We previously demonstrated that TNFα induces rapid apoptosis of human peripheral blood neutrophils. To understand better the in vivo significance of neutrophil apoptosis, we examined using rats TNFα induced apoptosis of neutrophils obtained from various sources. Normal peripheral blood neutrophils (PBN) underwent significant apoptosis, but peritoneally exudated neutrophils (PEN), peripheral blood neutrophils obtained from rats i.p. injected with inflammation inducing agents (inflammatory PBN), and bone marrow neutrophils were resistant to apoptosis induced by TNFα.
Addition to TNFα of cycloheximide in a low concentration that alone were barely able to induce neutrophil apoptosis, enhanced apoptosis of PBN, inflammatory PBN and bone marrow neutrophils but not of PEN, suggesting that in the former three sources of neutrophils certain apoptosis inhibitory protein (s) were synthesized by stimulation with TNFα, and in PEN these protein (s) had been already synthesized in vivo during migration through endotherial spaces.
We further examined in vivo clearance of neutrophils by i.v. injection with ⁵¹Cr labelled neutrophils and found that PBN are promptly scavenged in the liver and the spleen.

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Chronic marginal periodontitis is characterized by dense infiltration of lymphocytes in periodontal connective tissue. In this study, we investigated whether adhesive interactions between human gingival fibroblasts (HGF) and lymphoid cells may cause activation of the HGF. In vitro cell lines of HGF were co-cultured with human lymphoid cell lines for 3 hours. After the culture, IL-1β mRNA expression in HGF was evaluated by reverse transcription-polymerase chain reaction. We found that IL-1β mRNA expression was clearly increased when HGF physically interacted with human lymphoid cell lines, K562 and Ramos, which constitutively demonstrated significant binding ability to HGF but not IL-1β mRNA expression. Moreover, exogenous addition of culture supernatants of the lymphoid cells, which had been cultured in the presence or absence of HGF for 3 hours, did not induce IL 1β mRNA expression in HGF.
These findings indicated that the adhesive interactions between lymphoid cells and HGF should cause signal transduction into HGF and induce the increased expression of IL-1β mRNA in HGF. The present study suggests that HGF would modulate the inflammatory responses in periodontal lesions by interacting with the locally infiltrated lymphocytes.

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Human umbilical vein endothelial cells (EC) in culture were exposed to hypoxiareoxygenation. Superoxide generation in EC was observed quickly after reoxygenation almost within one minute and this was inhibited by allopurinol. In the same experiments, EC injury and neutrophil adherence to EC were measured. EC injury after reoxygenation was detected by both ⁵¹Cr release and cell detachment methods. EC injury was more easily detected by cell detachment method and significantly enhanced by the presence of neutrophils, which was inhibited by allopurinol. Neutrophil adherence to reoxygenated, but not to normal or hypoxic EC was, again, significantly reduced by allopurinol treatment of EC.
These results indicate that superoxide which was produced in reoxygenated EC mediates enhanced neutrophil adherence to EC and EC injury following reoxygenation. Through these mechanisms, superoxide would play an important role in the formation of reperfusion injury besides the possible direct toxic effect to the reperfused tissues.

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Both monocyte chemotactic and activating factor (MCAF) and N-formyl-methionyl-leucyl-phenylalanine (FMLP) stimulated an increase in cytoplasmic free Ca²⁺ ( [Ca²⁺] i) and changes in intracellular pH (pHi) in human monocytes in parallel at lower concentrations, and stimulated superoxide (O^-₂) release and changes in transmembrane potential in parallel at higher concentrations. The changes in pHi were characterized by initial rapid acidification followed by sustained alkalinization, and the changes in transmembrane potential were characterized by initial depolarization followed by partial repolarization. The time-courses of all responses stimulated by MCAF and FMLP were similar to each other, although the magnitude of all responses was less in MCAF-stimulated cells. MCAF by itself was a very weak stimulus for inducing O^-₂ release. However, MCAF primed monocytes and enhanced O^-₂ release stimulated by FMLP. The priming effect of MCAF was maximal within 5 min of preincubation, and the doseresponse curves for priming were identical to those for triggering of an increase in [Ca²⁺] i and intracellular acidification. Intracellular acidification was induced at lower concentration than O^-₂ release by MCAF stimulated monocytes. MCAF further potentiated FMLP-induced O^-₂ release in tumor necrosis factor (TNF) -, granulocyte-macrophage colony-stimulating factor (GM-CSF) -or IL-3-primed monocytes.
These findings suggest that MCAF, alone or in concert with other cytokines, primes monocytes for enhanced release of O^-₂, and that stimulus induced acidification is closely associated with an increase in [Ca²⁺] i, but not O^-₂ release.

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Successful transfer of murine Sjögren's syndrome from MRL/lpr mice into SCID mice and prevention of lesions by anti-CD 4 and -T cell receptor Vβ8 anti body treatment are described. Mononuclear cells is isolated from the inflammatory salivary gland tissues of MRL/lpr were transferred intraperitoneally into SCID mice. Autoimmune lesions resembling those seen in Sjögren's syndrome developed in the salivary and lacrimal glands of SCID mice 8 weeks after the injection. A major proportion of these infiltrating cells in transferred SCID mice were CD 4⁺ and Vβ8⁺ Moreover, we found that the lesions were prevented by administration of the isolated cells treated in vitro with anti-CD 4- and Vβ 8-bearing monoclonal antibodies. These results suggest that CD 4- and Vβ 8-bearing T cells are involved in recognizing an unknown autopetide and triggering autoimmunity in the salivary and lacrimal glands.

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We examined whether apoptosis-associated antigens were expressed in synovial tissues from nine patients with rheumatoid arthritis (RA) and five patients with osteoarthritis (OA) using immunohistochemical staining and DNA nick end labeling.
Paraffin sections of synovial tissues were stained with various antibodies, such as anti-PCNA monoclonal antibody, anti-Fas monoclonal antibody, anti-Le^y monoclonal antibody and anti-bcl-2 polyclonal antibody. DNA nick end labeling was also performed on each of the paraffin sections.
In RA specimens, the PCNA positivity ratio and Fas positivity ratio of synovial lining cells were 16.85±4.28% and 1.97±0.99%, respectively. On the other hand, in OA the corresponding ratios were 4.25±0.66% and 1.58±1.24%, respectively. The degree of proliferation of synovial lining cells was significantly different between RA and OA, but Fas antigens on the synovial lining cells did not differ significantly. DNA nick end labeling and staining with anti-Fas monoclonal antibody were positive in synovial lining cells, macrophages and fibroblasts. Anti-Le^y monoclonal antibody produced positive staining in inflammatory cells. On the other hand, antibcl-2 polyclonal antibody gave a positive reaction in synovial lining cells, macrophages, fibroblasts, inflammatory cells and vascular endothelial cells.
We conclude that apoptosis-associated molecules (for enhancement and inhibition) are expressed in synovial tissues from patients with RA.

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Cyclooxygenase (COX), or prostaglandin (PG) H synthase, is the first exzyme of the pathway in which arachidonic acid is oxidized to PGs. We previously reported that high levels of immunoreactive COX were present in synovia from patients with rheumatoid arthritis (RA), but not in synovia from patients with osteoarthritis (OA) or normal subjects. An inducible isoform of COX, COX2, has recently been identified.
So we examined the expression and modulation of both COX1 and COX2 in rheumatoid synovial tissues and cultured synoviocytes. COX1 and 2 mRNA were present in synovia from RA patients by RT-PCR method. Immunostaining with anti-COX1 and 2 antibodies showed that high levels of COX1 and 2 polypeptides were expressed in rheumatoid synovial tissues (synovial lining cell layer, inflammatory mononuclear cells, fibroblast-like cells, vascular endothelial cells) . Significantly less staining of COX1 and 2 was noted in OA synovia. In cultured rheumatoid synoviocytes under basal condition, both COX1 and 2 mRNA were present at low levels. COX2 mRNA was markedly increased by treatment with IL-1β or PMA. Dexamethason (Dex) suppressed the induction of COX2 mRNA. In sharp contrast, COX1 mRNA was not induced with IL-1β, and Dex did not have suppressive effects.
We suggest that the regulation of COX2 by IL-1β and corticosteroids may be important mechanisms to regulate inflammation in synovial tissues from RA patients.

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The effect of direct irritant action of anti-inflammatory powder drugs (about 2.5mg/kg) in canine gastric mucosa studied using an endoscopic compressed air drug delivery system technique. Aspirin and loxoprofen sodium induced mucosal lesions were limited to the target site of gastric mucosa in all dogs. Indomethacin (30%) and diclofenac Na (40%) induced superficial gastritis at the pyloric antrum, but did not appear to spray sites. Mefenamic acid, ketoprofen and proglumetacin maleate did not induced mucosal lesions. Predonisolone caused the erosion and edema throughout the stomach. No lesions were induced by lactose and sucralfate.
In the present study, we could observe the experimental gastric mucosal lesion at the target site of the gastric mucosa by an administration of a small amount of powdered drugs via an endoscope. The administration of powdered drugs by endoscopy is useful for the investigation of direct effects of those on the gastric mucosa.

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Concentrations of diclofenac in serum and tissues were studied after single oral administration of Voltaren SR capsule in patients with rheumatoid arthritis.
Samples of blood, synovial fluid, synovial membrane, fat and muscle tissues were taken 6 hours or f hours after Voltaren SR administration.
(1) The concentration of diclofenac in the synovial fluid was higher than that of the serum.
(2) The higher concentration of diclofenac in the synovial fluid compared to that of serum lasted for at least 12 hours after administration.
(3) The concentration of diclofenac in the synovial membrane was higher than that in fat and muscle tissues.
These results show that Voltaren. SR capsule has a good distribution to the synovial membrane and synovial fluid, and that it remains there for at least 12 hours.

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The strong microbicidal activity of gold compounds towards Helicobacter pylori (HP) has been recently demonstrated in vitro, raising its potential clinical therapeutic application. Accordingly, we investigated the in vitro activity against fungi and bacteria of anti-rheumatic drugs (auranofin, GST, d-penicillamine, bucillamine, and salazosulfapyridine) . For the assessment of anti-bacterial and anti-fungal activity, an agar dilution test was performed. In brief, 25.6mg of each drug was dissolved in 1 ml of dimethyl sulf oxide, and then the minimum bactericidal concentration and the minimum fungicidal concentration (MIC) were determined by inhibition of the visible growth of organisms during subsequent incubation for 48hrs at 37°C.
Auranofin was found to show some activity against 3 strains of Staphylococcus (S) (S. aureus 1, ug/ml, MRSA 8μg/ml, S. epidermidis 0.5μg/ml), 7 strains of Streptococcus, 2 strains of Enterococcus, 4 strains of clostridium and Candida albicans (4 μg/ml) . A weak activity of GST against gram (-) bacterias was also recognized, but the other antirheumatic drug did not show any anti-bacterial activity.
The mechanism of the antibacterial and antifungal activity of gold compounds is not known, but it is possible to speculate that gold compounds usually combined with active chemical sulfhydryl groups on proteins including enzyme and these binding caused the death of bacteria.

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