplakia (n=4) and oral squamous cell carcinoma (OSCC) (n=2) were analyzed using the Affymetrix GeneChip system and the results were confirmed with RT-PCR. Eight genes were up-regulated (>2.0-fold) and ten were down-regulated (<0.5-fold) in all leukoplakias analyzed with the GeneChip. In particular, loricrin and keratins displayed greater differences between normal tissue and leukoplakia. Some of the 18 alternatively expressed genes were markedly down-regulated in squamous cell carcinoma compared with leukoplakia. Our data suggested that gene abnormalities in cytoskeleton network components might be responsible for the development and progression of oral leukoplakia.

Accelerative effect of a selective cyclooxygenase-2 inhibitor on Fas-mediated apoptosis in human neutrophils.

Inflammatory stimuli, such as cytokines, can induce cyclooxygenase-2 (COX-2) expression in neutrophils. Selective, anti-inflammatory COX-2 inhibitors have been developed for patients with acute inflammatory diseases. Recent work has shown that selective COX-2 inhibitors interfere with tumor cell growth. The purpose of this study was to examine the capability of selective COX-2 inhibitors on Fas-mediated apoptosis in cytokine-stimulated neutrophils. Tumor necrosis factor-alpha (TNF-alpha) and granulocyte-macrophage colony-stimulating factor (GM-CSF) enhanced prostaglandin E2 (PGE2) release through the induction of COX-2 in neutrophils. This effect was not seen with either interleukin (IL)-1beta or IL-8. TNF-alpha-and GM-CSF-induced PGE2 release was blocked by the addition of the selective COX-2 inhibitor, N-(2-cyclohexyloxy-4-nitrophenyl)-methanesulfonamide (NS-398; 1 microM). GM-CSF, IL-1beta and IL-8 suppressed Fas-mediated apoptosis in neutrophils; however, this effect was not seen with TNF-alpha. The anti-apoptotic effect of cytokines on Fas-mediated neutrophil apoptosis was attenuated by the addition of NS-398 (100 microM). These results suggest that NS-398 operates via two distinct mechanisms for regulating apoptosis and COX-2 activation in neutrophils. This distinction is indicated by the difference in concentration of NS-398 required for acceleration of Fas-mediated neutrophil apoptosis, and the inhibition of PGE2 synthesis. Moreover, NS-398 suppressed the anti-apoptotic activity of IL-8 and IL-1beta, but did not induce COX-2; therefore, the pro-apoptotic mechanism of the selective COX-2 inhibitor may be unrelated to COX-2 activity. Thus, a selective COX-2 inhibitor may contribute to the reduction of acute inflammation through the enhancement of neutrophil apoptosis.

Modulation of neutrophil apoptosis in plasma of patients after orthognathic surgery.

BACKGROUND: Human neutrophils undergo rapid apoptosis during in vitro culture. The aim of this study was to investigate the role of interleukin-8 (IL-8) on neutrophil apoptosis in surgery-induced inflammation. MATERIALS AND METHODS: Blood samples were drawn from 21 patients with mandibular prognathism 2 days before, and 1 and 5 days after orthognathic surgery. The IL-8 levels in the separated plasma were measured using an ELISA kit. The expression of two receptors for IL-8, CXCR1, and CXCR2, and their role in neutrophil apoptosis was evaluated using a flow cytometer. RESULTS: The IL-8 levels in the plasma were correlated with acute inflammatory markers, such as peripheral blood neutrophil counts and C-reactive protein levels. Both IL-8 receptors were markedly raised in patient-derived neutrophils 1 day post-operatively. Recombinant IL-8 (0-100 ng/ml) suppressed apoptosis in fresh-isolated neutrophils from healthy donors dose-dependently. Neutrophil apoptosis 1 day post-operatively was slightly accelerated in the presence of fetal bovine serum compared to the value 2 days pre-operatively and 5 days post-operatively. In contrast, in the presence of autogenous plasma, neutrophil apoptosis was significantly suppressed 1 day post-operatively compared to the value 2 days pre-operatively and 5 days post-operatively. Moreover, the anti-apoptotic effect of plasma on neutrophil apoptosis was partially decreased by the addition of anti-IL-8 neutralizing antibody. CONCLUSIONS: These results suggest that circulating neutrophils are susceptible to augmentation by IL-8 through the reinforcement of IL-8 receptors in acute inflammatory conditions. Furthermore, IL-8 may, in part, contribute to the regulation of neutrophil survival during the inflammatory response.

Regulation of Fas-mediated apoptosis in neutrophils after surgery-induced acute inflammation.

BACKGROUND: Neutrophils undergo rapid Fas-mediated apoptosis during in vitro culture. The purpose of this study was to investigate the effects of surgical stress upon the Fas-mediated apoptotic response in circulating neutrophils. MATERIALS AND METHODS: Blood samples were drawn from eight patients with a mandibular prognathism, and who had undergone a bilateral sagittal split ramus osteotomy, at 2 days before, and at 1 and 5 days after surgery. The circulating neutrophils in each blood sample were then evaluated for their susceptibility to Fas-mediated apoptosis in either the presence or the absence of autogenous plasma. RESULTS: Fas-induced apoptosis in the neutrophils of these surgically treated patients was found to be slightly accelerated at 1 day postoperatively in the presence of FBS, compared with 2 days preoperatively and 5 days postoperatively. However, we obtained different results for these experiments in the presence of autogenous plasma. The Fas-induced apoptotic response levels in the neutrophils at day 1 postsurgery following exposure to autogenous plasma were significantly suppressed compared with the levels at both 2 days preoperatively and 5 days postoperatively. The Fas expression levels on the cell surface of the neutrophils were not altered, but the levels of soluble Fas (sFas) in the plasma were reduced to almost inverse levels during the postoperative periods. The levels of granulocyte-macrophage colony-stimulating factor, interleukin-6, and interleukin-8 levels in the plasma were also markedly raised in the plasma from each of these patients at 1 day postoperatively. However, the anti-apoptotic effects of the plasma on the Fas-mediated neutrophil apoptosis were not influenced by the addition of their neutralizing antibodies for these cytokines. The suppressive effects of postoperative plasma on Fas-mediated neutrophil apoptosis were blocked by the phosphatidylinositol 3-kinase (PI 3-K) inhibitors, LY294002, and wortmannin. Additionally, these effects were also abrogated by the extracellular signal-regulated kinase (ERK) inhibitor, PD98059, but not by the p38 mitogen-activated protein kinase inhibitor, SB203580. CONCLUSIONS: The increase in sFas levels in the plasma of patients with acute inflammation may lead to the inhibition of Fas-mediated neutrophil apoptosis. Moreover, the activation of the PI 3-K and ERK signaling-dependent pathways may, in part, also contribute to the down-regulation of the Fas-mediated apoptotic response in neutrophils.

Enhancement of susceptibility to Fas-mediated apoptosis in HL-60 cells through down-regulation of cellular FLICE-inhibitory protein by phosphatidylinositol 3-kinase inhibitors.

Promyelocytic leukemia HL-60 cells are resistant to Fas-mediated apoptosis. The signaling pathway for Fas-mediated apoptosis in various cells, including HL-60 cells, is currently unknown. Here, we studied the role of survival/apoptosis associated phosphatidylinositol 3-kinase (PI 3-K)/Akt in this process. We found that both PI 3-K inhibitors, wortmannin and LY294002, markedly suppressed phosphorylation of Akt and Bad in HL-60 cells. PI 3-K inhibitors significantly accelerated not only spontaneous apoptosis, but also Fas-induced apoptosis in HL-60 cells. The pro-apoptotic effect of PI 3-K inhibitors favored Fas-mediated apoptosis rather than spontaneous apoptosis in HL-60 cells. The caspase-3 or -8 inhibitor reduced the pro-apoptotic effect of the PI 3-K inhibitors for Fas-mediated apoptosis in HL-60 cells, but the caspase-9 inhibitor did not. Although PI 3-K inhibitors did not affect Fas expression in HL-60 cells, cellular FLICE-inhibitory protein (c-FLIP) levels were markedly reduced by PI 3-K inhibitor treatment. Furthermore, antisense oligonucleotide of c-FLIP confirmed that down-regulation of c-FLIP enhanced sensitization to Fas-mediated apoptosis in HL-60 cells. These results suggest that the PI 3-K/Akt signaling pathway may, in part, regulate Fas-mediated apoptosis in HL-60 cells through c-FLIP expression.

Clinical evaluation of circulating interleukin-6 and interleukin-10 levels after surgery-induced inflammation.

BACKGROUND: It was previously reported that both pro- and anti-inflammatory cytokines are elevated in systemic inflammatory response syndrome (SIRS). Cytokine-mediated systemic neutrophil activation is a direct consequence of SIRS, and can lead to multiple organ dysfunction syndrome (MODS). This prospective study assessed the risk of SIRS and MODS after orthognathic surgery by measuring the circulating levels of inflammatory cytokines such as IL-6 and IL-10 as well as the neutrophil functions as a marker of organ failure. MATERIALS AND METHODS: Blood samples for the measurement of IL-6, IL-10, CRP, neutrophil counts, and neutrophil function were drawn from 21 patients with mandibular prognathism at 2 days before, and at 1 and 3 days after orthognathic surgery. The neutrophil function was estimated by superoxide production and elastase release under the stimulation of FMLP. RESULTS: Eight of the 21 patients were applicable to SIRS criteria 1 day postoperatively, and all of the subjects were excluded from SIRS criteria 3 days postoperatively. Although IL-6 and IL-10 levels were raised 1 day postoperatively, increased cytokine concentrations were decreased in most patients at 3 days postoperatively. The IL-6 concentration and the ratio of IL-6 to IL-10 were higher in the SIRS-matched group compared with the non-SIRS-matched group. Neutrophil priming for superoxide production and elastase release was discovered 1 day after orthognathic surgery, and differences in those values could not be distinguished between the groups. CONCLUSIONS: These results suggest that a few patients in whom high levels of circulating inflammatory cytokine and neutrophil-derived toxic factor continue may have a possibility of contracting severe diseases such as SIRS and MODS after orthognathic surgery. We conclude that the ratio of IL-6 to IL-10 may be a predictive factor in SIRS.

Multiple granulomatous inflammation in the minor salivary glands: a proposed new entity, allergic granulomatous sialadenitis.

We report a patient who presented with multiple small submucosal nodules with granulomatous inflammation in the minor salivary glands of the oral cavity. A 43-year-old woman presented with a 1-week history of multiple small submucosal nodules in her oral cavity after having taken medicine for abdominal pain. The patient did not have a history of fever, rectal bleeding, skin lesions or arthritis, but did have a history of drug allergy and bronchial asthma. Histopathological examination of the submucosal nodules showed sialadenitis with marked infiltration of lymphocytes, eosinophilic cells, macrophages and Langhans-type or foreign-body-type multinucleate giant cells. The macrophages tended to be aggregated and appeared to have caused immature granuloma formation without caseous necrosis. Degranulated eosinophilic cells were numerous. Sarcoidosis, Crohn's disease, tuberculosis and atypical mycobacterial infection were not identified by medical examination. Three weeks after discontinuing the medication the patient was seen again at a follow-up visit. Multiple submucosal small nodules and other symptoms were not evident at that time. This case report may represent a new entity of salivary gland disease that we tentatively refer to as 'allergic granulomatous sialadenitis'.

Recombinant human bone morphogenetic protein-2 promotes Indian hedgehog-mediated osteo-chondrogenic differentiation of a human chondrocytic cell line in vivo and in vitro.

We examined osteo-chondrogenic differentiation of a human chondrocytic cell line (USAC) by rhBMP-2 in vivo and in vitro. USAC was established from a transplanted tumor to athymic mouse derived from an osteogenic sarcoma of the mandible. USAC usually shows chondrocytic phenotypes in vivo and in vitro. rhBMP-2 up-regulated not only the mRNA expression of types II and X collagen, but also the mRNA expression of osteocalcin and Cbfa1 in USAC cells in vitro. In vivo experimental cartilaginous tissue formation was prominent in the chamber with rhBMP-2 when compared with the chamber without rhBMP-2. USAC cells implanted with rhBMP-2 often formed osteoid-like tissues surrounded by osteoblastic cells positive for type I collagen. rhBMP up-regulated Ihh, and the expression of Ihh was well correlated with osteo-chondrogenic cell differentiation. These results suggest that rhBMP-2 promotes chondrogenesis and also induces osteogenic differentiation of USAC cells in vivo and in vitro through up-regulation of Ihh.

RETRACTED: A human chondrogenic cell line retains multi-potency that differentiates into osteoblasts and adipocytes.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the author, Dr. Kimihito Yagami. Dr. Yagami's collaborators Yohei Uyama, Yasumasa Yoshizawa, Saburo Kakuta, Akira Yamaguchi, Masao Nagumo were not involved in the RT-PCR experiments and figure preparation The editor, Sundeep Khosla, was notified by an independent group that specific bands in Figure 3 of the paper appear to be duplicated. This was brought to the attention of the authors. Due to the long interval from the original publication of the paper, the raw data was not available; however, the authors subsequently chose to retract the entire manuscript, and the editor agreed.

Effect of 5-fluorouracil on G1 phase cell cycle regulation in oral cancer cell lines.

5-fluorouracil (5-FU) has been widely used for chemotherapy of head and neck cancer, and is known to affect the cell cycle and induce apoptotic death of cancer cells. However, the molecular actions of 5-FU on the cell cycle regulatory mechanism have not been fully explained. Herein we analyzed the effects of 5-FU on the expression of G1/S-related cell cycle regulators in oral cancer cell lines. In vitro 5-FU treatment of oral cancer cells resulted in an increase in G1/S phase cells. p21 expression was augmented by 5-FU without any notable changes in p53 expression. A remarkable up-regulation of cyclin E and a concomitant down-regulation of cyclin D were observed after 24 h 5-FU treatment. Our results suggest that 5-FU-induced changes in cell cycle regulation of oral cancer cells might associate with an alteration of G1 cyclins expression. p21 was remarkably up-regulated, but it was speculated that its activity might be cancelled by an increased binding to CDK4.

Inhibition of cyclooxygenase-2 suppresses invasiveness of oral squamous cell carcinoma cell lines via down-regulation of matrix metalloproteinase-2 and CD44.

Expression of cyclooxygenase-2 (COX-2) in tumors is known to be associated with enhanced angiogenesis, suppression of host immunity, and tumor invasion. In the present study, human oral squamous cell carcinoma (OSCC) cell lines NA and HSC-4 were used to evaluate the effects of NS-398, a selective inhibitor of COX-2, and COX-2 antisense oligonucleotide (COX-2 AS) on the invasion activity of OSCC cells. Matrigel invasion assay revealed that the invasiveness of NA and HSC-4 was suppressed by treatment with either NS-398 or COX-2 AS. These reagents down-regulated the secretion of matrix metalloproteinase-2 (MMP-2) to culture supernatant as well as the expression of MMP-2 mRNA and protein. Membrane-type 1 matrix metalloproteinase (MT1-MMP), an activator of proMMP-2, was also down-regulated by treatment with these reagents. Furthermore, expression of CD44 on the surface of these cells was reduced by treatment with either NS-398 or COX-2 AS. In addition, MMP-2 antisense oligonucleotides reduced the expression of CD44 on the surface of both OSCC cell lines. These findings suggest that NS-398 and COX-2 AS suppress the invasiveness of OSCC cells via down-regulation of MMP-2 and CD44. Genetic or pharmacological inhibition of COX-2 may therefore be a beneficial strategy in the treatment of OSCC patients.

Suppressive effect of selective cyclooxygenase-2 inhibitor on cytokine release in human neutrophils.

To clarify whether a selective cyclooxygenase-2 (COX-2) inhibitor can affect various functions in human peripheral blood neutrophils. For this purpose, the effects of selective COX-2 inhibitors, NS-398 and nimesulide, on the expression of COX-2, PGE2 release and respiratory burst, degranulation and cytokine release in activated neutrophils were examined. Peripheral blood neutrophils were stimulated with formyl-methionyl-leucyl-phenylalanine (FMLP; 100 nM) or opsonized zymosan (OZ; 200 microg/ml). Then, the expression of COX-2 at protein and mRNA levels was detected by Western blot analysis and RT-PCR. The concentration of prostaglandin E2 (PGE2) and cytokines in the culture supernatant of neutrophils was determined using ELISA. Superoxide generation was measured by the cytochrome c reduction method. Elastase activity was measured using a chromogenic substrate assay specific for human neutrophil elastase. FMLP and OZ enhanced PGE2 release through induction of COX-2 protein and mRNA expression. FMLP- or OZ-induced PGE2 release was abolished by the addition of NS-398 or nimesulide; nevertheless, even a high concentration of COX-2 inhibitor did not change FMLP- or OZ-induced expression of COX-2 at message and protein levels. Although FMLP- or OZ-induced superoxide generation and elastase release were not affected by the addition of COX-2 inhibitor, cytokine release such as interleukin (IL)-1beta, IL-6 and IL-8 was significantly inhibited by high concentration of COX-2 inhibitor, but tumor necrosis factor-alpha (TNF-alpha) was partially attenuated. These studies showed that selective COX-2 inhibitors, NS-398 and nimesulide, suppressed PGE2 and proinflammatory cytokine release in activated neutrophils. These results suggest that selective COX-2 inhibitors may contribute to resolution of acute inflammation through the reduction of inflammatory cytokine release in activated neutrophils.

Age-related increases in LPS-stimulated nitric oxide production from cultured rat bone marrow cells.

OBJECTIVE: To understand bone metabolism during senescence, we examined age-related change in nitric oxide (NO) production from bone marrow cells stimulated by lipopolysaccharide (LPS). METHODS: We evaluated the age-related change in the NO production and expression of iNOS protein and mRNA of LPS-stimulated bone marrow cells collected from the tibiae of young and retired female and young and retired male rats. In addition, we used flow cytometry to assess changes in the distribution of CD14, a cell surface receptor of LPS. RESULTS: The results revealed that NO production from bone marrow cells stimulated with LPS changed with aging. The NO levels in old rats were significantly higher (P<0.05) than those in young rats. Polymerase chain reaction (PCR) analysis indicated that the LPS-induced expression of iNOS mRNA was augmented in retired rats. Although the distribution pattern of the bone marrow cells was similar between young and retired rats, the percentage of CD14-positive cells in specific populations differed between the age groups. Specifically, in the granule-containing bone marrow cells, the percentage of CD14-positive cells was increased in retired rats. CONCLUSION: Our results indicate that LPS-stimulated NO production from rat bone marrow cells increased with age and that the difference in responsiveness might be due to changes in the percentage of CD14-positive cells in the bone marrow.