Interleukin-6 and tumour necrosis factor-α cooperatively promote cell cycle regulators and proliferate rheumatoid arthritis fibroblast-like synovial cells.

Objective: To elucidate the roles of interleukin-6 (IL-6) and tumour necrosis factor-α (TNF-α) in cell cycle regulation and proliferation of rheumatoid arthritis fibroblast-like synovial cells (RA-FLSs). Methods: Under stimulation with IL-6/soluble interleukin-6 receptor (sIL-6R) and TNF-α, we examined the expression of cell cycle regulators [p16INK4a, p21Cip1, p27Kip1, cyclin-dependent kinase-4 (CDK4), CDK6, Cyclin D, Cyclin E, and retinoblastoma protein (pRB)] by quantitative polymerase chain reaction, Western blotting, and immunofluorescence staining. The expression of pRB, with or without 10% foetal bovine serum, was examined by Western blotting. DNA synthesis and cell viability were examined by the BrdU assay and WST-8 assay, respectively. After transfection with siRNA/p16INK4a, siRNA/p21Cip1, siRNA/p27Kip1, siRNA/CDK4, or siRNA/CDK6, RA-FLSs were successively stimulated with or without IL-6/sIL-6R or TNF-α to determine cell viability. Results: IL-6/sIL-6R significantly decreased the expression of p16INK4a, and increased p21Cip1, Cyclin E1, CYCLIN D, and pRB. TNF-α decreased the expression of CDK4, and significantly increased p27Kip1, CDK6, Cyclin E1/E2, CYCLIN D, CYCLIN E, pRB, and phosphorylated pRB (phospho-pRB). By immunofluorescence staining, CYCLIN D and phospho-pRB were simultaneously stained in the single cell. In serum-free culture, the expression of pRB was apparently decreased. DNA synthesis and cell viability were significantly increased by IL-6/sIL-6R and TNF-α. Silencing of CDK6 attenuated the cell viability induced by IL-6 and TNF-α. Conclusion: The results indicate that IL-6 and TNF-α interact with each other in regulating the cell cycle and accelerate the proliferation of RA-FLSs.

TNF-α modulates expression of the circadian clock gene Per2 in rheumatoid synovial cells.

OBJECTIVES: To study the effect of tumour necrosis factor (TNF)-α, responsible for the inflammation and circadian rhythm of rheumatoid arthritis (RA), on the expression of circadian clock genes in primary cultured human rheumatoid synovial cells. METHOD: The expression of circadian clock genes, including circadian locomotor output cycles kaput (Clock), brain and muscle Arnt-like protein-1 (Bmal1), period (Per)1/2, and cryptochrome (Cry)1/2, and the proline and acidic amino acid-rich basic leucine zipper (PAR bZip) genes, a transcriptional activator of Per2, including D site of albumin promoter binding protein (Dbp), hepatic leukaemia factor (Hlf), and thyrotroph embryonic factor (Tef), and a transcriptional repressor of Per2, E4-binding protein 4 (E4bp4), in TNF-α-stimulated synovial cells was determined by real-time polymerase chain reaction (PCR). The D-box motifs in the Per2 promoter were mutated by site-directed mutagenesis, and the promoter activity of the Per2 gene was examined using the luciferase assay. RESULTS: TNF-α enhanced the mRNA expression of Bmal1 and Cry1 but did not affect that of Clock, Per1, or Cry2. However, TNF-α inhibited the mRNA expression of the Per2 gene, as well as Dbp, Hlf, and Tef, but enhanced the mRNA expression of E4bp4. Furthermore, TNF-α inhibited the transcriptional activity of the wild-type Per2 gene in a manner dependent on the D-box 1 and D-box 2 motifs in the Per2 promoter. CONCLUSIONS: TNF-α modulates the expression of the Per2 gene through the D-box binding proteins DBP, HLF, TEF, and E4BP4, in rheumatoid synovial cells, and thereby may contribute to the pathogenesis of RA.

Ganglioside GM3 promotes cell migration by regulating MAPK and c-Fos/AP-1.

Gangliosides have been proposed as modulators of transmembrane signaling. Recently, GM3, a glycosphingolipid containing monosaialic acids, is thought to be one of the key molecules of signal transduction in mammalian cells. In this study, we used mouse embryonic fibroblast cell lines (MEFs) established from sialyltransferase-I knockout mice (GM3 synthase KO mice) to evaluate the regulation of mitogenic signals by gangliosides. Cell proliferation assay revealed a higher growth potential of GM3 KO MEFs. Immunoblots showed upregulation of Ras/Raf/MEK/ERK pathway in GM3 KO MEFs, and these signals resulted in enhanced translocation of ERK into the nuclei. Further, both exogenous and endogenous add-back of GM3 decreased the activities of MAPK in GM3 KO MEFs. In addition, GM3 KO MEFs formed foci in high-density culture condition, and analyses of cell cycle modulators revealed the resistance of GM3 KO MEFs for entering cell cycle arrest. Finally, sustained expressions of c-Fos in GM3 KO MEFs were shown to correlate with DNA-binding activity between c-Fos and AP-1. These results demonstrate that the deletion of sialyltransferase-I changes the character of MEFs to a highly activated state of the MAPK pathway, indicating the critical role of GM3 as a regulator of membrane-transmitted signals.

15-deoxy-delta(12,14)-PGJ(2) induces synoviocyte apoptosis and suppresses adjuvant-induced arthritis in rats.

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily and have a dominant regulatory role in adipocyte and monocyte differentiation. PPAR-gamma agonists are also negative regulators of macrophage activation and have modulatory effects on tumorigenesis. In this study we demonstrate that synovial tissue localized expression of PPAR-gamma in patients with rheumatoid arthritis (RA). We detected markedly enhanced expression of PPAR-gamma in macrophages, as well as modestly enhanced expression in the synovial lining layer, fibroblasts, and endothelial cells. Activation of the PPAR-gamma by 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) and the synthetic PPAR-gamma ligand (troglitazone) induced RA synoviocyte apoptosis in vitro. Moreover, intraperitoneal administration of these PPAR-gamma ligands ameliorated adjuvant-induced arthritis with suppression of pannus formation and mononuclear cell infiltration in female Lewis rats. Anti-inflammatory effects of 15d-PGJ(2) were more potent than troglitazone. These findings suggest that PPAR-gamma may be an important immunoinflammatory mediator and its ligands, especially 15d-PGJ(2), may be useful in the treatment of RA.

Expression of cyclooxygenase-1 and -2 in human colorectal cancer.

Several studies indicate that nonsteroidal anti-inflammatory drugs including indomethacin, aspirin, sulindac, and piroxicam reduce the risk of colon cancer. Furthermore, nonsteroidal anti-inflammatory drugs that inhibit the cyclooxygenase (COX) enzyme were shown to inhibit the development of colon cancer in animal models of carcinogenesis. Non-steroidal anti-inflammatory drugs inhibit the enzymatic activity of both the constitutive (COX-1) and inducible (COX-2) isoforms of COX enzyme. We have investigated the expression of COX-1 and COX-2 polypeptides in human colon cancer tissues using immunohistochemistry. Enhanced COX-2 expression was observed in colon cancer tissues from 15 subjects with clinically diagnosed colorectal cancer. Marked COX-2 expression was observed in cancer cells, inflammatory cells, vascular endothelium, and fibroblasts of the lesional tissues compared with the nonlesional and normal colon tissues. The extent and intensity of the immunoreactive COX-2 in cancer cells was much greater than that of the other cell types. In contrast, the expression of COX-1 polypeptide was weak in both normal and cancerous specimens. These data suggest that the enhanced expression of the COX-2 gene in colon cancer tissues may contribute to the enhanced synthesis of prostaglandin E2 by the colon cancer tissues. Enhanced expression of COX-2 may play a role in the pathogenesis of colon cancer. Furthermore, selective inhibition of COX-2 may prove to be more efficacious in the retardation of colon cancer development.

Urocortin expression in synovium of patients with rheumatoid arthritis and osteoarthritis: relation to inflammatory activity.

Peripherally produced CRH acts as a local auto/paracrine proinflammatory agent. Urocortin is a new member of the CRH family that acts through the family of CRH receptors. In this study, we demonstrated that the expression of urocortin mRNA in synovia of patients with rheumatoid arthritis was greater than that of patients with osteoarthritis. Also, we detected urocortin and CRH receptor immunoreactivity in the synovial lining cell layer, subsynovial stromal cells, blood vessel endothelial cells, and mononuclear inflammatory cells from the joints of rheumatoid arthritis and osteoarthritis patients. The expression of immunoreactive urocortin was significantly greater in rheumatoid arthritis than osteoarthritis (P < 0.0001) and correlated with the extent of inflammatory infiltrate. CRH receptor immunoreactivity was strong in mononuclear inflammatory cells of rheumatoid arthritis synovia. Urocortin stimulated IL-1beta and IL-6 secretion by human peripheral blood mononuclear cells in vitro. These findings suggest that, like CRH, urocortin is present in peripheral inflammatory sites, such as rheumatoid synovium, and acts as an immune-inflammatory mediator.

Preferential inhibition of cyclooxygenase-2 by meloxicam in human rheumatoid synoviocytes.

The aim of this study was to evaluate the anti-inflammatory effect of 4-hydroxy-2-methyl-N-[5-methyl-2-thiazolyl]-2H-1, 2-benzothiazine-3-carboxamide-1,1-dioxide (meloxicam) using cultured rheumatoid synovial fibroblast-like cells (synoviocytes). Synoviocytes were treated with meloxicam in the presence or absence of interleukin-1beta. Meloxicam had no effect on both cyclooxygenase-1 and -2 expression as determined by Western blot analysis, immunohistochemical staining, and reverse transcription polymerase chain reaction (RT-PCR). Even the lower doses of meloxicam inhibited cyclooxygenase-2 activity, but only the higher doses of meloxicam inhibited cyclooxygenase-1 activity as determined by prostaglandin E(2) synthesis assay. So meloxicam had a preferential inhibitory effect of cyclooxygenase-2 relative to cyclooxygenase-1 on cultured rheumatoid synoviocytes without affecting cyclooxygenase expression. On the other hand, indomethacin had no selectivity and dexamethasone inhibited the expression of cyclooxygenase-2. Our data indicate that clinical efficacy and safety of meloxicam for rheumatoid arthritis may result from its preferential inhibition of cyclooxygenase-2 activity relative to cyclooxygenase-1 on rheumatoid synoviocytes.

Auranofin inhibits interleukin-1beta-induced transcript of cyclooxygenase-2 on cultured human synoviocytes.

The aim of this study was to characterize the effects of auranofin (2,3,4,6-tetra-O-acetyl-l-thio-beta-D-gluco-pyranosato-S) on cyclooxygenase expression and prostaglandin E(2) synthesis on cultured human synovial fibroblast-like cells (synoviocytes). Synoviocytes were treated with auranofin in the presence or absence of interleukin-1beta. Cultured supernatants were harvested for prostaglandin E(2) synthesis. Cyclooxygenase-1 and -2 expression was analyzed with Western and Northern blotting. Translocation of nuclear factor-kappa B p65 was determined by immunostaining. Cytotoxicity was measured with 51Cr release assay. Auranofin attenuated interleukin-1beta-induced prostaglandin E(2) production of the cells in a dose-dependent fashion. Auranofin selectively suppressed interleukin-1beta-induced cyclooxygenase-2 mRNA and protein expression of the cells without alteration of cyclooxygenase-1 expression. Also, auranofin interfered with interleukin-1beta-induced translocation of nuclear factor-kappa B. These inhibitory effects did not originate in the cytotoxicity of the agent. Our data indicate that auranofin inhibits interleukin-1beta-induced prostaglandin E(2) synthesis and cyclooxygenase-2 expression via suppression of nuclear factor-kappa B activation on synoviocytes.

[A case of severe neuropsychiatric lupus erythematosus treated by plasmapheresis: diagnostic values of serum antiribosomal P protein antibodies and interleukin-6 in cerebrospinal fluid].

We report here a case of neuropsychiatric lupus erythematosus with organic brain syndrome and transverse myelitis which was successfully managed by plasmapheresis. A 27-year-old female with facial rash, arthralgia and fever was diagnosed as having SLE and treated with oral prednisolone (PSL) in June 1996. After 6 weeks she demonstrated muscle pain and a spiking temperature. The dose of PSL was increased but clinical symptoms did not improve. In August, pulse methyl-PSL was performed and she subsequently-developed delirium, impairment of orientation, memory and perception, which were followed by paraplegia of the lower extremities and loss of sphincter control. Intravenous bolus cyclophosphamide was not effective, but liver dysfunction, bone marrow suppression and respiratory failure due to an infection of pneumocystis carinii were observed. We then performed plasmapheresis or immunoabsorption several times. After this treatment steady improvement was observed. High values of antiribosomal P protein antibodies in the serum and interleukin-6 in the cerebrospinal fluid decreased. Small foci of increased signal intensity detected on cranial magnetic resonance imaging and hypoperfused areas on single-photon emission CT diminished. The patient was maintained on low-dose PSL and no recurrence has been observed 15 months from the onset.

[A case of systemic lupus erythematosus complicated with marked intestinal edema and paralytic ileus].

A 43-years-old female was admitted to our hospital because of facial erythema and photosensitivity in 1983 and was diagnosed as systemic lupus erythematosus (SLE). She was treated with betamethazone 2.5 mg/day as an outpatient. Abdominal pain and diarrhea were developed in September, 1995. So she was admitted to our hospital and diagnosed as having paralytic ileus. Ultrasonography showed marked intestinal edema. Forbidden oral intake and given antibiotics, she was satisfactorily improved in a few days, but the symptoms got worse soon. We forbid oral intake again. We performed a pulse therapy with 1 g of methylprednisolone and increased a dose of betamethasone 2.5 to 4.0 mg/day, but the symptoms were not improved. Since October 6th, serum creatinine level (s-CRE) increased to 8.1 mg/dl at October, 20th. We suspected the worsening of SLE nephropathy or drug-induced nephropathy, so we stopped a medication of pravastatin and used 50 mg/day of azathioprine. Moreover, we did a pulse therapy of methylprednisolone and a plasmapheresis. By these treatments, s-CRE level returned to normal range and paralytic ileus was completely improved. The cause of hypercreatininemia was suspected to be rhabdomyolysis due to pravastatin. The main cause of paralytic ileus with intestinal edema was suspected to be vascular disturbance of superior mesenteric arteries. We consider that pulse therapy and plasmapheresis are useful for a patient of SLE with marked intestinal edema and paralytic ileus.

Anchorage on fibronectin via VLA-5 (alpha5beta1 integrin) protects rheumatoid synovial cells from Fas-induced apoptosis.

BACKGROUND: Rheumatoid synovial cells are resistant to apoptosis induction in vivo, whereas, fibroblast-like synovial cells in rheumatoid arthritis (RA-FLS) are vulnerable to Fas-induced apoptosis in vitro. OBJECTIVE: To clarify this discrepancy by studying the contribution of the interaction between cellular integrin and matrix fibronectin (Fn), which is significantly increased in the rheumatoid joints, to the induction of apoptosis in RA-FLS. METHODS: Integrin and Fas mRNAs were measured by reverse transcription-polymerase chain reaction in RA-FLS. Integrins expressed in rheumatoid synovial tissues were analysed by immunohistochemistry. RA-FLS plated either on Fn or on control poly-L-lysine were incubated with agonistic anti-Fas monoclonal antibodies (mAbs). Apoptosis induction was evaluated using terminal deoxynucleotidyl transferase mediated UTP nick end labelling (TUNEL) and immunoblotting for caspase-3 and poly (ADP-ribose) polymerase in the presence or absence of anti-VLA-5 mAb. RESULTS: VLA-5 (alpha5beta1 integrin), a major integrin expressed on RA-FLS, was required for the adhesion of RA-FLS on Fn. RA-FLS plated on Fn were more resistant to Fas-induced apoptosis than those plated on control poly-L-lysine. This protection by Fn was reversed by anti-VLA-5 mAb. CONCLUSION: Anchorage of RA-FLS on matrix Fn via VLA-5 protects RA-FLS from Fas-induced apoptosis, and Fn abundantly present in rheumatoid synovium appears to afford RA-FLS resistance against apoptosis induction in vivo.

Death receptor 3 (DR3) gene duplication in a chromosome region 1p36.3: gene duplication is more prevalent in rheumatoid arthritis.

The death receptor 3 (DR3) gene is a member of the apoptosis-inducing Fas gene family. In the current study, fluorescence in situ hybridization (FISH) and Fiber-FISH revealed the existence of a second DR3 gene approximately 200 kb upstream of the original DR3 gene. The existence of the duplicated DR3 gene was confirmed by sequencing the corresponding human artificial chromosome clones as well as with quantitative PCR that measured the ratio of the DR3 gene mutation (Rm), intrinsic to rheumatoid arthritis (RA) patients, by simultaneous amplification of the normal and mutated DR3 sequences. The DR3 gene duplication measured by FISH was found to be more frequent in patients with RA as compared to healthy individuals. We therefore surmise that the human DR3 gene can be duplicated and that this gene duplication is more prevalent in patients with RA.

Helicobacter pylori water extract induces interleukin-8 production by gastric epithelial cells.

In Helicobacter pylori-associated gastric mucosal injury, interleukin (IL) -8, a potent leukocyte chemoattractant, is produced by epithelial cells infected by H. pylori and directs neutrophils to the gastric mucosa. According to previous studies, the IL-8 production requires direct contact between the bacteria and epithelial cells. The aims of the present study were to determine whether an H. pylori water extract (HPE) induces IL-8 production by gastric epithelial cells and to characterize IL-8-inducing substances in HPE. Extracts were prepared from a standard strain and from strains obtained from patients with gastric ulcers. After addition of HPE to MKN 45 cells, a gastric cancer cell line, IL-8 in supernatants and IL-8 mRNA were measured by immunoassay and reverse transcription-polymerase chain reaction, respectively. For characterization, active fractions obtained by gel filtration of standard-strain HPE were treated by heating or trypsinization. To study the signal pathway leading to IL-8 production, inhibitors for protein kinase A (PKA), protein kinase C (PKC), or protein tyrosine kinase (PTK) were incubated with MKN45 cells before HPE stimulation. HPE from the standard strain and one of these clinical strains induced IL-8 production. Lipopolysaccharide or cagA in the strains showed no correlation with IL-8 concentration. Standard-strain HPE induced IL-8 mRNA expression in MKN 45 cells. Gel filtration localized activity to a low-molecular-weight fraction of about 7 kDa, which was resistant to heat and trypsin digestion. PKC inhibitors significantly blocked HPE-induced IL-8 production by MKN 45 cells; however, the PKA inhibitor or PTK inhibitors showed a partial inhibitory effect. HPE contains a nonprotein substance of low molecular weight that is responsible for IL-8 induction in gastric epithelial cells. This induction is mainly dependent on the activation of PKC but partially also dependent on PKA or PTK.

The expression and localization of fibroblast growth factor-1 (FGF-1) and FGF receptor-1 (FGFR-1) in human breast cancer.

Fibroblast growth factor-1 (FGF-1) is an inducer of angiogenesis, the growth of new blood vessels. The expression and localization of FGF-1 (acidic FGF) and FGF receptor (FGFR)-1 in mammary tissues from patients with breast cancer was investigated using Western blot analysis and immunohistochemistry. The affinity-purified FGF-1 antibody which did not have cross-reactivity to FGF-2 (basic FGF) was used in this study. Western blot analysis demonstrated the presence of FGF-1 protein in all of the samples from breast cancer, but not benign tumors such as mastopathy and fibroadenoma. To assess the localization of FGF-1 in cancer tissues, immunostaining with specific antibody was performed. All samples from breast cancer displayed significantly intense staining with FGF-1 antibody. The extent and intensity of immunoreactive FGF-1 polypeptides in cancer cells was statistically much greater than those of cells from fibroadenoma or mastopathy. Control immunostaining with normal rabbit serum or anti-FGF-1 antibody adsorbed with the recombinant FGF-1 polypeptide was completely negative. In contrast to FGF-1, Western blot analysis demonstrated the presence of FGFR-1 protein in all of the samples from breast cancer and benign tumors. By immunohistochemical analysis, the enhanced expression of FGFR-1 was observed in breast cancer cells. Benign tumor cells or interstitial cells displayed a faint expression of FGFR-1. These results demonstrated that breast cancer cells not only generated FGF-1, but also expressed FGFR-1, and FGF-1 might play a role in the proliferation of breast cancer cells not only by paracrine but also by autocrine mechanism.

Polymorphisms of the tumor necrosis factor alpha locus among autoimmune disease susceptible and resistant inbred rat strains.

Inbred rat strains manifest remarkable differences in susceptibility/severity to autoimmune disease. MHC alleles strongly influence the pathogenesis of autoimmune disease in rats, but the precise mechanism(s) remain inadequately defined. The TNFalpha gene is located in the class III region of the MHC. Polymorphisms, influencing either the structure or expression of the TNF protein, might contribute to differences in autoimmune disease susceptibility/severity. We therefore sequenced the Tnf locus using genomic DNA from ACI, BB(DR), BN, DA, F344, and LEW rats that vary in susceptibility/severity to autoimmune diseases. We found 42 polymorphisms among these six strains. Although none of these polymorphisms are predicted to change the amino acid sequence of the TNF protein, several reside in potential non-coding regulatory regions and may influence expression levels. These polymorphisms may serve as good candidates for analysis of TNF expression to elucidate the mechanism(s) by which the MHC regulates susceptibility and/or severity of autoimmune diseases.

ACTH expression in synovium of patients with rheumatoid arthritis and Lewis rats with adjuvant arthritis.

Abstract Adrenocorticotropic hormone (ACTH) and another pro-opiomelanocortin-derived neuropeptide, ß-endorphin (ß-End), are stimulated by corticotropin-releasing hormone (CRH) at the anterior pituitary. CRH and ß-End have predominantly proinflammatory effects in peripheral inflammatory sites. We have supposed that inflammatory stimuli develop ACTH as well as ß-End. In this study, we investigated the expression of ACTH in inflamed synovial tissue from patients with rheumatoid arthritis (RA) and osteoarthritis (OA), and at inflammatory joints with adjuvant-induced arthritis (AA) in female Lewis (LEW/N) rats. The expression of ACTH immunostaining was significantly greater in synovium of RA patients than in that of OA patients (P < 0.0001), and correlated with the extent of inflammatory mononuclear cell infiltration. Extensive and intense intracellular ACTH immunostaining, which correlated with the advance in arthritis score, was observed in the synovial lining layer, inflammatory mononuclear cells, and fibroblast-like cells of synovium and chondrocytes in LEW/N rats with AA. In addition, we performed double immunostaining of the same sections from arthritic joints in rats with anti-ACTH and anti-CRH antibodies. ACTH and CRH colocalized in inflammatory mononuclear cells and fibroblast-like cells. ACTH may play a role in the pathogenesis of RA as well as CRH.

Selective inhibition of cyclooxygenase-2 with antisense oligodeoxynucleotide restricts induction of rat adjuvant-induced arthritis.

The effects of cyclooxygenase (COX)-2 antisense oligodeoxynucleotide (ODN) in induction of adjuvant-induced arthritis were investigated. Female Lewis rats were injected with Mycobacterium butyricum intradermally at the base of tails to induce arthritis. Synthetic 18 mer phosphorothioate ODNs corresponding to the translation initiation site of rat COX-2 mRNA were prepared. The antisense (AS), sense (S), and "scrambled" (Sc) ODNs were intraperitoneally administered. Arthropathy was evaluated with arthritis score, paw edema, and histological examination. Expression of COX-1 and -2 protein and mRNA were examined with immunostaining and reverse-transcription polymerase chain reaction, respectively. COX-2 AS ODN significantly suppressed induction of arthritis in a dose-dependent manner without severe adverse effects, whereas S and Sc ODNs did not show significant inhibitory effects. COX-2 mRNA and protein expression were also suppressed only by COX-2 AS ODN without any alteration of COX-1 expression. These data suggest that selective inhibition of COX-2 with AS ODN may have a therapeutic potency in the treatment of rheumatoid arthritis.

C-myc antisense oligodeoxynucleotides can induce apoptosis and down-regulate Fas expression in rheumatoid synoviocytes.

OBJECTIVE: To investigate the role of c-myc in the pathogenesis of rheumatoid arthritis (RA) and the mechanism of synovial apoptosis. METHODS: Using cultured human synoviocytes from patients with RA and c-myc antisense oligodeoxynucleotides (AS ODN), we examined the inhibition of cell proliferation by the MTT assay and the induction of apoptosis with TUNEL staining and fluorescence microscopy. In addition, the effect of c-myc on down-regulation of Fas expression was analyzed by flow cytometry, cytotoxicity assay, and reverse transcriptase-polymerase chain reaction. RESULTS: Treatment with c-myc AS ODN induced inhibition of cell proliferation, along with down-regulation of c-Myc protein and c-myc messenger RNA (mRNA) expression. The morphologic changes of synovial cell death were typical of apoptosis. In addition, c-myc AS ODN treatment down-regulated expression of Fas mRNA but not Fas antigen. Analysis of the involvement of the caspase cascade revealed that the cytotoxic activity of c-myc AS ODN was completely blocked by inhibitors of both caspase 1 (YVAD-FMK) and caspase 3 (DEVD-FMK). CONCLUSION: Our results strongly suggest that c-myc AS ODN might be a useful therapeutic tool in RA and clarify that cell death by c-myc AS ODN is induced through the caspase cascade, similar to Fas-induced apoptosis. In addition, combination therapy with anti-Fas antibody and c-myc AS ODN reduced Fas-dependent cytotoxicity.

Morphological study of cytotoxicity produced by PSK-induced polymorphonuclear leukocytes (PMNs) and Nocardia rubra cell wall skeleton.

The morphologic changes in PMNs induced by an i.p. injection of PSK, a polysaccharide from the mycelia of Coriolus versicolor, and tumor cells undergoing cell death, were evaluated by immunohistochemical staining and electron microscopy. Male C3H/He mice, 8-10 -weeks old, received an i.p. injection of 125 mg/kg of PSK. Their PMNs were obtained 6 h after the PSK injection by peritoneal lavage. N-CWS (Nocardia rubra cell wall skeleton) was added at the start of the chromium release assay using the MM46 mammary carcinoma cell line, which is syngeneic to C3H/He mice, as target cells. During the cytotoxic assay, the cells were fixed at various time points. The MM46 cells expressed ICAM-1 while the PMNs expressed both ICAM-1 and LFA-1 as determined by immunohistochemical staining and immunoelectron microscopy using anti-ICAM-1 and anti-LFA-1 antibodies. PMNs with ruffle-like microvilli adhered to the MM46 tumor cells 30 min after the addition of N-CWS. Immunoelectron microscopic findings suggested that the adhesion molecules were LFA-1 on the PMNs and ICAM-1 on the MM46 tumor cells, but cell fusion between the PMNs and tumor cells was not observed. The MM46 tumor cells gradually lost their microvilli, which showed cell damage, and died 6-7 h after the addition of the N-CWS. This time course of tumor cell death is compatible with the results of the cytotoxic assay. Pretreatment of PMNs by anti-LFA-1 antibody suppressed 1% lysis of MM46 tumor cells from 90% to 10% (p < 0.01). These data suggest that adhesion molecule on the surface of PMNs such as LFA-1 might play an important role on signal transduction of these PMNs cytotoxic function in this experimental system.