Fourth European Workshop on Inflammation Wilrijk March 1982

Clinical rheumatology, 1982, 1, N ~ 2, 140-152

Fourth European Workshop on inflammation Wilrijk March 1982

At the fourth European Workshop on the role of the complement system, endotoxins and intravas- cular coagulation in inflammation held at Wilrijk Belgium on 15-16 March 1982, the following abs- tracts were submitted.

Inflammatory stimuli augment synthesis of complement component C3 by macrophages. H.P. Hartung, D. Bitter-Suermann, U. Hadding, Inst.f.Med.Microbiology, Hochhaus Augustplatz D-65 Mainz, FRG

Macrophages (mo) are major producers of complement. We investigated whether inflammatory stimuli applied in vivo affect synthesis and release of complement components C3, C2 and C4. Peritoneal mo were harvested from guinea pigs either untreated or intraperitoneally injected with serum albumin or C. parvum 4/7 days before sacrifice, ma cultured under serumfree conditions for 12 hrs, and supernatants tested for C3 by ELISA, and C2/C4 by hemolytic titration employing serum fron C2/C4 deficient guinea pigs. In addition, secretion of N-acetylglucosaminidase was determined. Mo from untreated animals released about 70 ng/106 cells C3 into culture su- pernatant, albumin elicited mo 88ng and C.parvum stimulated ma 183 ng. Secretion of C2 ranged from 0.99 x 108SFU to 2.66 x 108 in albumin - and 3.93 x 108SFU in C.parvum-stimulated mo, corresponding values of C4 release being 1.9 x 108; 3 x 108; 4.1. x 108 SFU/106 cells. By employing an ELISA we could demonstrate that in vivo application of inflammatory stimuli augments synthesis and release of C3, the most abundant complement component. These changes were paralleled by alterations in C2, C4 and Iysosomal enzyme secretion. Our findings show that ma can be induced to enhance synthesis and release of complement at sites of inflammation.

Human platelets, after their contact with influenza virus, activate the complement system in autoiogous serum. F. Maillet, C. Lambrd, M. Kazatchkine. INSERM U 28 and U 139. H6pital Broussais 75014 Paris, France.

Guinea pig erythrocytes that have been exposed to influenza virus activate the human alternative pathway through virus-induced desialation of the cells. Neuraminidase treatment of rabbit platelets enhances their clearance in vivo. Washed human platelets were prelabeled with 51Cr and resuspended in Tyrode's solution containing Influenza virus Hong-Kong/1/68 for 15 min at 4~ After elution of the virus at 37~ the platelets were washed and resuspended in autologous serum that had been dialyzed against Veronal buffered saline containing Ca + § and Mg § § (VBS § +), VBS containing 8mM/EGTA and 2mM Mg § § (VBS-MgEGTA) or VBS containing 20mM ED- TA (VBS-EDTA) for 45 min at 37~ Four point nine per cent 51Cr release and no complement consumption were observed in VBS-EDTA serum. In contrast, 9 ~ 51Cr release with 50 ~ decrease in C3 and B hemolytic activities occured in VBS-MgEGTA serum and 20,5 ~ 51Cr release with 60 070 decrease in C2 hemolytic activity occured in VBS +§ serum. These results suggest that Influenza virus may alter the platelet surface in such a way that both complement pathways might be recruited and the cells be lyzed in autologous serum.

Contact activation by bacterial fragments. E.S. Kalter, W.C. van Dijk, A. Timmermans, J. Ferhoef, B.N. Bouma, University Hospital, 3511 GV Utrecht, The Netherlands.

To investigate whether bacterial cell wall fragments activate the contact system of plasma we incubated a mixture of purified human Factor XII, Prekallikrein (PK) and High Molecular Weight kininogen each at its plasma concen-

tration with increasing amounts of bacterial fragments (Final conc. 0,02-5 mg/ml) for 30 minutes at 4~ Thereafter kallikrein amidolytic activity was measured and expressed as percentage of the maximal activation induced by Dextran sulfate. Lipopolysaccharide of 4 different E. coti strains induced 50-90 07o activation, depending on their concentration. The liped A fraction also induced 80 ~ activation at optimal conc. () 0.312 mg/ml) but the polysaccharide fraction induced ( 15 07o activation at all conc. tested. Fragments of Sta- phyloccus aureus, peptidoglycan and teichoic acid induced 70-10007o activation but protein A did not generate kallikrein activity. In normal human plasma to which 125I PK was added, bacterial fragments induced the formation of 125I-kallikrein-inhibitor complexes, suggesting that these fragments also in plasma can induce the activation of the contact system. Our findings may explain the decrease of these factors, observed in patients during bacterial shock.

Fixation and activation of the first component of complement by soluble lgG-aggregates. G. Doekes, M.R. Daha, L.A. van Es, Dept. Neph- rology, Univ. Hosp., 2333 AA Leiden, The Ne- therlands.

Soluble immune complexes (slcx) may activate the classical pathway of complement, dependent on their size and composition. Using stabilized aggregates of normal human IgG as a model for sIcx, we studied the influence of their size on the fixation and activation of the first complement component (C1). Purified human C1 was incubated with 7S IgG and with aggregates containing 5, 10, and 20 molecules IgG, separated by su- crose-density centrifugation. Fixation of C1 was measured as the consumption of C 1 hemolytic activity and activation as the appearance of C4-con- suming ability. The four preparations tested were all able to fix and to activate C1, but the efficien- cy of both processes was strongly dependent on the aggregate-size : on a weight basis (IgG)20 was about 1000 times as efficient as 7S IgG. Further- more, it appeared that significantly more C1 could be activated than was found to be fixed to the aggregates. From these results we conclude that C1 can be activated during a trainsient binding to an immune complex, after which the activated form (CI) is released, making the immune complex available for the activation of another C 1-molecule.

Clinical rheumatology, 1982, 1, N ~ 2, 141

The effect of dapsone on complement activation. L. Drummond, D.K. Gemmell, Scientific Devel- opment Group, Organon Laboratories Ltd., Newhouse, Scotland.

Both the rapid therapeutic effect of Dapsone (D) in Dermatitis Herpetiformis (DH) and its marked inhibitory effect on the Reversed Passive Arthus response in the rat have been explained by the drug's claimed ability to inhibit complement activation. Using two classical pathway assays and one alternative pathway assay (employing rabbit red blood cells and C4 deficient guinea pig serum) we have attempted to confirm the effect of Dapsone and some other drugs, both in vitro and ex vivo, on complement activation. We have found that D, unlike the well-established anti- complement drug Suramine and the anti- rheumatic sodium aurothiomalate, demonstrated no anti-complement effects. The anti-infiamma- tory activity of D in DH and in animal models would appear therefore to be due to mechanisms other than complement activation inhibition.

Complement (C) activation stimulates prostacyclin (PGI 2) production by rabbit aorta and peritoneum. M. Rampart, H. Bult, and A.G. Her- man, Department of Experimental Pharmacol- ogy, University of Antwerp, B-2610 Wilrijk, Bel- gium.

C activation was achieved by incubation (120', 37~ of pooled human serum with Cobra Ve- nom Factor (CVF, 10 U/ml) or endotoxin (LPS, W.E. coli O l l l B4, 5 mg/ml). Rabbit peritoneum (35-40 mg), incubated in CVF- or LPS-activated serum, produced significantly more PGt z (measured by radioimmunoassay of 6-oxo- PGF1~ ), when compared with incubations in fresh serum, and heat-inactivated serum (15', 56~ containing the same amounts of CVF or LPS. Stimulation of PGI: production correlated well with loss of total hemolytic activity and consumption of factor C3. Decrease in concentrations of factor C5 and C4 (the latter only upon incubation with LPS), indicated that split products of these factors possibly contributed to the complement-induced stimulation of PGI a production by isolated rabbit peritoneum. Further- more, using the "Well-technique,, 1 we have shown that trypsin-digested factor C5 dose- dependtly increased the PGI 2 production by the endothelium of rabbit aorta. The finding that

142 Clinical rheumatology, 1982, 1, N ~ 2

complement activation and individually activated complement factors stimulate PGI 2 biosynthesis is of particular interest for the regulation of vascular tone in shock situations, and vascular permeability during inflammatory reactions.

REFERENCE

1. Weksler et al. J. Clin. Invest. 1981

Stimulation of PGE and TXB 2 release from macrophages by C3b. H.P. Hartung, U. Hadding, D. Gemsa x, Inst. f. Med. Microbiol., Univ.Mainz; Inst. Immunol., Univ. Heidel- berg, • FRG.

Release of prostaglandins and thromboxanes from macrophages (mo) can be elicited by various inflammatory and immunological stimuli. C3b has been discussed to modulate mo function. We investigated its influence on the liberation of PGE and TXB 2 from mo. Albumin induced guinea pig peritoneal mo (1.5. • 106/well) were cultured in the absence of serum for 18 hrs. Puri- fied homologous C3b was added at concentrations form 10-80 ug/ml. Supernatants were tested for PGE and TXB 2 by radioimmmunoas- say. C3b dose dependently stimulated the release of PGE (6.5. ug/ml in reponse to 75 ug/ml C3b). The effect was comparable to that of zymosan. Also significant amounts of TXB 2 were liberated (16-20 ug/ml following addition of 80 ug/ml C3b). Kinetic studies revealed that TXB 2 release started to rise above spontaneous secretion after 2 hrs. approaching a plateau at 12 hrs. (2 hrs : 2.2 ug/ml ; 6 hrs: 10.8; 12 hrs. ; 18; 18 hrs: 19.8). 19.8). Indomethacin totally blocked the stimula- tory action. To prove specificity C3b was passed down an immunosorbent column to which mono- clonal anti-C3 lgG was coupled, and cultures exposed to the effluent. Immunoabsorption almost completely abrogated the effect of C3b. Consid- ering the action of arachidonic acid derivatives in inflammatory responses our findings lend further support of the view that C3b is involved in the pathogenesis of inflammation: by causing PGE and TXB 2 release as well as secretion of lysoso- mal enzymes.

The ability of H to modulate C5 utilization by the cellbound alternative pathway C5 convertase differs on sheep and rabbit erythrocytes. E. Fischer,

M.H. Jouvin, M. Kazatchkine INSERM U 28 H6pital Broussais, 75014 Paris, France.

The ability of H to inhibit formation of the alternative pathway C3 convertase differs on activating and non activating cells. C3 convertase sites are converted to C5 convertase sites by deposition of additional C3b molecules and C5 binds to C3b in a reaction that is inhibited by H.C5 conver- tases sites P (C3b)nBb were formed on the non- activating cells sheep erythrocytes (E s) and on the activating cells rabbit Er and desialated E s (E des) by sequential deposition of C3b by fluid phase and amplification convertase; the sites were revealed by incubation of the cells with purified C5 and with rat serum treated with KSCN and hy- drazine. When incremental amounts of H were added to the reaction, a dose-dependent inhibition of C5 utilization was observed. The amount of H that inhibited 50 ~ of C5 utilization was the same for a given cell regardless of the number of C5 convertase sites that were expressed on the cell. However the 50 ~ inhibitory dose of H was 46ng/107Esp(c3b)nBb, 45 and 63 ng/107Edesp(c3b)nBb while it was at least 170ng/107Erp(C3b)nBb. Since the amount of H and the time of incubation with H (4mn) were insufficient to irreversibly decrease C5 convertase sites on the cells, these results suggest that H mo- dulates in a sialic-acid independent manner utilization of C5 on activators and non activators of the alternative pathway by competing for uptake of C5 within the convertase site.

Activation of macrophages by platelet activating factor (PAF). H.P. Hartung, M.J. Parnham +, J. Winkelmann +, U. Hadding, Inst. Med. Mi- crobiol., Univ. Mainz; Pharmacol. Res.Lab. § A. Natterman & Cie., K61n, FRG.

Platelet activating factor (PAF) influences platelet and neutrophil functions. We set out to study whether PAF activates macrophages (mo) as indicated by induction of the oxidative burst. The response of resident or C. parvum elicited guinea pig peritoneal mo to synthetic PAF (1 - o-octadecyl 2-acetyl-sn-glycero-3 phosphoryl choline) was measured as luminol dependent chemilumine- scence (CL) and H202 release into culture supernatants. PAF dose-dependently stimulated the oxidative burst in C. parvum elicited mo at concentrations as low as 3.8 • 10 -9 M / I (3.5. nM H202/106 m o / h ; 3.8 • 10 -5 M / l : 20.3). Peak


CL values were reached 6 min after addition of PAF (3.8 • 10 -8 M / l : 18.000 RLU = relative light units ; 1.9 • 10 -5 M/1 : 65.000 RLU). Kinet- ic studies revealed that most of the H202 release occurred within 30 min of adding PAF. Resident mo exhibited only little response to PAF which could be enhanced by incubating ma in the presence of 1% gelatine. Our results demonstrate a new biological effect of PAF : stimulation of the oxidative burst in m~. Subsequently released oxygen radicals are microbicidal and cytotoxic, and may cause vascular endothelial injury. In view of these findings mo-platelet interaction in inflammation takes on a new significance.

Macrophage mitogenic activity of exudate or serum during acute non immunological inflammation. M. Pelletier and J.P. Giroud D6parte- ment de Pharmacologie, CHU Cochin Port-Roy- al, Paris France.

During the early phase of an acute inflammatory reaction a mitogenic factor for macrophages was observed in inflammatory exudate and serum. In exudate the highest activity was found 4 hours after the induction of inflammation and in serum only 1 hour after. In inflammatory serum a sec- ond peak was seen at 24 hours. At the same time there is no exudate in the pleural cavity. This mitogenic activity is still detectable over a large range of concentrations : in inflammatory serum from : 1:2 up to 1:8192, in exudate from 1:2 up to 1 : 2048. These results suggest that the macrophage mitogen present in inflammatory exudate could be different from that present in serum of animals suffering acute inflammation; or, at least, that the mitogenic factor from serum is not the result of exudate factor released into the serum. Inflammatory exudate and serum of athymic mice undergoing an acute inflammatory reaction exhibited a mitogenic activity for macrophages. This result suggests that the release of both mitogenic factors is not thymo-dependent.

Bacterial endotoxins decrease the number of guinea pig lung B-adrenoceptors. A.J.M. Schreurs and F.P. Nijkamp. Rudolf Magnus In- stitute for Pharmacology, Vondellaan 6, 3521 GD Utrecht, The Netherlands.

Haemophilus influenzae is a bacterium common- ly found in the deeper respiratory airways of patients with asthmatic bronchitis. We previously reported that vaccination of guinea pigs with Haemophilus influenzae results in an attenuated isoproterenol induced relaxation of isolated guinea pig tracheal spirals. In the present study we determined the number of /3-adrenoceptors (Bmax) in guinea pig lung homogenates by means of a 3H-dihydroalprenolol binding assay. Follow- ing vaccination with Haemophilus influenzae the Bm~ decreased significantly by 19% from 1920 + 80 to 1560_+ 70 fmol/mg protein, while the binding affinity of the sites was not changed. Al- so. B. pertussis and an E. coli strain (e. coli OlllB4) but not S. aureus, influenza A virus and E. coli J5 were able to induce similar effects. Fur- thermore the purified lipopolysaccharide (LPS, endotoxin) of E. Coli Ol11B4 decreased the number of lung/3-adrenoceptors when injected intraperitoneally four days before the experiments. The purified LPS of E. coli J5 had no effect on these receptors. From this study it can be con- cluded that endotoxins from gram negative bacteria, can modulate receptor densities in the lung.

Subsidized by the Dutch Asthma Foundation (NAF nr. 300).

Possible association of thromboxane A2 and endotoxemia in liver cirrhosis. R.J.T. Ouwendijk, F.J. Zijlstra, J.H.P. Wilson, LL. Bonta & J.E. Vincent. Departments of Internal Medicine and Pharmacology, Erasmus University, Rotterdam, The Netherlands. Systemic endotoxemia is found in approximately 45 % of patients with advanved liver cirrhosis and is associated with thrombocytopenia, renal failure and a poor prognosis. As some of the effects of endotoxins are thought to be mediated by thromboxane A2, we measured the metabolite TXB2 in plasma from 9 patients with cirrhosis and 34 controls. Raised plasma TXB2 levels ()200 pg/ml) were found on one or more oc- casions in 6 of the cirrhotics. All 6 had thrombocytopenia, whereas the 3 with normal TXB2 levels had normal platelet counts. A raised serum creatinine was found in I of the 6. Raised TXB2 levels were also associated with low levels of anti- plasmin and antithrombin III. In conclusion we observed that liver cirrhosis with complications is associated with raised plasma TXB2 levels. This may represent a conse- quence of endotoxemia.


Clinical relevance of chromogenic endotoxin determination in blood.L.L.M. Thomas, H.R. Biiller, A. Sturk, K. Joop, J.W. ten Cate, De- partment of Haematology, Div. of Haemostasis, Univ. Hospital "Wilhelmina Gasthuis,,, Amster- dam, The Netherlands.

We developed a sensitive method (detection limit 10 pg/ml) lot the quantitative determination of endotoxins (lps) in blood using the chromogenic substicite S 22 221. A pilot study in 400 patients samples showed a good correlation between lps- assays and bacterial cultures. In 66 % of gram- negative cultures the corresponding lps assay was positive. No false-positive lps-assays were found when gram-positive micro-organisms (m.o.) were cultured. Previous studies from our laboratory 2 revealed that post-operatively persisting low antithrombin III (AT III) and plasminogen (PLG) levels are early indicators of impending septi- caemia. In a prospective study (56 patients) the following results were obtained: frequent low grade endotoxaemia after major surgery, good correlation between low AT III and PLG levels and endotoxaemia, 75 % association between positive lps-assays and clinical symptoms of septi- caemia no gram-negative m.o. without a positive lps-assay.

REFERENCES

1. Clin. Chim. Acta 1981, 116, 63 2. Throm. Research 1981, 24, 73.

Effect of lipopolysaccharide (LPS), Lipid a (LA), and polysaccharide (PS) on phagocytic cell function. P. Henricks and J. Verhoef, State University Utrecht, The Netherlands.

The LPS that contains only core glycolipid (mainly LA) was isolated from E. coli J5 and UDP-gal-epimerase deficient mutant of E. coli 0111B4 and complete LPS from the parent strain E. coli 0111B4. Polymorphonuclear leukocytes (PMN ; 107/ml) were incubated with and without LPS J5 (PMN-J5) or LPS 01lIB4 (PMN- 0111B4). The ability to produce superoxide (0-2) and phagocytic and chemotactic activity by the PMN were measured. Control PMN (PMN-C) produced 40.6 + 2.0 nmol, PMN-J5 17.0 + 2.4 nmol, (p(0.001) and PMN-0111B4 44.7 _+ 2.2 nmol 0- 2 107 PMN/30 (p(0.001) upon stimulation by opsonized staphylococci. The phagocytic and chemotactic activity of PMN-J5 was significantly

diminished compared to unchanged phagocytic and chemotactic activity of PMN-0111B4. PMN were also incubated by the LA and PS fraction of LPS. PMN-LA produced 14.3 _+ 2.2 nmol compared to PMN-Ps 40.0 _ 2.4 nmol O-2/107 PMN/30 min upon stimulation by opsonized staphylococci. When PMN were directly stimulated by LPS or LPS fractions, cells stimulated by LPS J5 or by LA produced twice as much O- 2 as PMN stimulated directly by LPS 01 lIB4 or PS. When thioureum, a scavenger of radicals, was used during incubation with LPS J5 the capacity of the PMN to produce 0- 2 upon stimulation by staphylococci remained normal. Therefore PMN are stimulated by the LA region of LPS. During the process toxic radicals are produced which destroy phagocytic and chemotactic activity of the PMN.

Cutaneous responses to platelet activating factor (PAF-acether) in the guinea-pig. C.P. Page & W. Paul, Department of Clinical Pharmacology, Cardiothoracic Institute, Fulham Road, Londen SW3 6HP England.

Intradermal injection of PAF-acether (0.1-100 ng/site) into guinea-pig skin caused dose related changes in both plasma protein (125-I-human serum albumin) extravasation and accumulation of l 11-Indium oxine labelled platelets. Extrava- sation produced by PAF did not necessitate the presence of an exogenous vasodilator as has been reported in the rabbit 1. The dose of PAF-acether which produced a threshold cutaneous response was similar to that reported by Stimler et al. 2. PAF-acether induced a dose related accumulation of 111-1ndium oxine labelled platelets. Prelimina- ry experiments have shown no exudation of 111- Indium labelled red blood cells, with these doses of PAF-acether.

REFERENCES

1. Wedmore, C.V. & Williams, T.J. Br. J. Pharmac (1981) 74: 916P-917P.

2. Stimler, N.P. et al. Am. J. Pathol. (1981) 105: 64- 69.

lntracellularly generated, cyciooxygenase products of arachidonic acid (AA) elevate cyclic-amp (cAMP) levels in resident peritoneal cells. G.R. Elliott, M.J.P. Adolfs and LL. Bonta, Depart-


ment of Pharmacology, Medical Faculty, Eras- mus University, POBox 1738, 3000 DR Rotter- dam, The Netherlands.

Previous workers have suggested that the ability of cells to respond to exogenous prostaglandins (PGs) with an increase in cAMP levels is inversely related to their ability to synthesise PGs. Thus, resident peritoneal cells synthesise more PGE 2 than elicited cells but produce less cAMP in response to exogenous PGE 2. We have demonstrated that resident rat peritoneal cells incubated with 3 x 10-6M AA dissolved in saline synthesised 50 070 more cAMP than control cells. This increase was inhibited by 3 • 10-SM indomethacin, a concentration which by itself bad no effect on cAMP levels. It was observed that when AA was dissolved in ethanol (10 ~1 volume) there were permeability changes in cells incubated with greater than 1.5x 10-6M as shown by trypan blue uptake. Such changes were only seen with 6 x 10-SM AA or greater if it was dissolved in saline. Cells incubated with 3 x 10 -8 M AA contained 25 07o less cAMP than control cells. Further addition of AA resulted in almost complete loss of cellular cAMP. Intracellularly generated AA cyclooxygenase metabolites stimulated cAMP formation in resident peritoneal cells but AA presented in a form favouring penetration of the celle membrane caused permeability changes resulting in the loss of cellular cAMP.

Exogenous prostaglandin E z (PGE2) inhibits the cyclic-AMP elevating effect of prostacyclin (PGI 2) elicited peritoneal macrophages. LL. Bon- ta and M.J.P. Adolfs. Department of Pharma- cology, Erasmus University Rotterdam, POBox 1738, 3000 DR Rotterdam, The Netherlands.

Inflammatory granuloma macrophages(Mo) are, in terms of elevation of cylic-AMP (c-AMP), un- usually sensitive to PGE 2 but insensitive to PGI 2. In contrast, elicited peritoneal M~ are equally re- sponsive to PGE 2 and PGI 2. Because M~ embed- ded in granuloma tissue are exposed to higher concentrations of endogenous PGE 2 than are elicited l~eritoneal Mo, we examined in these cells the influence of PGE 2 on elevation of c-AMP by PGI 2 or dl-5E-13, 14-didehydro carbo-prostacyclin (DDH-carbo-PGI2). PGE2, within the range of 1.4x 10 -9 to 1.2x 10-8M caused a concentration-dependent inhibition of the rise in c-AMP induced by 2.8 • 10-6M PGI 2. Similar low concen-

trations of PGE 2 also inhibited the c-AMP elevating effect of DDH-carbo-PGI 2. With higher concentrations of PGE 2 the inhibition is either non-existent or masked by the effect of PGE 2 per se on c-AMP levels. The low responsiveness of granuloma Mo to PGI 2 is probably due to perma- nent exposure of these cells to environmental endogenous PGE 2. It is conceivable that endogenous PGE 2 rather than endougenous PGI 2 is a major negative feedback regulator of inflammatory Mo.

Microvascular injury during gastric mucosal damage by anti-inflammatory drugs in pigs and rats. K.D. Rainsford, C.M. Willis & P.G. Ro- bins. Lilly Res. Ctr. Ltd., Windlesham, Surrey, England.

Electron-microscopic studies were performed to establish the relationship of damage of the mucosal capillaries to the development of gastric mucosal damage by non-steroid anti-inflammatory (NSAI) drugs. Oral administration of 5mg/kg indomethacin to pigs resulted in opening of endothelial junctions and extravasation of red blood cells into the interstitial space at 10-15mins following dosage, which preceeded surrounding mucosal damage. Similar changes were observed 10- 60 mins following single oral dosing of 50- 200mg/kg aspirin to rats and pigs or aspirin 250mg/kg/d for 14 days to rats. The weak prostaglandin (PG) synthesis inhibitor, benoxaprofen (20mg/kg/d) caused very little vascular injury and less mucosal damage than observed with aspirin-a potent PG synthesis inhibitor. No vascur lar damage of this kind was observed following repeated oral dosage of these drugs to pigs. These results suggest that microvascular injury by NSAI drugs may (1) be related to PG synthesis inhibition, and (2) adaptation of mucosal capillaries of different species to injury.

Leukocyte proteins as markers of disease activity in rheumatoid arthritis. E. Munthe, M. Fagerhol, Oslo, K. Willard, Argonne USA and E. Jellum, Oslo, Norway.

A new set of proteins (the L1 proteins) released mainly from granulocytes, monocytes and macrophages have been quantitated in plasma from more than 50 patients with classical and other form of arthritis and in paired samples of plasma


and joifit fluids from more than 10 patients with RA and other arthritides. Patients with the highest plasma levels had the most progressive disease as measured by clinical, radiological and other laboratory parametres. Strongly elevated joint fluid levels corresponded to progressive local joint disease with erosions. L1 protein levels in plasma and joint fluids showed a better correlation to clinical and radiological parametres of disease activity than the corresponding levels of CRP, ceruloplamin, haptoglo- bin and orosomucoid. LI proteins were also superior to ESR as indicator of disease activity and drug response. When proteins from peripheral blood leukocytes were analyzed using the ISO-DALT technique of two-dimentional polyacrylamide electrophoresis, the synthesis of seven new proteins, two of them identical to L1 proteins, was shown to increase in the cell-preparations from patients with active RA.

A pharmacological analysis of the responses of isolated aorta from rats with adjuvant arthritis. J. Fontaine, A. Herchuelz, J.P. Famaey. Labo- ratory of Pharmacology, University of Brussels, 1000 Brussels, Belgium.

In order to see if chronic inflammation can alter the reactivity of vascular smooth muscle, the effects of various agonists have been compared on isolated aorta from Wistar rats with and without adjuvant arthritis (26 days after the injection of Freund's adjuvant). The responses to 5-hydroxy- tryptamime (5-HT), noradrenaline (NA) and po- tassium chloride (KCI) were recorded isometri- cally. Cumulative dose-response curves to these agonists were determined in the absence (control curves) and in the presence of various concentrations of verapamil. In other experiments, curves were determined after removal of calcium ions from the physiological solution. No significant differences in the ED50 values and maximal responses for 5-HT, NA and KCI were observed between the aortic strips of both groups of rats. Moreover verapamil had the same inhibitory effect on the responses in both groups. However when calcium was removed from Krebs solution the responses to 5-HT and NA were significantly higher in the preparations from arthritic rats than in those from control rats. This suggests that the reactivity of the aortic smooth muscle from arth-

ritic rats is tess dependent on extracellular calcium than that of the control preparations.

A new model of acute inflammation: cobra venom factor induced paw oedema. S. Leyck, E. Etschenberg, U. Hadding, J. Winkelmann, A. Nattermann & Cie. GmbH, Pharmacol. Res., P.O. Box 350120, D-5000 K61n 30, F.R.G. ; Inst. of.Med. Microbiol., D-6500 Mainz, F.R.G.

Cobra Venom factor (CVF) maximally activates the alternative complement pathway, inducing a rapid depletion of complement components. In this connection, we have investigated the induction of acute inflammation by CVF, in re- lation to decomplementation, and have compared the response to the established carrageenin paw oedema. For induction of oedema, 0.1 ml CVF solution (10 U/ml A. bid.) was injected into the left hind paw of Han-Wistar rats. The development of the inflammatory process was monitored by plethysmography. Prior decomplementation of the rats prevented the development of oedema after CVF injection, while carrageenin injection still induced normal oedema. These results suggest that CVF induces a specific and functionally different type of acute inflammation from that induced by carrageenin. The effects of some antiinflammatory drugs on this newly developed model of acute inflammation are reported.

Hydroxyapatite chrystal-induced membrane damage in granulocytes and erythrocytes. J.G.R. Elferink and J.C. Riemersma, Laboratory of Medical Biochemistry, University of Leiden, The Netherlands.

Hydroxyapatite (HyAp) crystals may cause severe inflammations in vivo. Because granulocytes are able to release inflammation-promoting con- stituents, we studied the interaction of HyAp with granulocytes. For comparative purposes the effect of HyAp on erythrocytes was studied because these cells are simple in the sense of hav- ing only one membrane. Membrane damage was measured in rabbit exudate granulocytes as the release of the cytoplas- mic enzyme lactate dehydrogenase, and in erythrocytes as hemoglobin release. HyAp causes time- and concentration-dependent cell iysis. Inhibitors of phagocytosis, such as deoxyglucose, iodoace- tate and ethacrynic acid, strongly reduce HyAp-


induced lysis in granulocytes. Cytochalasin B- treated granulocytes are not lysed by HyAp; in- stead exocytosis occurs. Both granulocyte and erythrocyte lysis are strongly reduced by nega- tively charged polymers e.g. poly-D-glutamic acid. The results suggest that HyAp crystal- induced lysis in granulocytes is only possible to- gether with phagocytosis. Positive charges on the crystals play a predominant role in HyAp crystal- induced lysis of both cell types.

In vivo aggregation of platelets by platelet activating factor. W. Paul, C.P. Page & J. Morley, Department of Clinical Pharmacology, Cardio- thoracic Institute, Fulham Rd. Londen SW3.

Platelet activating factor (PAF) has been studied for its ability to affect platelet function in vivo using a novel technique as described by Davies et al. ~ Anaesthetised guinea-pigs with circulating 111-Indium oxine labelled platelets were maintained in a supine position, with scintillation detectors located over the thoracic (CI) and ab- dominal (C2) regions. The method is simple, re- quires the use of minimal amounts of anticoagu- lant and responses to ADP have been shown to be repoducible. Intravenous administration of PAF (30-100 ng/kg) causes an increase in counts in Cl and a concomitant fall in counts in C2 wich are expressed as a ratio C1/C2, consistent with the histological observation that platelets are ag- gregated in the pulmonary vasculature. The time to achieve maximal C1/C2 is within 60 secs after administration, the C1/C2 ratio returning to normal values within 3 minutes. The response of guinea-pig platelets to repeated doses of PAF in vivo exhibited tachyphylaxis ; as has been reported in vitro 2.

REFERENCES

1. Davies, O.J., Morley, J., Page, C. & Paul, W. (1981). In vivo aggregation of guinea-pig platelets, J. Physiol. In Press.

2. Lalau Keraly, C., Tence, M. Heymans, F. & Ben- veniste, J. Specific desensitization of washed rabbit platelets by PAF-acether and derivatives,. T-hrombos, Haemostas., 1981, 46: 270.

Chromogenic endotoxin determination in cereb- rospinal fluid. H. ten Care, L.L.M. Thomas, A. Sturk, K. Joop, J.W. ten Cate. Department of Haematology, Div. of Haemostasis, Univ. Hos- pital "Wilhelmina Gasthuis,,, Amsterdam, The Netherlands.

A sensitive method we developed for the quantitative determination of endotoxins (lps) in blood ~ has now been optimized for application in cereb- rospinal fluid (csf). The method is fast (75 min), sensitive (detection limit 15 pg/ml) and reprodu- cible. In preliminary experiments the assay was correlated with csf-bacterial cultures. 2 patients (p) grew N. meningitidis and 2 p H. Influenzae. The lps content ranged from 150 pg to ) 1 ng/ml. In 1 p the csf gram-stain was incorrectly consider- ed gram-positive whilst the lps-assay was correct- ly positive, 0 10 ng/ml). Only after 36 hrs the csf grew N. meningitidis. No gram-pos, micro-organisms were grown. 2 p with a viral meningitis were negative in the lps-assay. 43 p had a negative csf culture and lps assay. Summarizing an excellent correlation between csf cultures and lps-assay was found in the patients tested. This assay may enable early detection of lps in csf.

REFERENCES

1. Clin, Chim. Acta 1981, 116, 63-68

Effect of antisera on the induction of shock by E. coli, E.S. Kalter, W.C. Van dijk, P.A. Henricks, F. Nijkamp, J. Verhoef, University Hospital Utrecht, 3511 GV Utrecht, The Netherlands

We investigated the induction of hypotension (A MAP) in anesthesized rats (N = 5) by heat killed E. coli bacteria (0.2 x 101~ g I.V.) with different surface structures (SS) 5 minutes following the administration of normal rabbit serum (NRS) or antiserum (AS), 0.3 ml/100g I.V., raised in rabbits. Prior administration of NRS never influenced AMAP. AS-J5, directed against E. coli J5 (SS : lipid A) did not alter AMAP caused by J5 or by E. coli 078K80 (formaline killed, SS : O and K antigen), but significantly increased AMAP caused by E. coli 0111B4 (SS :O antigen) and E. coli 078K80 (heatkilled SS :O-antigen). AS-0111B4 increased AMAP caused by E. coli 0111B4 but not by JS. AS-078, directed against heatkilled (HK) E. coil 078K80 and AS 078K80, directed against formaline killed (FK) E. coli 078K80 increased AMAP caused by 078K80 both the HK and FK form's, but not by 011B4. E. coli 078K80 could not induce hypotension when formaline killed, with surrounding K-antigen, but induced shock after heat killing, with K-antigen destroyed. We conclude that antibodies, raised against the common core glycolipid, as present


on the surface of E. coli J5 enhance the induction of hypotension when additional 0-antigen is present but cannot overcome the blocking effect of K-antigen. Only type specific antisera can enhance the host-pathogen interaction even when the K-antigen is present.

Antiinflammatory effect on carrageenan, RPA and zymosan oedema of LA 2851, a new an- tiasthmatic compound. J.L. Junien, 34. Guillaume, C. Lakatos - S.E.R.P.A. Research Center of ARON Laboratories F - 92152 Suresnes CEDEX France

LA 2851 (2-4-Diamino-7-methyl Pyrazolo (1,5-a) 1,3,5 triazine), a bronchodilator and antiallergic compound in type I hypersensitivity, which inhibits AMPc phosphodiesterase (PDE) has been tested orally for activity against carrageenan oedema and complement dependent, reverse passive arthus (RPA) and Zymosan (Z) oedema in rat. LA 2851 was found active in the first test but showed a more marked effect in RPA and Z, in contrast to classic non steroidal antiinflammatory agents. The minimum active dose was 25 mg/kg (33 ~ inhibition (RPA), 49 070 (Z)), with plasma level concentrations (10 #g/ml) in the range of those required to relax contracted trachea in vitro. The maximum inhibition did not overcome 6007o up to 200 mg/kg indicating that LA 2851 did not suppress all the components of the reaction. PDE inhibition was excluded in the mechanism of action of LA 2851 since papaverine, a potent inhibitor of PDE was inactive. Using per- fused lung preparations and cascade bioassays, in conditions which excluded any antispasmic effect of LA 2851, products of Cyclooxygenase pathway from Arachidonic acid were not affected, but SRS material production using A 23187 iono- phore was inhibited. A direct effect on complement system has not yet been investigated.

Effect of 3,4-dehydroproline on complement activation, E.H. Karran, A.P. Green and F.R. Mangan, Beecham Pharmaceuticals, The Pin- nacles, Harlow, Essex, CMI9 5AD England.

The receptor component of Clq of the C1 complex has a region similar in structure to collagen (Reid and Porter, Biochem. J. 155, 1976, 19-23). We have shown that dehydroproline inhibits collagen production by cultured neonatal heart cells.

If dehydroproline is dosed at 100 mg/kg p.o. to rats 24 hrs and 1 hr before a reversed passive Ar- thus reaction, paw swelling is reduced by 27 07o. It is possible that dehydroproline is incorporated into the collagen-like portion of Clq and that its presence compromises Clq's function as initiator of the classical complement cascade.

The radical scavenging and pharmacological actions of MK-447 are not interdependent. T. Payne, B. Dewald, H. Siegl, H.U. Gubler, 1t. Ott & M. Baggiolini. Preclinical Research, San- doz Ltd., Basel, Switzerland CH-4002

MK-447 is known to enhance prostaglandin synthesis by scavenging the oxidative radical (OH.) formed during the conversion of PGG 2 to PGH 2 and which inactivates cyclooxygenase. Since MK- 447 also inhibits irritant induced inflammation in mice and rats, Kuehl and colleagues have suggested that PGG 2 and OH- are mediators of inflammation. The MK-447 analogues A and B which contain an asymmetric centre were prepared and tested. Both antipodes were equipotent radical scavengers and stimulators of prostaglandin syn- thetase but only the ( + ) antipodes showed antiinflammatory (carrageenin edema ; rat) and diuretic properties (Na § H20 excretion; rat). Both antipodes showed similar absorption, excretion anti-metabolism in the rat indicating that the observed differences in their pharmacological action were not due to distribution effects. The radical scavenging and pharmacological action of this class of compound are clearly not interdependent and the results question the pro-inflammato-

r y r ~ 1 7 6 ~ ' ~ ~ Y B r B Br

MK-447 HO NH 2 A HO NH 2 -- HO Nil 2

Interaction of diflunisal or sodium salicylate with aspirin and indomethacin in prostacyclin release by gastric mucosa. P. Conti, M.A . Continenza § M.G. Cifone, E. Alesse, G. Ianni and P.U. An- geletti. Institute of General Pathology and + Ana- tomy, School of Medicine, L 'Aquila , Italy, 67100.

The administration in rats of salicylates combin- ed with other non-steroid anti-inflammatory drugs (NSAIDs) has a weak effect on prostaglandin syntesis resulting in a low ulcerogenity on gastric mucosa. We tested wether the concurrent

administration of Diflunisal or Sodium Salicylate (SS) with Aspirin or Indomethacin reduced gastric prostacyclin (PGI2) in rat. PGI 2, a potent sti- mulant of mucosa blood flow, is known to pro- tect gastric wall of rat against ulcers. PGI 2 was determined in gastric mucosal slices by its ability to inhibit ADP-induced aggregation of human platelets as compared with authentie-P-~2. The results show that PGI 2 levels in rats treated by an association of SS or Diff. and Aspirin or Indome- thacin, are higher than those observed in rats treated with Aspirin or Indomethacin alone.

Raised plasma levels of thromboxane B2 in systemic mastocytosis. R.J.T. Ouwendijk, F.J. Zijlstra, J.H.P. Wilson, LL. Bonta, J.E. Vincent & E. Stolz. Departments of Internal Medicine, Pharmacology and Dermatology, Erasmus Univ- ersity, Rotterdam, The Netherlands.

The symptoms of patients with systemic mastocytosis cannot be entirely suppressed by antihistam- inics. Recently other workers found an increased excretion of a PGD2 metabolite in 2 patients with mastocytosis. To determine whether other products of the cyclooxygenase pathway might be involved in systemic mastocytosis, we measured plasma levels of TXB2 and 6-keto PGF1 by RIA in 6 patients with systemic mastocytosis and in 34 controls. Patients with mastocytosis had marked- ly raised TXB2 levels (530___105 pg/ml, X+s.e.m., vs 118+8.2 for controls p(0.001) and decreased 6-keto PGFlc~ levels (37+2.6 vs 48 _+ 1.6, p(0.05). In conclusion, we found that systemic mastocytosis is associated with raised plasma TXB2 and lowered plasma 6-keto PGF1 levels. As human mast cells do not produce large amounts of TXB2 in vitro, the present finding may be an indirect effect of leukotriene production.

A pharmacological analysis of thiol derivates on the responses of the guinea-PIG isolated ileum. J. Fontaine, J.P. Famaey, J. Reuse. Laboratory of Pharmacology, University of Brussels, 1000 Brussels, Belgium.

Several thiol compounds (d-penicillamine, dimer- captopropanol, cysteine) increase the responses of the guinea-pig isolated ileum to various agonists. This was also recently observed with leukotrienes. We have performed a comparative


study of the effects of 5 thiol compounds on the responses of the guinea-pig ileum to various agonists (acetylcholine, histamine, serotonin, nicotine, bradykinin, PGE2). A nondose-related re- versible increase of 20 to 60 % was obtained on the responses of the agonists in most of the cases with cysteine, glutathione, dithiotreitol, mer- captoethanol and monothioglycerol at concentrations of 10 and 40 mg/1. However the responses to nicotine are not significantly increased in all the cases and with dithiotreitol they have been even strongly inhibited, which suggests a ganglioplegic effect of this compound. Cysteine induced in some experiments an ileal contraction interfering with its sensitizing effect. It ~is con- cluded that these 5 thiols exert at the muscular level a non-cholinergic sensitization of the ileum to various agonists. This might be a common property of all thiol compounds which might affect the agonists receptor sites.

In vitro spasmogenic effect on rabbit lungtissue of 1-0-octa decyl-2-acetyl-sn-glyceryl-3-phospho- ryicholine (GPC) (platelet-activating factor): specific desensitization after in vivo l-0-octadecyl-2-acethyl-gpc infusion. Camussi G. Mon- trocchio G, Antro C, Tetta C, Bussolino F, Emanuelli G, Laboratorio di Immunopatologia, Cattedra di Nefrologia e Patologia Medica III. UniversitA di Torino. Italia.

1-0-octadecyl-2-acetyl-GPC(3.3 • 10-9M) induced a slowly developing contraction of rabbit lung parenchymal strips in vitro by a mechanism dis- tinct from those of histamine (His), acetylcholine (Ach) and C5a. Prior exposure of lung tissue to 1-0-octadecyl-2-acetyl-GPC resulted in desensitization to 1-0-octadecyl-2-acetyl-GPC, but not to His, Ach, C5a.l-0-octadecyl-2acetyl-GPC appeared more potent on a molarbasis (3:3 • 10-9M) than His (10-SM), Ach (10-SM) and C5a(10-8M). However, rabbit lung tissue appeared more sensitive in terms of entity of maximal contraction to His dependent agonists (His,C5a). In vivo infusion of l-0-octadecyl-2-acetyl-GPC (l.5ug/kg) into rabbits induced cardiovascular and respiratory changes characteristics of an anaphylactoid reaction. The lung mechanical alterations were related to platelet dependent mechanisms rather than to a direct action of l-0-octadecyl-2acetyl- GPC. However, the rabbit lung parenchymal strips obtained 30' after infusion with 1-0-octadecyl-2-acetyl-GPC appeared completely desensit-


ized to 1-0-octadecyl-2-acetyl-GPC, but not to His, Ach and C5a.

Quantum chemical analysis of structure-activity relationships in non-steroidal, antiinflammatory drugs. E.L. Mehler, J. Habicht, and K. Brune, Depts, of Structural Biology and Pharmacology, Biocenter, Univ. of Basel, Switzerland and Univ. of Erlangen, FRG

Ab Initio, quantum chemical methods were used to analyze structure-activity relationships in non- steroidal anti-inflammatory drugs. The biological data for this study derive from an assay of the inhibition of prostaglandin production in macro- phagesk Of about 80 compounds assayed 30 were selected for calculation. These compounds in- clude substituted phenols, benzoic acids, and sali- cyclic acids where the substituents are hydride, hydroxide, fluoride, and chloride. The geometrics of these compounds were assumed to be planar with standard bond lengths and angles. The results show a correlation between the pED50 of the active compounds and the orbital energy of the ~r-HOMO (Highest Occupied Mo- lecular Orbital) with a correlation coefficient of r -0.9. They indicate that potency increases with decreasing binding strength of the r -HOMO elec- trons suggesting that charge transfer may be im- portant for interaction with relevant binding sites. Additional results indicate that charge build-up on the substituent enhances activity whereas ex- cessive charge on the carboxyl group inhibits activity.

REFERENCE

1. K. Brune, K.D. Rainsford, K. Wagner, and B.A. Peskar, Arch. Pharm. 1981 315, 269.

Inflammatory bowel diseases: cellular reactivity mediated by serum factors ? A. Van Gossum +, M. Cremer +, M. Delronche ++, T. Ap- pelboom + +. Service de Gastro-Entdrologie + and Laboratoire de Rhumatologie ++. H6pital Er- asme Cliniques Universitaires de Bruxelles (ULB) Route de Lennik, 808 1070 Brussels Belgium.

The leucocyte adherence inhibition test was used to demonstrate, in the serum of patients with inflammatory and neoplastic bowel disease, a fac-

tor able to sensitize normal leucocytes against some intestine components. Indeed, leucocytes preincubated in serum of untreated patients suffering from Crohn disease, ulcerative colitis or colon neoplasia were unable to attach to glass tube when incubated with an intestine extract compared to incubation with a lung extract as control. Normal serum dit not have this effect. Moreover, the relative inhibition of adherence when quantified was particularly higher in colon cancer and ulcerative colitis than in Crohn disease. Steroid therapy seemed to alter the serum activity. This test could thus be used as a tool to early detect the various inflammatory or neoplastic conditions of the intestine. It also suggests that a specific response against immune or- gans exists in these diseases.

Effect of activated serum on mononuclear and polypmorphonuclear cells adherence. T. Ap- pelboom*, M. Pierart* and R. G6rtz**. Service et laboratoire de Rhumatologie*, H6pital Erasme, U.L.B. and Fondation Robert Debr6**, 808 route de Lennik 1070 Brussels, Belgium

Leukocyte chemokinesis, chemotaxis, adherence, occur sequentially depending on the type and the intensity of the signal. But the same signal could act not synergically on mononuclear and polymorphonuclear (PMN) cells. We therefore tried to compare the effects of C5a using zymosan treated serum (ZTS) and of Clq using heated serum (HS) with Clq inactivated, not only on PMN but also on monoculear cells adherence to glass. We observed that the adhesiveness after cell incubation in ZTS was increased for mononuclear cells but decreased for PMN. H.S. containing inactivated Clq showed an opposite effect: mononuclear and PMN adhesiveness were decreased. These results suggest that mononuclear cells respond to some inflammatory signals e.g. Clq and C 5 an by increased adhesiveness, but PMN response is different : Clq increases while Csa inhibits adherence. These data could contribute to explain why cells arrive sequentially on the site of inflammation.

Hydroxyl scavengers promote, and antilipoperox- idants inhibit cyclo-oxygenase activity. C. Deby and G. Deby-Dupont, Laboratoire de Biochimie Appliqu6e, University of Liege, 32, boulevard de la Constitution, 4020, Liege Belgium.

A self-inactivation mechanism limits the activity of cyclo-oxygenase, by a production of destruc- tive free radicals 1. It may be expected that free radical scavengers, extending the life of cyclooxygenase, must enhance the rate of prostanoids production. But many radical scavengers are si- multaneously antilipoperoxidant agents and slow down the prostanoid synthesis by this way. A bal- ance exists between the two properties, the resulting effect being inhibitor, indifferent, or stimu- lating, according to the prevailing property of the concerned compound. Few substances are free radical scavenger only, without action on lipoperoxidations. These scavengers will be expected to be potent cyclo-oxygenase activators. It is the case for uric acid, which is a strong cyclo-oxygenase activator : it is a potent hydroxyle scavenger, but does not affect lipoperoxidations.

REFERENCE

1. Egan R.W., Paxton J., Kuehl F.A., J.Biol. Chem. 1976, 251, 7329.

Enkephalins stimulate prostacyclin (PGI 2) production. M.A. Boogaerts, J. Vermylen, H. Deckmyn, C.F. Moldow, R. Verwilghen, University Leuven, Belgium.

Endogenous opioid peptides - endorphins and enkephalins - function in a variety of physiological systems. They have been implicated in the modula t ion of pain-percept ion and mood, in the release of various hormones and in the regulation o f respiratory and cardiovascular systems. We now report enkephal in action on basic inf lammatory processes and on haemostasis and thrombosis related phenomena. Human umbilical vein endothelial cells, cultured in vitro, are characterised and identified by their basal PGI 2 product ion, averaging 474 pg _+ 41 pg/3 • 105 cells/4 min. Af ter stimulation with arachidonic acid (AA, 20 uM) or thrombin (T, 10 U/ml ) PGI 2 product ion increases almost tenfold, averaging 3058 + 280, and 4069 -+ 710 pg/3 • 105 cells/4 min. re- spectively. Pretreatment of the endothelial cells with 10-8M methionine-enkephal in (physiological dose) further enhances the stimulated PGI2-product ion to 7135 +_ 1805 pg with A A and 6192 _+ 195 pg with T. ( 3 x 105 cells/4 min.). Fnkephalin stimulates PGIz-generat ion in a dose dependent manner , with maximum effect


at 10 -s for A A , and 10-1~ for T. Enkephal in alone, without A A or T, does not cause significant PGI2-product ion. Local inf lammatory processes, with microvascular occlusion and local th rombin gener- ation, thus might be modula ted by endo- genously secreted peptides. Enhanced endothelial PGI2-product ion possibly represents a negative feedback mechanism for stress-related thrombotic phenomena.

Endotoxin schock in the pig: cardiovascular effects and changes in intestinal serotonin content. E. Schrauwen, A. Weyns, Ch. Gillebert, A. Houvenaghel. Lab. of Vet. Physiol. and Lab. of Vet. Anat . , RUCA, Antwerp, Bel- gium.

The effects of a LD10 o dose of E. Coli endotoxin, 0.5 m g / k g intravenously, on hemody- namic parameters and on 5-hydroxytryptamine (5-HT) content of duodenal endocrine cells have been evaluated in pentobarbi ta l anesthet- ized young pigs ( + 15 kg). Mean survival t i m e amounted to 104+15 min. Total peripheral and superior mesenteric vascular resistance revealed an initial transient increase followed by a pronounced and sustained decrease, whereas pulmonary resistance displayed a pronounced and sustained increase. Systemic arterial pres- sure revealed a delayed decrease f rom a control value of 97 + 5 mm Hg to a preterminal value of 3 0 + 6 mm Hg, whereas cardiac output gradual ly declined f rom 2.4-+0.3 to 1 .3+0.2 1.min -1. The observed responses in the pig therefore resemble these described for primates. Cytof luorometr ic measurements of the duodenal 5-HT cells revealed no evidence for a decrease in the 5-HT content as compared to sham-shocked animals. However, a marked variabil i ty within both groups was present. So no indicat ion can be argued for a 5-HT depletion of the duodenal endocrine cells by endotoxin shock in the pig.

Further study of fluorescent free radical products in synovial fluid. D.G. Wickens and T.L. Dormandy.Department of Chemical Pa- thology, Whi t t ington Hospi ta l , London N19 5NF, England.

Considerable evidence suggests that fluorescence (Ex 340-370nm, Em 430-460 nm) may

152 Clinical rheumatology, 1982, lcrN ~ 2

reflect biological free-radical activity: it has been detected in synovial fluid from patients with rheumatoid arthritis (RA). By fractio- nation using gel-filtration chromatography we have shown that this fluorescence is associated with three protein fractions. 70-75 % of the total fluorescence is present in the albumin fraction and its characteristics point to a Schiff base structure. 15-20 ~ is due to an as yet un- characterised fraction of molecular weight approximately 20.000 daltons. The remaining 5- 10 ~ is associated with gamma-globulin and is almost certainly due to the free-radical oxid- ation of tryptophan residues. Significantly higher levels of fluorescence was observed in all three fractions in synovial fluid from RA patients compared to synovial fluid from de- generative arthrosis patients. This was espe- cially noted in the gamma-globulin fraction (p (0.001).

Activation of rabbit platelets by endotoxin : significance of thromboxane (TX) and release reaction. H. Bult, G.M. Laekeman and A.G. Herman, University of Antwerp, Department of Experimental Pharmacology, B-2610 Wil- rijk, Belgium.

Recalcified rabbit platelet rich plasma was exposed to 10 different preparations of endotoxin (Lipopolysaccharide, LPS) from smooth forms of Salmonella and E. coli bacteria. Changes in turbidity and the luminescence caused by a luciferin/luciferase mixture in the presence of released ATP were monitored in a lumi-aggregometer (Chronolog). After 15 min indomethacin (1M,28 M) was added and TXB 2 formation was assessed with radioimmunoassay. Responses were compared with incubations with saline, ADP and arachidonic acid. LPS (1 mg/ml) always caused a slow decrease in turbidity, probably representing ag- glutination. With some preparations of LPS this was followed by a more rapid increase in light transmission, which coincided with ATP release. These preparations of LPS were also active in lower concentrations. All LPS preparations caused a small increase in TXB 2 biosynthesis, which was more significant if the release reaction also occured. IM prevented TXB 2 biosynthesis, but hardly influenced ATP release and the turbidimetric pattern. Aggluti- nation may be followed by platelet activation, with the simultaneous occurrence of TX biosynthesis, release reaction and aggregation. However, TX biosynthesis is not a prerequisite for endotoxin induced release reaction and aggregation.

Fourth European Workshop on Inflammation Wilrijk March 1982

Documents

Transcript of Fourth European Workshop on Inflammation Wilrijk March 1982