Platelet-activating factor (PAF) is not only a potent proinflammatory compound, but also is involved in cell proliferation and differentiation. cDNAs coding for PAF receptor were isolated in our laboratory from human, guinea-pig, rat and mouse. They consist of 341-342 amino acids with 7 putative transmembrane domains, characteristic to a G-protein-coupled-receptor superfamily. The gene for human PAF receptor is localized on chromosome 1, having three exons separated each other. By 5'-alternative splicing, two transcripts (leukocyte- and heart-type) are expressed under the direction of two distinct promoters. Signal transduction of PAF receptor disclosed that it couples with multiple effector systems including phospholipase C activation, inhibition of adenylate cyclase, MAP kinase activation, and phospholipase A₂ activation. These multiple second messenger systems enable the versatile biological activities of PAF, in spite that PAF is a single molecule without any receptor subtypes.

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In an air pouch type allergic inflammation model in rats, PAF antagonists inhibit neutrophil migration into the pouch fluid although levels of PAF in the pouch fluid are less than the detectable amount. To clarify roles of PAF in neutrophil migration, effects of PAF antagonists (CV-6209, L-652, 731, Y-24180) on transendothelial migration of neutrophils were examined in vitro. Histamine and thrombin stimulated LTB4-induced transmigration of neutrophils through the confluent monolayer of human umbilical vein endothelial cells (HUVEC) . Treatment with PAF antagonists selectively inhibited the histamine- and thrombin-induced transendothelial migration, suggesting that PAF plays some significant roles in endothelial cell recognition by neutrophils.
Treatment of HUVEC with TNF-α or IL-1/β induced neutrophil adherence to HUVEC monolayer and expression of adhesion molecules, such as ICAM-1, ELAM-1 or GMP-140. However, PAF antagonists showed no effect on the cytokine-induced neutrophil adherence to HUVEC nor the expression of these adhesion molecules. Furthermore, histamine- and thrombin-induced neutrophil adherence to HUVEC was inhibited by PAF antagonists without altering the expression of these adhesion molecules.
Transendothelial migration of neutrophils was not inhibited by antibodies to GMP-140 and ELAM-1, but was completely inhibited by an antibody to LFA-1/β. An antibody to ICAM-1 partially inhibited the transendothelial migration of neutrophils.
These results suggest that PAF produced by HUVEC plays important roles in signaling for neutrophil adherence to endothelial cells without affecting the expression of these adhesion molecules.

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This study has analyzed the pathogenetic mechanism of the intimal thickening after baloon injury, which is currently a serious problem on the balloon angioplasty for coronary arteriosclerosis, from the aspect of wound-healing inflammation. The locally generated angiotensin II (AII), which is known for its aggravating effect on vascular hypertrophy, has been implicated to participate in the pathogenic mechanism. One month after the balloon injury, dog arteries showed myointimal hyperplasia as well as the increases in AII and AII-generating enzymes, angiotensin-converting enzyme and mast cell chymase, with the latter enzyme increasing more markedly. In the adventitia of lesioned vessels, the density of mast cells and fibrolasts was increased with apparent fibrosis and angiogenesis. These pathohistological findings suggest that the adventitial fibrosis reflects an excessive woundhealing response to the injury, and that the mast cell-fibroblast cross-talk may couple the adventitial fibrosis with the intimal hyperplasia. In this hyperplastic lesion, mast cell chymase seems to play a pivotal role by generating AII, and also by chymase's proinflammatory actions which are not yet fully defined. Thus, it is reasonable to regard the intimal thickening as a manifestation of exaggerated wound-healing response to an injury.

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Activation of human neutrophils by the chemotactic peptide formyl-methionyl-leucyl-phenylal-anine ( FMLP), phorbol myristate acetate ( PMA ) and calcium ionophore (ionomycin) consistently and predominantly induced tyrosine phosphorylation of a microtubule-associated protein kinase (MAPK) . The dose-resposne curves showed that tyrosine phosphorylation and O^-₂ release were stimulated in parallel by PMA, whereas tyrosine phosphorylation and an increase in [Ca²⁺] i, but not O^-₂ release, were stimulated in parallel by FMLP or ionomycin. The potency of inducing tyrosine phosphorylation was ionomycin>FMLP=PMA, whereas the potency of triggering of O^-₂ release was PMA>ionomycin = FMLP. Tyrosine kinase inhibitor (genistein) inhibited O^-₂ release induced by FMLP, but not by PMA, However, genistein did not impair FMLP-or PMA-induced tyrosine phospho-rylation of MAPK. UCN-01, a protein kinase C inhibitor, inhibited O^-₂ release and tyrosine phosphorylation of MAPK induced by PMA, but not by FMLP. Increased tyrosine phosphorylation of MAPK was also detected in immature myeloid cells (HL -60 cells) stimulated by PMA and FMLP, but not by ionomycin.
These findings suggest that tyrosine phosphorylation of MAPK is induced by the PKC dependent and independent mechanisms according to the stimuli, and the PKC-independent and ionomycin-sensitive mechanism is inoperative in HL-60 cells and tyrosine phosphorylation of MAPK is unlikely to be causally related to activation of the respiratory burst.

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Calcitonin gene-related peptide (CGRP) is known to be co-localized with substance P (SP) in sensory C-fibers. We investigated the interaction between these neuropeptides in the superoxide (O^-₂) production of human neutrophils. SP and SP_4-11 fragment (10-100μM) induced O^-₂ production of neutrophils in a dose dependent manner. Phospholipase C inhibitor, U-73122 and neomycin, suppressed the O^-₂ production by SP and SP_4-11 fragment. CGRP (10-20μM) reduced the SF induced O^-₂ production, and this effect of CGRP was blocked by CGRP receptor antagonist, CGRP_8-37 (10μM) . Dibutyryl-cyclic AMP (dBcAMP; 0.5 mM) and phosphodi-esterase inhibitors, 3-isobutyl-1-methyl xanthine (IBMX, 5μM) and theophylline (0.5 mM), also inhibited SP-induced O^-₂ production. The cAMP-dependent protein kinase (A-kinase) inhibitors, H-8 (10μM) and KT5720 (50 nM), suppressed the CGRP-induced effect. These data suggest that CGRP inhibits the SF induced O^-₂ production in neutrophils by interfering with phosphoinositide signaling through receptor linked activation of the cAMP/A-kinase-dependent pathway.

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Aerosol antigen challenge augmented the bronchial responsiveness to acetylcholine in guinea pigs actively sensitized with ovalbumin aerosol, which became significant 7 h and persisted for at least 120 h after antigen exposure as compared to saline treated animals. When orally administered to guinea pigs 1 h before antigen challenge, the specific thromboxane (Tx) A₂ receptor antagonist S-1452 (10 mg/kg) almost completely inhibited the development of brochial hyperresponsiveness, as assessed 24 h after antigen exposure. However, it was ineffective, when administered at 1 or 22 h post-challenge. Acute lung hemorrhagic vascular injury was observed immediately after antigen challenge, when marked production of TxB₂ was observed in the bronchoalveolar lavage fluid, and was dramatically suppressed by pretreatment with S-1452 at doses required to inhibit the bronchial hyperrespon-siveness. These findings suggest that acutely and transiently generated TxA₂ contribute to the onset of aerosol antigen-induced long-lasting bronchial-hyperresponsiveness in actively sensitized guinea pigs, probably by producing lung vascular damage.

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Nitric oxide (NO) has been identified as a potent and multifunctional mediator which is implicated in a wide range of biological functions. Besides its function as a physiological mediator, evidence is accumulating that NO participates in inflammatory and autoimmune-mediated tissue destruction. We have investigated the effects of various herbal extracts on the NO production in lipopolysaccharide (LPS) -stimulated macrophages using a marine macrophage cell line RAW264. Scutellariae radix extract was shown to be most effective in the suppression of inducible NO production from LPS-stimulated RAW264 cells. The extract suppressed the LPS-induced NO production from RAW264 cells in a dose-dependent manner at concentrations higher than 4μg/ml, with 50% inhibitory concentration of 20μg/ml. L-citrulline production was decreased in parallel with reduction of NO release. Treatment of LPS-stimulated RAW264 cells with Scutellariae radix extract resulted in decrease of inducible NO synthase (iNOS) mRNA levels, suggesting that the extract may effect on the iNOS gene transcription and/or stability of iNOS mRNA. Baicalein was demonstrated to be a major contributory component to the suppressive effect of Scutellariae radix extract on the inducible NO production from LPS-stimulated macrophages.

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Therapeutic effects of recombinant human erythropoietin (rHuEPO) in the treatment of anemia associated with rheumatoid arthritis (RA) were evaluated by multi center cooperative trial.
Thirty-nine patients with RA received rHuEPO intravenously at 200IU/kg once a week (Group 1) or 100IU/kg three time a week (Group 2) for 8 weeks.
The mean value of hematocrit (Ht) was 25.7% in Group 1 and 27.4% in Group 2 before the treatment, and increased to 29.6% and 32.1% respectively after 8 weeks. The treatment resulted in significant improvement of Ht in both groups. rHuEPO was more effective in higher serum iron group (>29μg dl) and/or lower plasma EPO concentration group ( ≤ 30mIU/ml) in the base line.
The effective improvement on anemia of “effective” or more was 50 and 65% in Groups 1 and 2, respectively. The global improvement of “improvement” or more was 32% in Group 1 and 55% in Group 2.
Side effects included hypertension in 2 of the 39 patients, presumably as a result of the rapid rise of Ht value. There were no other findings to be marked.
In conclusion, patients with RA showed excellent hemoglobin responses to rHuEPO, although a meaningful change in rheumatic manifestation was not seen.

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A treatment involving the removal of granulocytes from the peripheral blood was attempted in patients with rheumatoid arthritis (RA) using a newly developed extracorporeal adsorbing column containing cellulose acetate beads (G-1 column, Japan Immunoresearch Laboratories, Takasaki) .
A total of 63 patients with RA, from 4 hospitals, was treated from 2 to 10 times (a mean of 6.9 times) over a duration of 1 to 5 weeks (usually for 4 weeks) using the G-1 column. The G-1 column could remove up to 65% of granulocytes, based on comparison between blood entering and leaving the column.
For evaluating the therapeutic efficacy of the G-1 column, 59 patients were studied. The mean number of granulocytes in the peripheral blood decreased from 6754±358 before to 5913±293/μl after G-1 column therapy, a 12.5% reduction. The Lansbury index which measures 4 parameters, swollen and tender joints, morning stiffness, grip strength and erythrocyte sedimentation rate, showed an improvement from 60% to 48% in the 59 patients evaluated. In a subset of 40 patients who received therapy for 4 weeks, the Lansbury index decreased from 60% to 54% at 1 week (p <0.001), to 51 at 2 weeks (p<0.001), to 5100 at week 3 (p<0.01) and to 48% at week 4 (p<0.001) . The most prominent improvements were in tender joint scores from 79±5.6 to 36±5.0 (p< 0.001) and grip strength from 120±9.6 to 137±9.8 mmHg (p<0.001) . No clinically significant side effects were observed in the 59 patients.
In conclusion, patients with RA showed an overall improvement of 61.0% and the usefulness of the G-1 column was judged to be 61.0%.

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