Contribution of mast cells to edema formation in rat paws: a microdialysis study on mast cell deficient rats(Ws/Ws) Yancai Guo, Takatoshi Mochizuki, Eiiehi Morii*, Yukihiko Kitamura* and Kazutaka Maeyama Dept. of Phannacol., Ehime Univ. Seh. of Med., Ehime and *Dept of Pathol., Osaka Univ. Med. Sch., Osaka, Japan Potent bioactive mediators such as histamine, serotonin and cytokines are released from mast cells after stimulation with compound 48/80(C48/80) or antigen. Among them, histamine is known as a potent agent to elicit edema fonnation in rat paws. Using a microdialysis technique, we detennined the time course of changes in histamine release in the subplantar space of hind paws. Recently, mast cell defieient rats(WslWs) with a mutation of c-kit receptors were developed. We applied the mierodialysis technique to these mutant rats and c1arified the contribution of mast cells to the fonnation of rat pawedema. C48/80 inereased histamine release and edema fonnation in the rat paw dose-dependently. Its histamine level increased 426-fold in 20 min after stimulation with 50 J.lg of C48/80 and returned to the basal level in 4 hr in nonnal rats (+1+), whereas no histamine release and edema fonnation were observed in WslWs rats. Antigen stimulation eaused the same patterns as those indueed by C48/80. These results suggest that histamine released from mast eells plays a eritical role as a trigger for the development of edema.
SUBSTANCE P POTENTIATES THE IMMUNOLOGICAL ACTIVATION OF RAT PERITONEAL MAST CELLS Hang Yung Alaster Lau, Yeung Shing Ng Department ofPharmacology, Faculty ofMedicine, Chinese University ofHong Kong, Shatin, New Territories, Hong Kong. The present study has demonstrated that, in addition to directly activating rat peritoneal mast cells, suhstance P can potentiate the immunologie activation of rat peritoneal mast cells at concentrations which did not induce significant histamine release (0.3 )..iM to 3 )..iM). The selective non-peptide neurokinin receptor antagonists CP99994 (NK-I), SR48968 (NK-2) and SR 142801 (NK-3) failed to reduce the potentiating effect of substance P at concentrations which were previously reported to be effective at the respective neurokinin receptors (0.1 nM to 111M). The current findings hence suggested that substance P does not potentiate immunologie mast cell activation through neurokinin receptors. Instead, substance P may potentiate immunologie mast cell activation through the same peptidergic pathway which is responsible for the direct activating action of the neuropeptide on mast cells. This was confinned by the observations that similar potentiating action was obtained -with compound 48/80, which is another weH known mast cell activator working through the peptidergic pathway. Fwthennore, the putative inhibitor of the mast cell peptidergic pathway, benzylkonium chloride (BAC) was found to effectively suppress the mast cell potentiating action of substance P. Conclusion: This project has produced preliminary results which postulate that instead of directly activating the mast cells, substance P may allow mast cells to be activated by basal levels of immunologic stimuli during neuro genie inflammation. This kind of interaction between substance P and mast cells maybe more relevant to the pathogenesis of neurogenie inflammation in human since most human mast cells are not directly activated by substance P.
PHARMACOLOGICAL MODULATION OF TWO LEUKOCYTE POTASSIUM CURRENTS. Björn. E. Svensson. Dep. Cellular & Molecular Pharmacology, Preclincal R&D, Novum Unit, Astra Pain Control AB, S-141 57 Huddinge, Sweden. Several types of ion channels have been found in leukocytes, and drugs in the blood stream may have easy access to and may interact with these channels. Various functions of leukocytes may, in turn, be controlled by their ion channels. In the present study !wo potassium conductances were compared with
respect to their sensitivity to
modulation by known potassium channels blockers as weil as by local anesthetics in clinical use. Calcium-Bctivated end inwardly rectifying potassium conductances in promyelocytic HL-60 cells were studied using the patch clamping technique. Charybdotoxin (100 nM) and 4aminopyridine (5 mM) selectively inhibited the calcium-activated potassium conductance, whereas BaCI 2 (50 !IM) selectively inhibited the inwardly rectifying conductance. The potassium currents were not or only slightly modulated by apamin (100 nM) or triethylammonium chloride (5 mM). The conductances were inhibited by the local anesthetics lidocaine (3 mM) and ropivacaine (3 mM). The latter was, however, ten times more potent on the calcium-activated conductance. Conclusions: These studies support the feasibility of using ion channel modulators to investigate the control of various leukocyte functions by their channels. The previously reported perturbation of leukocyte functions, e.g. decreased respiratory burst, by local anesthetics, may be due to their modulation of potassium channels in these cells.
PHOSPHOUPfDS INCORPORATION BY CULTURED LEUCOCYTES AND RESULTING EFFLUX OF UPfD-DERIVED PRODUCTS. Nishiyama-Naruke, A.; CUri, R. Deparlment of Physio/ogy and Biophysics, University of Silo Pau/o, Brazil.
Phospholipids are important as structural element of membranes and in the functional activ~ of Iymphocytes (Iys) and macrophages (mcl>S). In the present study, ('C)phosphatidylcholine (PC) incorporation and ("C)pCderived exporting products by Iys and mcl>S were evaluated. Lys prepared from mesenteric Iymph nodes and peritoneal mcl>S from rats were cultured for 24 hrs in Eagle medium added of radiolabelIed PC. Radioactivity from PC was incorporated at a significantly greater rate by than by Iys. In both cell types, the radioactivity of L3-PC-1-stearoyJ..2-[1-"Clarachidonyl was distributed in various phospholipid classes determined by thin-Iayer chromatography (pC, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid and phosphatidylinositol). In contrast, lipids derived from L3-PC-[n-methyl14C] choline-1,2 dipalmitoyl, were found basically in PC fraction. In other experiment, after the incorporation period (24 hrs), mcl>S were washed and then cultured for another period of 24 hrs. Medium was analysed and the radioactivity was found mainly in fatty acids (65,90%±1,87) and phospholipids (25,95%±2,53) fractions. Therefore, Iys and mcl>S are able to incorporate extracellular phospholipids. McI>S export phospholipids and fatly acids possibly derived from its phosphOlipase activity. Phospholipids may play a role tor the interaction between Iys and in the immune and inflamatory responses.
S 227 P-II-2-03 KINETICS OF LEUKOCYTE RECRUITMENT AND CYTOKINE LEVELS IN ACUTE PHOTODAMAGED MOUSE SKIN Henrietta Moore & Edward Larnbert Novartis Research Institute, Vienna, Austria Exposure of mouse skin to a high dose of UVB radiation results in early erytherna and oederna formation followed, at 48 and 72h, by significant and parallel increases in myeloperoxidase (MPO; a neutrophil marker) activity and TNFa release. We have also rneasured the N-acetyl g1ucosaminidase activity (NAG; a macrophage marker) present in unirradiated skin and fuund it to be consistent with a resident monocyte/rnacrophage population. In three experiments comprised of groups of 5 fumale NMRI mice, UVB irradiation of skin induced a significant (p<0.05) decrease in this NAG activity at 24h from 19.3 ± 1.4 to 15.3 ± l.2mU/ear. Tbe activity at 72h was significantly (p<0.01) increased!Tom 19.2 ± 0.8 to 32.6 ± 2.9mUlear. This kinetic may be exp1ained !wo ways: first, if UVB can access the NAG enzyme within the resident macrophages it may directly inhibit activation at the 24h time point. Second, the number of cells present at 24h relative to the fluid infIux may be small and reflected by a lower NAG activity. At 72h monocyte recruitment is then reflected by increased NAG activity. Conclusion: Thi. model of cutaneous inftammatioo can provide information in the same skin sample 00 both the cytokine and leukocyte profile and cou1d therefore be used to examine a wide range of effects of anti-inflammatory compounds.
P-II-2-04 IL-15 STiMULATES OXIDATIVE BURST IN NEUTROPHILS IN THE "PRIMED" STATE E. Wojtecka-Lukasik, W.Masliilski, S.Masliilski Department of Biochemistry, Institute of Rheumatology, Warsaw, Po land In this study we have investigated the effect of IL-IS, a newly described cytokine exerting IL-2 like activities, on respiratory burst of PMN leukocytes isolated from healthy blood donors or patients with rheumatoid arthritis. The oxygen metabolism of PMNs \\as measured by luminol-enchanced chemiluminescence. We found that human IL-15 has no effect on resting neutrophils. In contrast, IL-15 significantly enhanced oxygen production in neutrophils activated in vitra with N-formyl-methionylleucyl-phenylalanine (FMLP), phorbol 12 myristate 13 acetate (PMA) or opsonized zymosan. The effect is dose and time dependent. Moreover, neutrophils isolated from blood of patients with rheumatoid arthritis ("primed in v;vo) or healthy individuals ("primed" in vitra) respond to exogenously added IL-15 by enhanced production of
P-II-2-06 EVALUATION OF LEUKOCYTE ROLLING IN RAT MESENTARY VENULES BY NON-INTRA VITAL MICROSCOPIC HISTOLOGICAL METHOn Kohji Yamaki, *JohanRaud, *Lennart Lindborn, *Per Hedqvist, and Sachiko Oh-ishi. Department of Pharmacology, Sehool of Pharmaceutical Sciences, Kitasato University, Tokyo 108, Japan and * Department of Physiology and Pharmacology, Division of Physiology I, Karolinska Institutet, Stockholm S17717, Sweden. We have deve10ped non-intravital mieroscopic histological method for analyzing leukocyte rolling in the stimulated rat mesentery venules. Using this method, we have found that intraperitoneal injection of histamine increased leukocyte number inthe venules. At the same time we provedthat the polymorphonuclear leukocytes (PMN) were rolling but not adhesive on the venule wallsunder intravital microscope using fucoidin whichis knownas an inhibitor of selectin-induced leukocyte rolling. In this communication we report that a mechanism oftumor necrosis factor-a (TNF-a) induced increase of PMN at mesentery venulesin rats. A peak increaseof PMN rolling wasa! 3 hOUT afterTNF-a injection, and itsmaximum response wasa! the dose of200 Ulral. Fucoidin and dextran sulfate (10 mgikg, i.v. 5 min before stopping blood Dow) inhibited this increase by about 40%. Dexamethasone (0.1 mglkg i.v. simultaneouslywithTNF-a injection) alsocompletely inhibitedthis PMN increase. These resulls suggeste that the increased PMN at mesentery venule by TNF-a mayaIso be rolling and this phenomenon may probably relate to both of P-selectin and E-selectin.
P-II-2-07 COMBINED DEFICIENCY IN NEUT20FHIL FUNCTIONS AFTER BONE ~jARRo-w TRANSl'LANTATION (BMT) AND THE IN VITRO EFFECT OF G-CSF Masako Katoh, Nakayama M., Umeda ~i., Tsuji A_' Flrst Dept .. Intern. Med., Sch. Med_, and Coll. Health Frof.', Toho University, Tokyo 1~3 Japan We evaluated the neutrophil functions in a patient with AML who recieved allogeneic BMT. Chemotactic responses, random mObility, phagocytosis, 0 - release and bactericidal activity were sever~ly impaired early after reconstitution._ These neutrophil functions gradually improved with time course after B~T, all of which normalized at 12 months after reconstitution. Both 0 - release and bactericidal activity of neutfophils were significantly enhanced from 6 and 12 months after reconstitution with the pretreatment of 50 ng/rnl of G-CSF in vitro. These findings suggest that the com-
neutrophils and therefore may play an important role in inflammation.
bined disorders in neutrophil functions in Bsrly phase R.fter BhT may pl'1.Y an importlint role in the susceptibility to bacterial infections until one year after B~T. Administration of G-CSF should be recornrnended for preventing and treating the prolonged and unmanageable infections.
EFFECTS OF FLOW ON NEUTROPHIL-MEDIATED CA2+ RESPONSE IN HUMAN VASCULAR ENDOTHELIAL CELLS
Sustained anti-inflammatory effects of G-CSF in volunteers on monocytes and Iymphocytes under 12 days of treatment
Masahiko Yamaguchi Hiroshi Machida, Risa Korenaga*, Noriko ToyamaSorimachi**, Joji Ando*, Hiroshi Nakano, KaofU Kumada, Department of Surgery, Showa University Fujigaoka Hospital, Yokohama, *Department of Cardiovascular Biomechanics, Faculty of Medicine, University of Tokyo, and * *Department of lmmunology, Tok)'o Metropo!itan Institute of Medical Seience, Tok)'o, Japan Lcukocyte-vascular endothelial ccll (EC) interactions which promote inflammatory reaction, involve cell adhesion and bidirectional signaling. We tested effects of flow on neutrophil-mediated changes of intracellular free Ca 2+ levels ([Ca2+]i) in ECs. Cultured human umbilical vein ECs stimulated b)' lipopol)'saccharide (LPS) were labeled with Fura-2, and exposed to fluid flow with or without neutrophils. Simultaneous ehanges in fluorescence intensit)' reflecting [Ca 2+ ji were monitorcd. Application of flow with neutrophil to LPS-stimulated ECs Icd to an increase in [Ca 2+ J, in some of the cclls although either flow without neutrophil or neutrophil without flow rarely induced an apparent rise in [Ca 2+ Ji. Results suggest that both flow and neutrophils are essential to induce a rise in [Ca 2+Ji in stimulated ECs. Flow application either with neutrophils to unstimulated ECs or with erythrocytes to stimulated ECs did not promote an)' rises in [Ca 2+].. Conclusion: Flow and stimulation of ECs, ma)' pla)' a significant role in neutrophil-mediated c)'tosolic free caleium response in ECs.
T. Hartung, A. Wendel, H.-D. Volk, P. Stevens and the G-CSF study group Biochemical Pharmacology, Univ. Konstanz, and Clinical Immunology, CharM, Berlin, Germany, and Amgen, Thousand Oaks, U.S.A.
oxygen radicals. Conclusion: 11-15 stimulates oxygen radicals production in "primed"
G-CSF is known as a granulocyte growth and maturation factor with antibactericidal effects. In previous studies we demonstrated that short term treatment of volunteers with G-CSF has anti-inflammatory effects on monocytes. We performed another volunteer study in order to establish whether these effects can be maintained under prolonged treatment, whether they extend to Iymphocyte functions as weil as to establish a dose-relationship. 24 male volunteers (20-40 years old, 70-90kg weight) were double-blindly treated 12 times daily with either placebo 75, 150 or 300 ~g Filgrastim (G-CSF, Neupogen"). G-CSF resulted in attenuation of TNF production of monocytes in response to various LPS concentrations ex vivo. The effect was seen in all G-CSF groups and became more pronounced during the treatment. Lymphocytic IFNy release in LPSstimulated wh oie blood, decreased earlier than TNF in the G-CSF groups, i.e. 24h after initiation of treatment. Lymphocyte proliferation induced by PHA or anti-CD3antibodies was markedly suppressed after an initial phase of augmentation. Natural killer cell activity of Iymphocytes was not affected. Conclusion: The pharmacological effects of G-CSF can be sustained under prolonged treatment periods even at subclinical dosages. This unique combination of anti-bactericidal and anti-inflammatory properties might have bearing for new indications such as non-neutropenic infection, sepsis prophylaxis, chronic
inflammation and suppression of transplant rejection.
S 228 P-II-2-09 HOW DOES AN EXTRA-CORPOREAL PERFUSION DEVICE, G-I, SUPPRESS RAT ADJUVANT ARTHRITIS? Hiromichi Takase, Masaaki Takai, Yasuo Yanagihara, Satoru Nakazato, Jun Okumura, and Masahisa Kyogoku Fujii Memorial Res. Inst., Otsuka Pharmaeeutieal Co.,Ltd., Otsu, Japan G-I ameliorates arthritic symptoms in rat adjuvant arthritis (The Sth APLAR in Australia, 1996; The Ist ICFA in Japan, 1996). The mechanisms were investigated on the neuro-endocrine-immunological aspeets. Acclimated female Lewis rats were used. Ten ml of venous blood was perfused through G-I for I hr following the injection of adjuvant into right hind paw under anesthesia. Paw swelling was plethysmometrically observed. Hyperalgesia was evaluated as a shortening of withdrawal latency against thermal stimulation by Hargleaves' s method. Plasma eorticosterone level was estimated by RIA. IL-Iß in plasma or in supematant of eell suspension was measured by ELISA. G-I signifieantly suppressed adjuvant-induced hyperalgesia (G-I: -6.S±0.4 sec, eontrol: -7.7±O.2 sec) and paw swelling (G-I: 0.75±0.07 ml, eontrol: 1.40±O.05 ml). Increase in plasma eorticosterone level was observed du ring G-I perfusion (G-I: 233.5±S9.6 nglml, eontrol: -21.7±S9.4 nglml) and lasted for 60 min. IL-I ß, which is undeteetable in perfused blood, beeame detectable in the sup of eell suspension recovered from used G-I. Modulation of hypothalamus-pituitary-adrenal axis with the cytokine induced by G-I, in addition to elimination of inflammatory eells, would contribute to the meehanisms underlying the anti-arthritic action in adjuvant arthritis model.
P-II-2-11 HYPOCHLOROUS ACID-MEDIATED CELL INJURY: IDENTIFICATION OF CELLULAR TARGETS AND LETIIAL CONSEQUENCES Margret Vissers, Juliet Pullar, Anitra Carr, Christine Winterbourn. Free Radical Research Group, Christehurch School of Medieine, PO Box 4345, Christehureh, New Zealand. Hypochlorous acid (HOCI) is the major oxidant produced by stimulated neutrophils. This oxidant can penetrate cell membranes and will react with many eell constituents, and it therefore is likely to be a significant cause of tissue injury at sites of inflantmation. We have investigated the consequence ofHOCI exposure on red blood cells and cultured mnbilical vein endotheliaI cells. HOCI is toxie to and causes lysis ofboth cell types. At low levels ofHOCI there is significant loss of glutathione and membrane thiols, and in endotheliaI cells there is a decrease in the activity ofthiol-dependent enzymes, particu1arly giyceraldehyde-3-phosphate dehydrogenase. However, thiol oxidation is reversible under some conditions and does not necessarily equate weil with toxieity. In red blood cells lysis is not related to thiolloss, but some of these reactions may contribute to cell death in endothelial cells. Our studies indicate that less weil documented reactions of HOCI are responsible for cytotoxicity. Significant among these are the rapid formation of protein erosslinks and of ehlorohydrins resulting from reaction with unsaturated fally acids. In red cells erosslinking of the cytoskeletal proteins correlates closely with lysis and OCCUTS at low levels ofHOCI exposure. Tbe nature ofthe erosslink is as yet unidentified, but does not involve disulphides or Schiff's bases. Chlorohydrin formation can also be demonstrated, bot may be significant only at higb HOCI concentrations.
AN EXTRACORPOREAL PERFUSION DEVICE, G-I, SPECIFICALLY ADSORBS INFLAMMATORY CELLS IN HUMAN AND RAT Yasuo Yanagihara, Masaaki Takai, Hiromiehi Takase, Satoru Nakazato, Jun Okumura,
A 'NOVEL' X--CGD PATIENT WITH A NORMAL SUPER OXIDE GENERATING ACTIVITY IN PERIPHERAL EOSINOPHILS.
Fujii Memorial Res. Inst., OtsukaPharmaceuticai Co,Ltd., Otsu, Japan, *Kobe University Sehool of Medicine, Kobe, Japan G-I eolumn, having anti-rheumatic action, is a extracorporeal perfusion deviee to selectively remove granuloeytes. The characteristie of leukoeyte adsorption was further analyzed. Small G-I eolumn eontaining of I g beads was used for analysis. Ten ml of heparinized human blood, taken from S patients suffered from definitive and aetive rheumatoid arthritis(RA) or S normal subjeets, was perfused onto G-I for I houT. Ten ml of arthritie rat venous blood was extraeorporeally perfused onto the eolumn for I hour under anesthesia and heparinized eondition. Blood sampies were analyzed by flow eytometer witheell surfaee marker antibodies. G-I adsorbed human inflammatory leukoeytes. High adsorption effieieneies on granuloeytes (19.5%), monoeytes (21.1%), and platelets(16.3%) were observed. B cells(CDl9+, 6.3% ) were also adsorbed, but T eells(CD3+, 2.2%) and NK eells(CDI6+/56+, 2.3%) were exaetly not absorbed in normal subjeets. RA patients had higher numbers of peripheral granuloeytes (6390 eellSJ).tl vs normal, 2950 eell~!l). Adsorption of inflammatory eells by G-I was prominent in the patients. Upregulation of Mae-I(CDllb) expression on the granuloeytes and monoeytes passed through G-I was observed. These adsorption and aetivation properties wereobserved in arthritie rats in same way. The adsorption and aetivation of inflammatory cells were abolished in eomplement-depleted rats with eobra venom faetor. G-I specifically eliminates inflammatory eells by the aid of eomplement aetivation. Additionally, eonsiderable number of aetivated eells return to the host.
Dcpartmcnt 01' Pediatrics, Nagasaki University, Nagasaki, Japan. and Tsuji Y *Department of ßiochemistry, Institute of TropicaJ Medicine, Nagasaki University,
Nagasaki, Japan. In X-linked Chronic granulomatous disease (eGD) patients, gp91 Phox wao;; usually
defisient in common in neutrophils, eosinophils, monocytcs, and B lymphO(.)'tes, suggesting a L'ommon mechanism fm thc expression uf gp91P hox in Ihose cclls. We now show a novel eGD patient who exceptionally has a normal superoxide generating aL1ivity and expresses thc sudacc cytochmme b558 10 norrna1 extent in periphcral eosinophils, and present the case as an example of a ncw subtype of X--CGD. He is 64 years old and the o]d!st eGD patient so far we know. He had suffered from mild to severe reLllrrent infet.1ions, which disappearcd completely by interferon-gamma at 2.5x l05U/m 2/week wh ich is equivalent 10 onc-twentieth of (bse usually used The treatment recreased 19E level significantly. We found a point mutation in the promotor region of gp91 P1lOx gene. This mutation is involved in the element oC<..l1picd by a similar fat.1OI in human periphcral neutrophils, monocytes and B lympho<.:ytes but not in eosinophils for the expression of gp91P /lOX • This patient is very precious because he is the only patient who indicates a promotor element working in common in neutrophils, monocytes and ß Iymphoc)'tes, and suggest a particular element effective onl)' in eosinophils tor the expression of gp91 P11Ox •