SECTION ONE - CUNICAL ASSAY AND BIOINCOMPAnBIUTY 1.

A flow cytometrlc assay for measuring complement receptor 1 (CR1) and the complement fragments C3d and C4d on erythrocytes. Jona Frevsdottir and Asbjorn Sigfusson. Dept. of Immunology, The National University Hospital, Landspitalinn, 101 Reykjavik, Iceland.

A flow cytometric assay (FCA) has been developed to measure complement receptor 1 (CR1) and the complement fragments C3d and C4d on erythrocytes. It is possible to measure these parameters accurately with intra- and intertest coefficient of variation of 2.0% and 6.5% respectively. The method discriminates between low and high levels of erythrocyte CR1, C3d and C4d. Comparison to a previously described RIA method shows an excellent correlation where r2 is 0.94, 0.93 and 0.91 for CR1, C3d and C4d respectively. The flow cytometric assay has been used to measure CR1, C3d and C4d on erythrocytes from 98 healthy individuals and 95% upper limits for C3d and C4d have been established. There is a wide distribution of CR1 levels amongst the healthy individuals but their C3d and C4d levels are low and often not over background level. The possible application of this method in clinical medicine will be discussed.

2.

Adhesive properties of S-protein and clusterin: A particular challenge for quantitative enzyme immunoassays (EIAs). K. HoLgsen, T.E. Mollnes, J. Tschopp1 and M. Harboe. Institute of Immunology and Rheumatology, University of Oslo, Norway, 1Institute of Biochemistry, University of Lausanne, Switzerland.

S-protein (vitronectin) and clusterin (SP-40,40/CLI) are multifunctional, adhesive proteins which inhibit complement-mediated lysis. Double antibody EIAs for these sticky proteins are technically difficult since considerable amounts bind unspecifically to the plastic surface in spite of blocking agents and detergents. The validity of such assays may therefore suffer if this problem is not overcome. The Dynatech Immulon 2 In plate, 0.2% Tween 20 and pH 6.0 eliminated the unspecific binding of S-protein, but not of clusterin. presence of 0.2% Tween 20 clusterin bound almost selectively to uncoated plastic and was successfully quantitated in a single antibody assay. The 95% reference range of S-protein concentration among blood donors was 0.23-0.80 g/L (median 0.41). The corresponding range for clusterin was 78-128% of the median value.

3.

Immununoradiometric assay for human complement component C8. A. Abraha and J.P. Luziol. Department of Chemical Pathology, John Radcliffe Hospital, Headington, Oxford OX3 9D4 and 1Department of Clinical Biochemistry, University of Cambridge, Addenbrooke's Hospital, Hills Road,

Cambridge, CB2 2QR, UK. In a variety of rheumatic diseases measurement of terminal complement components in body fluids may provide a useful indicator of disease activity and help evaluate the response to therapeutic manipulation. In Behcet's disease and rheumatoid arthritis elevation of plasma C9 concentration has been shown to be a better indicator of disease activity than CRP or ESR [1,2]. Using monoclonal antibodies prepared against different subunits of C8 [3,4] we have recently established a two site immunoradiometric assay for C8. C8 was captured on 96well plastic plates coated with an antibody to the p subunit (Bl) and measured with an 1251-labelled antibody to the a subunit (E2). An assay was established with a working range of 1-10 ng/100pl (plasma range 20-185 mg/L) at a CV of 12%. C8 concentration in plasma from 81 blood donors (34 male, 47 female) with a mean age of 46 yrs (ranging 21-67) was 116 ± 25 mg/l (range 55-183). Preliminary data on plasma concentrations of C8 in patients with rheumatic disease has been obtained. 1. 2. 3. 4.

Morgan, B.P. et al. (1983) Clin. Chim. Acta 34, 85-94. Rumfeld, W.R. et al. (1986) Br. J. Rheumatol. 25, 266-270. Abraha, A. et al. (1988) Biochem. J. 251. 285-292. Abraha, A. & Luzio, J.P. (1989) Biochem. J. 264, 933-936.

1

4.

Colorimetric micro-assay and rapid Isolation of human complement component C9. Hester J. Bootsma, Carmen W. van den Berg and Hans van Dijk. Eijkman-Winkler Laboratory of Medical Microbiology, Section of Experimental Microbiology, Utrecht University, University Hospital G04.614, P.O. Box 85,500, NL-3508 GA Utrecht, The Netherlands. A novel, functional micro-assay of human complement component C9 is presented. The assay is based on the principle of reactive (C5b6-initiated) lysis of chicken erythrocytes and uses a commercially available C9depleted serum as reagent for C9. The method is sensitive, since serum samples can be tested in high dilution (up to 1/3000). The assay is also specific, because rapid, activity-guided isolation of the haemolytic component from human serum yields a single protein with the characteristics of C9. Advantages of our assay over immunochemical and other haemolytic assays are its functional character, its relatively high sensitivity, its specificity in terms of minimal interference by C8, and its general accessibility since all materials are commercially available. C9-titres in a normal population of students (n = 38) showed an exponential distribution pattern with a mean value t SE of 103-955 t 0.404 (mean ± 2SE - 1,403 - 57,943) units per ml.

5.

Activation of the alternative pathway of complement by an Immunoadsorption cartridge used In ABOincompatible kidney transplant candidates. J.R. Stubbs. B.A. Benson, J.A. Loch and A.P. Dalmasso. Minnesota, USA.

V.A. Medical Center and U. of Minnesota, Minneapolis,

The BIOSYNSORBtm Immunoadsorption Cartridge (Chembiomed Ltd., Edmonton, Alberta, Canada) is an investigational device used in the removal of ABO blood group antibodies from the blood of ABO-incompatible kidney transplant candidates. Various adverse reactions have been observed during these procedures. Complement activation is postulated as playing a pathophysiologic role in some of these reactions. Therefore, the complementactivating characteristics of this device were analyzed. Twenty-eight procedures were performed on 16 Blood was collected from the patients pre-treatment, at 15 minutes and 60 minutes during the patients. The samples were assayed for CH50, AP50, C3a, C5a, Bb, hemolytic procedures, and immediately post-treatment. The results show early and marked complement activation. C4, and SC5b9 levels. Significant elevations of C3a and C5a levels peaked at 60 minutes, however significantly C3a, C5a and SC5b9 were seen by 15 minutes. lower post treatment C3a and C5a levels suggest a rapid decline after 60 minutes. Mean CH50 and AP50 levels decreased 26% amd 39% from pre- to post-treatment, respectively. Most striking, however, was the >40 fold pre- to post-treatment increase in mean Bb level (0.2 pg/mL to 10.6pg/mL), contrasted with a modest (20%) decrease in mean hemolytic C4 level. In vitro studies consisted of a 60-minute incubation of type 0 or type A blood or plasma with the immunoadsorbant carrying type A specific oligosaccharide. Marked increases in C3a levels occurred regardless of blood type. These in vitro results, in combination with the in vivo studies showing marked elevation of Bb levels and marginal changes in hemolytic C4 levels, suggest that complement activation during immunoadsorption with the BIOSYNSORBTM Cartridge is predominantly through the alternative pathway and due to a process other than the binding of ABO antibodies.

6.

Complement activation and cytokine release during haemodialysis (HD). M. Gardinali, A. Anelli1, A. Rosti2, A. Calcagno, L. Conciato, U. Zonio, M. Del Pretel, P. Cori2, G.A. Moroni2, D. Brancacci1 and A. Agostoni. Clinica Medica, Div. di Nefrologia1 e Immunoematologia2 Ospedale San

Paolo-Universitat di Milano, Italy. Recent evidences showed Complement system is activated during HD, particularly with cuprophane membrane. C5a acts synergistically with bacterial that cytokine levels are also increased during HD. Such a condition could exist also lipopolysaccharides (LPS) in promoting IL-1 and TNF production in vitro. The aim of this study is to evaluate plasma levels of in vivo when bacterial contamination occurs during HD. various cytokines (IL-1p, IL-2, TNF) and IL-2 receptors (IL-2R) during HD performed with membranes which activate complement system to a different extent as cuprophane (CU). polysulfone (PS), polyacrylonitrile (PAN) Bacterial contamination was minimized using sterile bicarbonate dialysis and polymethylmethacrylate (PMMA). Blood bath. Forty patients were dialyzed (10 for each membrane) three times weekly for three months.

2

samples were serially drawn during HD (0, 15', 120'. 360'). As expected, complement was activated most by CU (C5a peak after 15' - 56.0 ± 28.1 ng/ml). PMMA (6.2 ± 4.9), PS (5.6 ± 2.9) and PAN (5.4 ± 0.9) determined In all samples (before and during only a mild activation and/or absorbed anaphylatoxins on their surface. HD) IL-1p. IL-2 and TNF levels were below the assay detection limits (respectively 15 pg/ml, 10 I.U./ml, 60 pg/ml). IL-2 receptors (IL-2R) were slightly elevated in uremic patients comparing to controls (525 - 710 I.U./ml) but did not change during HD. Pre-dialysis cytokine and IL-2R levels after three months of dialysis with CU were not equally increased. Our data indicate that if precautions are taken to minimize bacterial contamination, complement activation per se does not determine a significant increase in plasma cytokine levels.

7.

Activity of recombinant soluble Decay Accelerating Factor. Ingrid W. Caras. Genentech, Inc., San Francisco, Cal. 94080,

USA.

Decay Accelerating Factor (DAF) is normally a membrane-bound glycoprotein, anchored to the cell surface by a We have engineered a soluble, secreted form of DAF (SeDAF) by glycophospholipid (GPI) membrane anchor. removing the signal for attachment of the GPI membrane anchor, located at the COOH-terminus of the protein. Purified SeDAF appears to retain the activity of native, membrane-bound DAF, inhibiting both the classical SeDAF is capable of inhibiting complementpathway and alternative pathways of complement activation. mediated lysis of antibody-sensitized sheep red blood cells (classical pathway) and of non-sensitized rabbit In addition SeDAF blocks fluid phase activation of complement, red blood cells (alternative pathway). The assessed by measuring C5a formation following activation by soluble immune complexes or endotoxin. ability of SeDAF to inhibit complement activation in vivo was tested in guinea pigs, using the Reversed Passive Arthus reaction. SeDAF may be useful in treating complement-mediated diseases of inflammation.

8.

High molecular weight complement InhlbItor(s) of Symphytum officinale. F. Van den Dungen, C.J. Beukelman. A.J.J. Van den Berg, H.C. Quarles van Ufford, H. Van Dijk and R.P. Labadie. Research Centre for Natural Products and Phytopharmaceuticals, State University of Utrecht, Faculty of Pharmacy, The Netherlands.

Our research aims at the isolation and characterization of medicinal plant components with immunomodulatory One of the plants studied is SvmDhvtum officinale, which is said to have wound healing activity. activity. An ethanolic extract of the roots, which gave a strong inhibition of the classical pathway was used as The extract was centrifuged starting material to study interference with individual complement components. (150,000 x g), and the supernate was subsequently subfractionated by use of amicon ultrafiltration with cutoff levels of 300, 100, and 10 kd. The activity resided in the fraction with a relative molecular mass of over 300 k. The > 300 Complement component C4 was inhibited by this fraction in a dose-dependent manner. Kd fraction was ninhydrin and naphto-resorcinol positive, pointing at the presence of amino acids and sugars, Elucidation of the molecular structure of the active principle is respectively in macromolecular entity. subject of current investigation.

SECTION TWO - INHIBITORY PROTENS AND NON-LETHAL EFFECTS 9. Analysis of the activities of recombinant mouse CR1, CR2 (Cr2) and p65 (The Crry gene product), a family of molecules with structural and functional homologies to the human membrane RCA gene family. V.M.Holers. T. Kinoshita, W. Wong, C. Brenner and H. Molina. HHMI at Wash. Univ. Sch. Med., St. Louis, MO 63110, USA, Osaka Univ. Med. Sch., Osaka, Japan and BASF Bioresearch Corp., Cambridge, MA., USA. In order to fully utilize mouse models of the immune response in the analysis of RCA receptor and membrane regulatory protein functions, an understanding of the endogenous family is necessary. In studies by ourselves and others, three mouse cDNAs homologous to human CR1 and CR2 have been defined: MCR1 and MCR2 (also known as Cr2) and Crry. We have expressed recombinant forms of each in the human K562 erythroleukemia cell

3

line. Analysis of these molecules reveals that: 1) MCR1 and MCR2 have C3b and C3d binding activities which in general parallel human CR1 and CR2; however, they are likely the products of alternative splicing from a common gene, and 2) Crry encodes a protein known as p65 which has intrinsic complement regulatory activity and acts to protect cells from C3 deposition. Manipulation of the activities of these proteins in vivo should lead to substantial insights into the biologic roles each type of protein and activity plays in the immune response.

10. In vitro formation of polymeric S-protein and its effects on platelet aggregation. K. Hogasen, M. Roger, T.S. Halstensen, T. Hovig, T.E. Mollnes and M. Harbg&.

Institute of Immunology and

Rheumatology and Department of Pathology, University of Oslo, Norway.

S-protein was purified by heparin-affinity chromatography. The purified protein polymerized spontaneously when dialyzed against PBS. SDS-PAGE, western blot and gel filtration showed a slightly heterogeneous population of disulphide linked polymers with MW of approx. 1000 kD. Aggregometry of washed platelets showed that polymeric S-protein had platelet aggregating properties, in contrast to the reduced and alkylated monomeric form. Electron microscopic studies were performed on washed thrombin stimulated platelets incubated with S-protein. The platelets were then fixed in paraformaldehyde and stained with monoclonal antibodies to S-protein and gold-conjugated secondary antibodies. These experiments revealed S-protein binding to released a-granule material and an apparent crosslinking of platelets. We therefore suggest Sprotein to be a cofactor in platelet aggregation.

11. Genetic deletion of bases in HRF20-gene in a patient deficient in HRF2O. E. Okada, N. Motoyama, S. Furue, N. Okada1, M. Yamashina2 and T. Takami2. Dept. Mol. Biol. Nagoya City Univ. School Med., Nagoya 467; 1Dept. of Microbiol., Fukuoka Univ. School of Med., Fukuoka 814-01, 2Dept. Pathol., Gifu Univ. Sch. Med., Gifu, 500, Japan.

HRF20 (CD59) is deficient in a patient suffering from paroxysmal nocturnal hemoglobinuria (PNH). This deficiency in HRF20 is not only on blood cells but also other tissues including endothelial cells and peripheral nerves. His parents are cousins and both have decreased HRF20 expression on blood cells suggesting that the deficiency could be genetic. We have established a cultured cell line of EBV-transformed B mRNA was purified from the cells and Northern blot lymphoid cells (NCU-ls) which are deficient in HRF20. analysis detected an HRF20cDNA signal indicating that HRF20 mRNA was transcribed. On the cDNA prepared from the mRNA, the HRF20 gene was amplified by the PCR method. DNA sequencing of the cDNA from the patient's mRNA showed base deletions at alanine (#16 and #95 from the N terminal). The deletion in the genomic DNA of peripheral blood lymphocytes was confirmed by the DNA sequence of an HRF20-open reading frame containing the #16 amino acid amplified by PCR. Furthermore, patient's parents and sister posessed intact and deleted These findings prove that the HRF20 deficiency was genomic DNA of HRF20 while his brother's DNA was intact. a genomic event.

12. The CD59 antigen protects spermatozoa against membrane attack complex mediated damage. Isabelle A. Rooney. Alexandra Davies and B.P. Morgan. Department of Medical Biochemistry, UWCM, Cardiff,

CF4

4XN, UK. Complement-fixing anti-spermatozoal antibodies (ASA) have been detected in the sera of both men and women and are associated with infertility. Complement has been implicated as a mediator of spermatozoal damge but the Here we have mechanisms by which complement causes immobilisation and lysis of spermatozoa remain unclear. utilised depleted sera to examine the requirements for complement-mediated immobilisation and lysis of The results demonstrated that these effects are dependent on formation of the complete spermatozoa in vitro. membrane attack complex (MAC). Using specific monoconal antibodies in immunofluorescence microscopy, flow cytometry activated cell sorting and western blotting we have examined the expression of the MAC regulatory The cells stained strongly for CD59 antigen but not for proteins MIP (HRF) and CD59 antigen on spermatozoa. MIP. Spermatozoal CD59 antigen had a similar molecular weight to the erythrocyte protein and was, at least

4

in part, GPI anchored in the membrane. The protein was also present in seminal plasma. Neutralisation of spermatozoal CD59 antigen using Fab2 fragments of specific antibodies rendered the cells much more susceptible to immobilisation and lysis by the homologous MAC indicating that the protein was of potential functional relevance in maintaining spermatozoal viability.

13. The complement Inhibitory protein CD59 Ag Is present and functionally active on second trimester foetal

fibroblasts. Isabelle A. Roonev and B. Paul Morgan.

Dept. of Medical Biochemisty, UWCM, Heath Park, Cardiff, CF4 4XN, UK.

We have Little is known of the function and control of the complement system in foetal development. investigated the presence and function of three GPI linked complement inhibitory proteins, Decay accelerating factor (DAF), CD59 Antigen (CD59 Ag) and MAC inhibitory protein (MIP) on 3 strains of foetal skin fibroblasts routinely cultured for diagnostic purposes following spontaneous abortion at 16 - 20 weeks gestation. None of CD59 Ag was found by immunofluorescent and flow the strains studied showed evidence of genetic abnormality. In contrast, cytometric techniques to be strongly expressed on the surfaces of cells of all three strains. SDS PAGE and Western blotting of foetal levels of MIP and DAF were very low or absent on these cells. fibroblast membranes revealed a CD59 reactive species with electrophoretic mobility identical to that of erythrocyte membrane CD59 Ag (molecular weight 18 - 2OkDa), suggesting that the foetal protein is very similar or identical to the adult form of this protein. Blocking of CD59 Ag with F(ab)2 fragment of monoclonal antiCD59 antibody rendered the cells markedly more susceptible to lysis by human complement, demonstrating that this protein may function in the protection of foetal cells from MAC attack in utero.

14.

Role of Inhibitory membrane proteins In the complement dependent killing of trophoblast cells. F. Tedgj=, G. Narchil, S. Meri2, C. Betterle3 and 0. Radillol. Instituto di Patologia

Generale and lInstituto per l'Infanzia, Trieste, Italy, 2Department of Bacteriology and Immunology, Helsinki, Finland, 3Instituto di

Semeiotica Medica, Padova, Italy. Whether they exhibit a Trophoblast cells are known to be highly resistant to killing by decidual NK cells. similar resistance to C lysis and use C inhibitory membrane proteins for their protection has not been We have evaluated the susceptibility of trophoblasts to C killing using cells sensitized by established. antibodies from a patient with Addison disease and also unsensitized cells exposed to C activated through the In the antibody dependent system, the extent of trophoblast killing was related to reactive lysis system. the amount of antibodies, but the percentage of dead cells did not exceed values of about 40%. The results obtained with the reactive lysis were essentially similar and were related to the amount of C56 used. Immunohistochemical analysis of trophoblast cells for the presence of the C inhibitory proteins showed that all the syncytiotrophoblasts express CD59 and MCP, whereas DAF was detected only in about 30% of the cells. The number of dead cells increased substantially in the presence of mAb to CD59 and, to a lesser extent, in the presence of mAb to MCP, but was essentially unchanged when the trophoblast were incubated with mAb to DAF.

15. Loss of expression of CD59-antigen is associated with complement membrane attack complex deposition In myocardial Infarction. Vaveka1, P. Laurila2 and S. Meril. 1Department of Bacteriology and Immunology, University of Helsinki, Finland. 2Department of Pathology, University of Helsinki, Finland.

A.

CD59-antigen ("protecting. HRF20, MACIF, MIRL) is an inhibitor of the membrane attack complex of complement (MAC). It inhibits complement mediated cell lysis by interacting with the C8 and C9 components of MAC. Immunohistochemical examination of human heart specimens showed expression of the CD59-antigen in normal myocardial cells. In patients who had died of myocardial infarction (n=10) CD59 expression was lost or diminished in infarcted lesions. Loss of CD59 expression was accompanied by deposition of components of MAC: In control heart specimens without infarction no similar deposits of MAC were observed. C5, C6, C8 and C9. As the expression of CD59-antigen in normal myocardium was found to be sensitive to treatment with phosphatidyl-inositol specific phospholipase C (PIPLC) it is possible that loss of the protective CD59-antigen

5

Complement membrane attack against myocardial cells may thus be was caused by an endogenous phospholipase. due to an acquired loss of resistance to autologous complement and contribute to tissue damage during the reperfusion stage of myocardial infarction.

16. Regulation of expression of complement inhibitory proteins on thyroid follcular cells. B.P. Moraan, 1N. Tandon and 1A.P. Weetman. Department of Medical Biochemistry, UWCM, Cardiff and 1Department of Medicine, University of Sheffield Clinical Sciences Centre, Sheffield, U.K.

We have previously described the presence of heavy deposits of the membrane attack complex (MAC) in thyroid tissue from patients with autoimmune thyroid disease and have suggested a role for the MAC in disease pathogenesis1l2. In the present study we have investigated the distribution of MAC inhibitory molecules in Two MAC inhibitory thyroid tissue and their functional relevance on thyroid follicular cells (TFC). molecules have been described, MAC-inhibiting protein (MIP, also termed HRF and C8bp) and CD59 antigen Both proteins are present on circulating cells and the latter has (protectin. HRF-20, MIRL, MACIF, etc). Here we report that both proteins are also been localised to numerous epithelial and endothelial cell types. present in the thyroid follicles from normal and diseased tissue. Staining was strongest in the apical brush border region of the cellsin contrast to the MAC which was localised to the basement membranes of the thyroid TFCs in culture stained strongly for CD59 antigen and folicles and was only present in diseased tissue. weakly for MIP. Incubation of TFCs with T cell conditioned medium, thyroid stimulating hormone or with recombinant cytokines (TNF, IL-1, y -IFN) markedly upregulated the expression of both inhibitors and Antibodies against either of the inhibitory concomitantly rendered the cells less susceptible to MAC lysis. This phenomenon was proteins rendered both unstimulated and stimulated TFCs more susceptible to MAC lysis. most marked on stimulated cells where an additive effect of neutralising the two inhibitors was apparent. We suggest that expression of these regulatory proteins may limit the severity of tissue injury produced by complement-fixing antibodies in autoimmume thyroid disease. References: 1. Weetman, A.P., Cohen, S.B., Oleesky, D.A. and Morgan, B.P. (1989) Clin. Exp. Immunol. 77, 25. 2. Weetman, A.P., Freeman, M. and Morgan, B.P. (1990) Clin. Exp. Imunol. 82, 69.

17. Enhanced surface expression of the complement regulatory proteins C8bp and CD59 In stressed cells and in cells

undergoing apoptosis. G. Schieren, 0. Janssen and G.M. Hansch.

Institut fur Immunologie der Universitat Heidelberg, Germany.

Recently, we have shown that the surface expression of C8bp is upregulated when cells e.g. monocytes, U937, glomerular mesangial- or epithelial cells, synovial fibroblasts are cultivated in the presence of cytokines. An upregulation was also seen, when cells were exposed to Together with C8bp also CD59 is upregulated. Most interestingly, when cells e.g. U937 were subjected to heat shock (41.50C,2h) phorbol ester or LPS. Moreover, U937 which were cultivated in the presence of surface expression of C8bp was also enhanced. cycloheximide for 24h, drastically increased C8bp on the cell surface. At the same time, the cells were When apoptosis was induced with mistletoe lectin again enhanced surface expression of undergoing apoptosis. Taken together our data C8bp was seen, indicating a close link between apoptosis and upregulation of C8bp. indicate, that the surface expression of C8bp and CD59 is not only modulated by inflammatory stimuli, but also Thus, at least in functional terms, C8bp and CD59 appear to be stress proteins. by insults to the cell.

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18. Inhibition of the C(56)a7-9 Induced Interleukin 1 (1-i) synthesis of U937 by erythrocyte-derived C8 binding protein (C8bp). G. Schieren and G.M. Hansch.

Institut fur Immunologie der Universitat Heidelberg, Germany.

Acid activation of the complement system achieved by briefly lowering the pH to 6.4 at 40C resulted in the formation of C(56)a, which together with C7. C8 and C9 formed a lytic complex on erythrocytes. Incubation of acid activated serum with the human histiocytic cell line U937 also resulted in an uptake of C(56)a7-9 on the The cells, however, responded to C(56)a7-9 with release of 11-1. surface, but not in cell killing. Serum deficient in C8P was not stimulatory. To test whether the C(56)a7-9 dependent stimulation of U937 may also be controlled in a species-specific manner, C8bp, purified from human erythrocytes, was added to the U937 prior to complement stimulation. An up to 80% inhibition of the complement induced I1-1 release was seen. In conclusion, the stimulation of IL-1 release from U937 under attack by sublytic amounts of (C56)a7-9 was modulated by C8bp. Species restriction by C8bp thus appears not to be confined to the lytic process.

19. Expression of complement regulatory proteins and Integrins on human mesanglal cells In culture. Valerie Caudwell, Philippe Lesavre and Lise Halbwachs-Mecarelli.

INSERM U90, Hopital Necker, Paris, France.

Mesangial cells have been compared to different cell types: macrophages, fibroblasts, smooth muscle cells. In this study, we They have different functions in the glomeruli, but their characterization is uncomplete. analysed the expression of complement receptors (CR1, CR2), complement regulatory proteins (factor H. DAF, MCP) and ingegrins (CR3, P150-95, LFA1) at the surface of human mesangial cells in culture. Cells from human normal kidneys were grown in RPMI medium supplemented with 10% heat-inactivated fetal calf serum and used in the experiments between the 3rd and 7th passage. We performed immunocytochemical assays using monoclonal The results showed antibodies, the results were confirmed by RIA, ELISA assays, immunoblot or facs analysis. the absence on mesangial cells of CR1, CR2, factor H and LFA1. MCP and DAF were present on the cells, DAF molecules are visualized as "vesicle-like" formations in the cytoplasm. The presence of CR3 and P150-95 on mesangial cells is an original feature, while those molecules have been described only on PMN, NK cells and monocytes/macrophages.

20.

Cells lacking GPI-llnked proteins have Impaired ability to vesiculate. M. Whitlow, P. Marshall, K. Iida, R. Silber, W. Rosse and V. Nussenzweig. Departments of Pathology and Dermatology, NYU Medical Center, NY, and Durham, NC, USA. We have recently found that erythrocytes can protect themselves from the terminal complement proteins C5b-9 by releasing, in a calcium-dependent manner, vesicles containing the membrane-attack complexes. Others have shown that human erythrocytes (E) release from their membrane small vesicles (60-150 nm diameter) in response to calcium influx, and that these vesicles are enriched in glycan phophatidylinositol (GPI)-linked proteins [such as decay-accelerating factor (DAF) and acetylcholinesterase (AchE)], relative to the intact red cell membrane. To gain further insight into the mechanisms of vesiculation, and into the role of GPI-linked proteins, we studied vesiculation of E from patients with paroxysmal nocturnal hemoglobinuria (PNH), when subjected to the calcium ionophore A23187 . PNH E that did not contain GPI-linked proteins, released 50% less vesicles than normal red cells, although the 45Ca++ influx was normal. We also studied vesicle release from the lymphoblastoid cell line JY25 as compared with the mutant JY5, which does not express GPI-linked molecules. JY5 produced half the amount of vesicles than JY25 when these cells were treated with Ca+tionophore. These findings suggest that GPI-linked molecules may participate in the process of vesiculation, and that their absence from PNH cells may contribute to their enhanced susceptibility to complement mediated

damage.

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21.

Homologous C5-9 Induces a large flux In human erythrocytes. J.A. Halperin and A. Nicholson-Weller, Dept. of Cellular and Molecular

Physiology, and Dept. of Medicine, Havard Medical School, Boston, MA, USA. We have previously shown that sheep erythrocytes (E) sensitized with antibody and exposed to heterologous Cl-9 can undergo a large, but transient increase in cell membrane permeability in the absence of lysis. We now report the effects of human C5-9 complexes on fresh human E, an approach that isolates the effects of the C5-9 lesion, and also includes the effects of the species restricting complement regulatory factors found in the E Human C5-9 was made from purified C56, and an excess of C7, C8, and C9. EC5-7 were formed at 370C, membrane. and then the cells were chilled to 40 for the addition of C8 and C9. After lh, the cells were either washed in the cold to remove the excess C9 (E5-91im) or kept in the presence of excess C9 (E5-9ex) and then warmed to

370C.

This method has been shown by Bhakdi and Tranum-Jensen to generate mono or poly C9 complexes respectively, when sheep E are the targets of heterologous complement. Lysis of E5-9ex was dependent on the In contrast E5-91im showed no lysis at any C5b-6 concentration, but did experience a concentration of C5b-6. Our results indicate that homologous C5-91im dose-dependent 5 to 10-fold increase in the uptake of 22Na. complexes can generate short lived transmembrane channels and transiently alter the permeability of the E membrane. The nonlytic but large permeability changes induced by the homologous C5-9 membrane attack complex may alter E in vivo at the site of complement activation.

22. Ughting up the Intracellular signalling sequence Induced by the membrane attack complex (MAC) of complement. A.X. Campbell, M.B. Hallett, G. Sala-Newby, S. Frith, K. Taylor, B.P. Morgan, M. Knightl, S.M. Smithl

and Department of Medical Biochemistry, UWCM, Heath Park, Cardiff, CF4 4XN and lInstitute of Cell and Molecular Biology, University of Edinburgh, Edinburgh, EH9 3JH, U.K.

A.J.

Trewavasl.

A new approach, using single cell imaging and recombinant bioluminescent proteins, has been established to follow the molecular sequence inside cells attacked by the MAC. A rise in cytosolic free Ca2+ is the earliest However, fluorescence ratio imaging of oligodendrocytes has shown that it detectable intracellular change. In some cells this is followed by a can take as long as 30 min before a rise in cytosolic Ca2+ is observed. The number of permeability thresholds threshold in the permeability to propidium iodide, and then lysis.

crossed in a particular cell depends on the rate of removal of the MAC by endo- or ecto-cytosis. Release by Using recombinant aequorin expressed in E. coli the MAC of Ca2+ from internal stores has also been observed. it has been shown that in bacteria also a rise in free Ca2+ is the earliest intracellular change following MAC attack. Firefly luciferase has been engineered using PCR to measure protein phosphorylation in live cells. The molecular sequence inside cells is being correlated with events within the MAC by engineering C9.

SECTION THREE - COMPLEMENT AND KILUNG OF PATHOGENS 23.

Ecoli and S. tyhimurium as a model for complement recognition. V. Dlabac

and H. Tlaskalova.

Institute of Microbiology, Czech. Acad. Sci., Prague 4. Czechoslovakia.

S. tvphimurium are special to us because more is known about them than about any other life" of form cellular (M. Schaechter and F. Neidhardt, 1987). This statement holds true also for surface structures of these two species of gramnegative bacteria. Using a collection of well defined mutants of Both qualitative E.cii and S. tvphimurium the sensitivity to complement dependent killing was estimated. and quantitative differences in sensitivity were found, which were clearly dependent on the defect of LPS structure. However, attempts to directly correlate the structure of LPS with the type of sensitivity were successful. Using the sera of antibody deficient newborn piglets most of the strains were partially only usually killed only by one mechanism. The analysis of results obtained with human adults were more complicated probably due to the presence of antibacterial antibodies.

"Today, E.colU and

8

24. Inhibition of the capacity of selected C5-9 complexes on complement-resistant bacteria to bind additional C9 molecules. J.R. Dankert.

Health Science Center, University of Florida, Gainesville, FL 32610, USA.

The dissociation constant (Kd) of the C9 molecule for the C5b-8 site (X2000 sites/cell) on a complementresistant strain of Es colU (LP1395) was measured to be 1.1 nM at OOC, conditions that permit the binding of The C5b-8,91 sites could bind another C9 molecule one C9 molecule per C5b-8 site, forming a C5b-8,91 site. The cells were returned to OOC and the Kd Of the binding of C9 only after a warming step (2-3 min at 370C). The Kd was 1 nM, however 50% of the C5b-8,91 sites were lost or unable to for these C5b-8,91 sites measured. bind another C9 molecule after this short period of time (2 to 3 min). This is in contrast to the C5b-8 site on these cells, and to the C5b-8,91 site on a complement-sensitive strain where no such inactivation occurred. It is concluded that on C-resistant cells the presence of a single C9 molecule per C5b-8 site (C5b-8,91) at 370C lead to a rapid inactivation of the complexes such that they were unable to bind additional C9. This could be due to an inherent instability of this site on resistant cells or to an inactivation process of the cell. The process was se~letive as C5b-8 sites were not similarly affected and remaining C5b-8,91 complexes (Supported by NIH AI22912). (=1000/cell) retained the ability to bind C9.

25. Immune lysls of Trypanosoma congolense: Variant surface glycoprotein is shed and forms a soluble covalent complex with complement component C3b. E.-W. Liu, E.B. Otesile and H. Tabel. Dept. of Veterinary Microbiology, University of Saskatchewan, Saskatoon,

S7N OWO, Canada.

Organisms of T. congolense, variant antigenic type (VAT) TC13, were incubated with fresh bovine serum in the In the presence of specific antibody, two forms of presence or absence of specific antibody to VAT TC13. variant surface glycoprotein (VSG) were found in the supernatant fluid of the incubation mixture: (1) Free soluble VSG (54 kd); (2) A soluble complex (225 kd) of VSG (54 kd) covalently bound to bovine complement VSG-C3b complex was demonstrated by Western blot with rabbit anti-bovine C3 as well component C3b (175 kd). Free VSG and C3bVSG complex were not detected when VSG-specific antibodies were as anti-TC13 VSG antibodies. It is speculated that in vivo formation of soluble covalent complexes of C3b-VSG might exert absent. pathogenic effects on the host: (1) C3 consumption and (2) blocking of C3b receptors of phagocytic cells.

26. Virulence of pneumococcal strains correlates with their complement-resistance. S. Geelen, P.C. Aerts, J. Verhoef, A. Fleer and H. Van Diik. Eijkman-Winkler Laboratory of Medical Microbiology, Utrecht University, University Hospital G04.614, P.O. Box 85,500, NL-3508 GA Utrecht, The Netherlands. The complement (C)-activating abilities of encapsulated S. pneumoniae serotype 3 (S3) and an unencapsulated mutant strain (obtained from Dr. W.A. Watson et al., Houston), as such or after passage through mice, were

studied. The original encapsulated S3 and mouse-propagated, unencapsulated strains appeared to be poor C A high correlation between the Cactivators, whereas the laboratory mutant strain activated C rather well. The activating abilities and LD50's of pneumococcal strains (r = 0.980; n - 6; P 5 0.001) was observed. Therefore, we suggest decreased C activation after mouse passage could not be ascribed to capsule formation. the ability to be a property of the cell wall. Pretreatment of the strains with proteolytic enzymes indicated that the factor mediating the C resistance of virulent, unencapsulated pneumococci, and even that of the encapsulated S3 is of protein nature. The characterization of the anticomplementary protein will be subject of further study.

9

SECTION FOUR - COMPLEMENT DEFICIENciEs 27. Acquired deficiency in C1-inhibitor associated with an undifferentiated gastric adenocarcinoma. and A. Pecoud. Division of Allergy and Immunology, University Hospital, Lausanne, and lCentral Laboratory, Blood Transfusion Service, Swiss Red Cross, CH-3000 Bern-22, Switzerland.

J.-B. Wasserfallen, P. Spath1

Acquired deficiency in Cl-inhibitor (Cl-INH) clinically manifests similar to hereditary angioedema and is usually associated with a B cell malignancy or is sometimes characterized by an autoantibody (afb) towards the We report the case of a 76-year old female patient with inconspicuous past medical history. Cl-INH molecule. In July 1989, she experienced a first episode of angioedema involving the mouth and the neck with dyspnea that A second edematous episode occurred in lasted for about 1 week. Routine blood analysis was normal. Blood tests revealed an increased erythrocyte sedimentation rate (88 November and a third in December 1989. mm/h), slight anaemia (Hb = 115 g/l), leukopenia (2.9 g/l), and decreased levels in complement proteins Cl-INH Low levels of these complement proteins were (< 0.02 g/l), C4 (< 0.03 g/l), as well as Clq (0.03 g/l). An aAb to Cl-INH was not detectable. ascertained by functional assays. Specific clinical investigation did not reveal a lymphoid malignancy. However, we found signs for a haemolytic anaemia (warm Ab IgG C3d). A gastrectomy and Endoscopy disclosed the presence of a gastric tumor (undifferentiated adenocarcinoma). The splenectomy were performed, with incomplete resection of the tumor (10/15 metastatic lymph nodes). evolution was characterized by total disappearance of the direct antiglobulin test's positivity as well as the complete disappearance of symptoms of angioedema and haemolytic anaemia, despite unchanged serum levels of A local recurrence of the gastric adenocarcinoma was diagnosed 5 months later and complement components. could not be resected, and the patient's condition is declining at the present time (15 months after initial To the best of our knowledge this surgery), without recurrence of either angioedema or haemolytic anaemia. is the second case of an adenocarcinoma associated with an acquired Cl-INH deficiency and is the first report of a gastric manifestation of malignancy in association with complement abnormalities.

28.

Cleavage of high molecular weight kininogen and piasmin generation during acute attacks in hereditary angloedema. Hackl, M. Cicardi and A. Agostoni. Clinica Medica, Ospedale S. Paolo, Universita di Milano, Italy and lCentral Laboratory of the Netherlands Red Cross, Amsterdam, The Netherlands.

M. Cugno, C.E.

We studied 23 patients with hereditary

angioedema (HAE) (18 in basal conditions and 5 during

acute

attacks).

We measured by RIA methods plasma levels of Cl-, factor XIIEighteen healthy subjects served as controls. and kallikrein-Cl-inhibitor complexes: plasmin-alpha 2 antiplasmin (PAP) complexes: tissue type (t-PA) and SDS PAGE and immunoblotting analysis of high molecular weight urokinase (u-PA) plasminogen activators. Patients with HAE in basal conditions had high plasma levels of Cl-Clkininogen (HMWX) were also performed. inhibitor complexes that further increased during acute attacks (P=0.0002 yvj normals); the plasma levels of Factor XII-, PAP complexes, normal in basal conditions, significantly increased during attacks (P=0.0009). About 95% of the total HMWK kallikrein- Cl-inhibitor complexes, t-PA and u-PA remained in the normal range. In HAE patients a positive was cleaved in patients during attacks yv 14% in basal conditions (P=0.0008). Neither PAP complexes nor cleaved correlation was shown betwen PAP complexes and cleaved HMWK (P(0.001). Our data indicate that during attacks in HAE, along with complement HMWK correlated with t-PA or u-PA. This phenomenon activation, there is an activation of the contact system and the generation of plasmin. and activators known the two u-PA). (t-PA apparently takes place without involving plasminogen

29.

Hereditary angioneurotic edema: study of 203 patients. M. Cicardi, L. Bergamaschini, D. Frangi, M. Cugno , G. Bisiani, S. Guzzoni and A. Agostini. Clinica Medica, Ospedale S. Paola, Universita di Milano, Italy.

Fifty one families have patients affected with hereditary angioneurotic edema (HANE). type I HANE (low antigenic and low functional Cl-INH plasma levels) and 7 have type II HANE with low Cl-INH function and antigenic levels between 50 and 80% in 3 families and clearly above 100% in the last 4. Forty

We are following 203

10

five percent of patients have one or more attack a month. Laryngeal attacks are reported by 70%. Autoimmune diseases are not recorded in our case list, but 5 patients have hypothyroidism. Prophylaxis with androgen derivatives was required by 30% of patients (half of them are in treatment for more than 5 years) and in 97% led to total remission of symptoms. No important side effects were registered, but one patient had to withdraw the drug for appearance of laboratory signs of hepatic cell necrosis. Prophylaxis with antifibrinolytic agents was employed in 14% of the patients resulting in a reduction in the frequency of the attacks in 56% of them. Short term prophylaxis was successfully performed with androgen derivatives to prevent laryngeal attacks in 32 patients undergoing dental surgery. Cl-INH plasma concentrate was used in 88 acute attacks. Sixty seven of them were laryngeal attacks and they started to revert within 30 min. from the end of the infusion, the same behavior was registered in the 15 bowel attacks that were treated while the 6 subcutaneous attacks only slowly decreased after the infusion.

30.

Anti-Cl-inhibitor autoantibodies and acquired angloedema. G. Bisiani, M. Cugno, M. Cicardi and A. Agostoni. Clinica Medica, Ospedale San Paolo, Universita di Milano,

Italy. We studied 6 patients with acquired Cl inhibitor (Cl-INH) deficiency [acquired angioedema (AAE)]. One had chronic lymphocytic leukemia (CLL), one breast carcinoma, one an IgM M component, one liver hydatidosis, and two no associated diseases. Autoantibodies against Cl-INH were measured by an ELISA method. They were

present in 5 patients (3 IgGl, 1 IgA, 1 IgM) and were absent in the patient with CLL. The autoantibodies were totally removed from plasma by absorption on Cl-INH coated Sepharose from which they could be recovered The interference of rheumatoid factor was ruled out by preabsorption of patients' plasma on IgG by elution. coated Sepharose. Autoantibodies from each patient were purified from plasma by affinity chromatography on The plasma levels of the autoantibodies were determined by an ELISA method referred Cl-INH-coated Sepharose. to Ig standards. The effect of the autoantibodies on Cl-INH function was tested by incubating for different times and at different temperatures the patients' plasma or the affinity purified autoantibodies with normal pooled plasma or with purified Cl-INH. No changes in Cl-INH function were detected in any experiment, even when contact system or fibinolysis activators (kaolin or urokinase) were added to the samples. Our data demonstrate, in 5 out of 6 patients with AAE, the presence of autoantibodies which bind to normal Cl-INH, but we could not demonstrate a direct influence on Cl-INH function in vitro.

31. A frameshift mutation giving rise to a premature stop codon In exon 8 of the C1-inhibitor gone of a type I HAE patient. Z.M. Siddicue, A.R. McPhaden and K. Whaley. Department of Pathology, Western Infirmary, Glasgow, UK.

Initial Southern blot analysis identified a Bgl II restriction fragment length polymorphism (RFLP) in a type I hereditary angio-oedema (HAE) kindred. Localisation studies indicated that the mutation giving rise to the RFLP occurs at the 5' end of exon 8. The polymerase chain reaction (PCR) was used to amplify exon 8 Clinhibitor patient DNA and the amplified product sequenced using asymmetric PCR. Sequence analysis revealed deletion of a single C nucleotide at position 16698 (Carter et al 1991, Eur J. Biochem) which is part of the Bgl II recognition sequence, AGATCT. This deletion produces a frameshift with the formation of a premature translation stop codon (TGA) at position 16738-16740, which would be predicted to give a truncated protein, 50 amino-acid residues short of the normal Cl-inhibitor protein.

32.

Identification of type 11 hereditary angio-oedema (HAE) mutations. Z.M. Siddi.ue, A.R. McPhaden and K. Whaley. Department of Pathology, Western Infirmary, Glasgow, UK.

Systematic PCR-direct genomic sequencing of type II HAE patient DNA was performed in five separate kindred. In patient A. aa Pl-site mutation changing Arg 444 -> His (CgC->CjC) as well a a P9-site Alq 436 -> Val (GCA>GTA) was identified in exon 8 of the Cl-inhibitor gene. In kindreds B. C and D a single Pl-site, Arg -> His mutation was observed. In kindred E, three mutations were found in exon 7.8. A silent mutation occurs at the P12-site: C&G->GA& which does not alter the normal glutamic acid residue at this position. The second

11

mutation at the Pll site result. in a substitution of the normal alanine (GAG) to glutamic acid (GaG). The third mutation is the previously reported, HgI Al polymorphism (Bock et al 1986, Biochem, 25: 4292-4301); Val 458 (DITG)-> Mct (&TG). The mutations at the P9, Pll and P12 sites have not been described previously. In the P11 mutation the hydrophobic alanine residue is substituted by the more polar, hydrophilic glutamic acid. The significance of the P9 mutation is not immediately apparent considering the presence of the Pl-site mutation on the same allele. An earlier description of a P9 mutation substituting the normal alanine for threonine has been implicated as a pathogenetic mutation in two type II kindred (Levy et al 1990, PNAS, 265268) However in contrast to the two pedigrees described in this earlier paper, our patients had normal levels of non-functioning Cl-inh. These data emphasise the heterogeneity of type II HAE.

33.

Characterisation of a splice-site mutation in type I hereditary angio-oedema. Z.M. SiddigUe, D.F. Lappin, A.R. McPhaden and K. Whaley. Department of Pathology,

Western Infirmary, Glasgow,

UK.

The presence of a Sty I restriction fragment length polymorphism (RFLP) was identified by southern blot analysis and the lesion localised to the boundary between exon 6 and intron 6. The Sty I recognition sequence is 5'CCAAGG3'. The sixth nucleotide (G) in this recognition sequence is the first nucleotide of the donor splice site GT. The mutation (G ->T) was characterised by PCR-direct sequencing and confirmed by M13 cloning and single strand sequencing of the amplified product. Northern blot analysis of patients' monocyte RNA showed a 50% reduction in Cl-inhibitor mRNA level as compared with a normal control. Abnormally large mRNA species were not identified. Single stranded cDNA was prepared from patient mRNA using reverse PCR amplification of the resultant transcriptase and exon 8 and exon 7 specific oligonucleotide primers. cDNA was performed using exon 6/7 and exon 6/8 specific oligonucleotide primers. On agarose gel electrophoresis a single species of cDNA was observed for each PCR reaction (exon 6/7; exon 6/8). Both PCR products were completely cleaved by Sty I and direct sequencing of each revealed a single nucleotide sequence which was identical with normal Cl-inhibitor mRNA. These data show that the mutant mRNA is undetectable in patients' monocytes probably due to its instability.

34. Discordant bactericidal responses to immunization with meningococcal vaccine in two C2-deficient sisters: correlation with disease. B. Selander, E. Holstrom, C. Soderstrom1, H. Kaythy2, and A.G. Sjoholm. Departments of Medical Microbiology and linfectious Diseases, University Hospital, Lund, Sweden and 2National Public Health Institute, Helsinki,

Finland. C2 deficiency was identified in a 14-year old girl (A) with meningitis caused by N. meningitidis serogroup W135 and a history including neonatal infection with serogroup B streptococci, and in her 17-year-old healthy sister (B). Both were immunized with tetravalent meningococcal vaccine. B showed distinct serum bactericidal A showed no bactericidal activity for serogroup W-135 after clinical infection, responses to vaccination. There was no evidence of blocking antibodies in nor did she develop bactericidal responses to vaccination. Addition of polyclonal IgG resulted in killing of meningococci in the prevaccination sera of the serum of A. Measurement of antibodies to the vaccine polysaccharides by ELISA confirmed the poor antibody both sisters. Further analysis, including investigation of IgG subclass levels and IgG responses in A as compared with B. allotypes provided no explanation of the different antibody responses. The results emphasize the potential importance of anticapsular antibodies capable of activating the alternative pathway. Development of more efficient vaccines, as well as passive immunization of some patients with complement deficiency might be

considered in this context.

35. Human complement C3 deficiency due to defect In secretion. Y. Katz, M. Schlesinger1, E. Lahat and Z. Fishelson2. Assaf Harofeh Medical Centre, Zerifin, Medical Center, Ashkelon and 2The Weizmann Institute of Science, Rehovot, Israel.

lBarzilai

The molecular basis of C3 deficiency in a 20y old newly diagnosed male patient was studied.

Peripheral blood

12

monocytes (PBM) and skin fibroblast cultures (F) of the patient and healthy individuals were metabolically Cell lysates and supernatants were then analyzed by immunoprecipitation labeled for 2 h with [35S]-Met. Normal levels of C3 using monospecific antibodies and protein A. followed by SDS-PAGE and autoradiography. were found in lysates of PBM and F of patient and control. However, unlike the control cells, the patient's Secretion of Clr, Cls, C1 inhibitor, C2, Factor B and Factor H by the patient's cells did not secrete C3. In pulse-chase experiments we observed a long delay in the disappearance of intracellular cells was normal. LPS and IL-1 increased C3 synthesis in patient's fibroblasts but had C3 and appearance of extracellular C3. Our results suggest that this C3 deficiency is caused by a specific defect in C3 no effect on C3 secretion. SDS-PAGE analysis of the secreted C3 and the pattern of trypsin-cleaved intracellular C3 secretion. indicated some abnormality in the structure of the patient's C3. Whether or not the defective secretion is causally related to the observed abnormal structure of the patient's C3 remains to be determined.

36. Factor I deficiency In two siblings, one with pyogenic Infection, the other healthy. T. VYan, K.A. Davies, B.J. Morley, C.M. Giles, A.D.B. Websterl and M.J. Walport.

RPMS, Hammersmith Hospital,

and 1CRC, Northwick Park Hospital, London, UK. A 14 year old male presented with a history of recurrent pyogenic infections, incuding a S. e*idermidis septic arthritis of the shoulder aged one year, recurrent sinusitis, orbital cellulitis, and meningococcal meningitis He had a There was no family history of recurrent sepsis and no consanguinity. at the age of 14 yrs. sister aged 19 who was well. Both the patient and his healthy sister were found to have homozygous deficiency The complement profile of the propositus (with similar findings in his sister) was: C3 28% of Factor I. normal human serum (NHS), C4 78% NHS, CH50