Truns/usinn Medicine. 1992. 2, 1-6

REVIEW A R T I C L E

The immune destruction of red cells*

c. p. Engelfriet, Deporrnienl

of Inimunoheniatolo~y,Cenrrul Loboratory of the Netlierlunh Red Cross Blood TransJiusion Service.

Anisterdom, The Neiherlunds

membrane which activates C3. Upon its activation C3 is split into C3a and C3b, which is bound to the cell. C3b may activate C5, which is then split into C5a and C5b and the activation of C5 may lead to the activation of C6, 7, 8 and 9 and the formation of the membrane attack complex (MAC). C3b can react with factor B to form a second C3-converting enzyme C3b Bb, thus activating what is called the amplification pathway by which many more C3 molecules may be activated. However the activation of C3 is strongly inhibited by the decayaccelerating-factor (DAF). DAF is a phosphatidylinositol-linked protein, which acts on the activation products of C2 and factor B, i.e. C2a and Bb and dissociates them from C4b and C3b thus preventing the assembly of the two C3-converting enzymes C4bC2a and C3bBb (Fujita et al., 1987). Once C3b is formed, it can only activate C5 before it is turned into iC3b, which is the first step in its inactivation. Factor H renders C3b susceptible to the activity of factor I (the C3b-inactivator), which turns C3b into the haemolytically inactive iC3b. Factor I, together with the CRI, then splits iC3b into C3dg and C3c. Finally C3dg may be reduced to C3d by proteolytic enzymes such as trypsin. It is not surprising that by the activity of DAF and the inactivation of C3b, frequently the activation of complement does not proceed beyond the activation of C3. Even if it does, the formation of the MAC and the direct lysis of the cell by the MAC is by no means certain because there are two other inhibiting proteins which may prevent this. The C8-binding protein interferes with the C8-C9 interaction and the polymerization of C9 into the MAC (Zalman et al., 1986; Schonermark et al., 1986) and the P18/CD59 protein inhibits the incorporation of C9 into the MAC and reduces C9 polymer formation (Sugita et al., 1988; Rollins & Sims, 1990). Thus it is not surprising that although the interaction of red cells with complement-binding antibodies always leads to the fixation of C4b and C3b on the red cell membrane, it often does not cause direct complement lysis of the cell. This may even be the case when red cells react with haemolysins such as anti-A or anti-B.

It is a great honour that the British Blood Transfusion Society has considered me for the James Blundell Award and I thank the Society very much for this honour. It is also a particular pleasure for me because there has always been a special relationship between the British and Dutch Blood Transfusion Services. This relationship began shortly after World War I1 when my previous boss and friend Joghem van Loghem was invited to the Lister Institute to learn typing for RhD from Ruth Sanger and Rob Race, because discovery of RhD was unknown in occupied Europe until after the war and there was no knowledge of typing for RhD in the Netherlands. Our relations with Rob and Ruth, and also with many other colleagues in the United Kingdom, have had a special quality ever since that first contact. I M M U N E D E S T R U C T I O N O F R E D CELLS Since the binding of an antibody to an antigen on the red cell membrane does not affect the life span of the cell, the immune destruction of red cells is only brought about by two mechanisms which may be activated secondarily to the antibody-antigen interaction. These mechanisms are the activation of complement and adherence to Fc receptors on cells of the monocyte/ macrophage system. In this paper I would like to give a survey of what is known about these mechanisms and of the way they may be influenced in uivo. ACTIVATION O F C O M P L E M E N T When a red cell reacts with a complement binding antibody, complement is activated along the classical pathway. The activation of CI leads to the formation of the C3-converting enzyme, C4bC2a, on the red cell *Based on the James Blundell Award Lecture given at the IX Annual Scientific Meeting of the British Blood Transfusion Society in Nottingham, 3-6 September 1991. Correspondence: Professor C. P. Engelfriet, Department of Immunohematology, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, P.O.Box 9190, 1006 A D Amsterdam. The Netherlands.

I

2

C . P . Engelfric~t Table 1. Complement receptors

Receptors

Reactive with

Expression

CRI

C3b>C4b>iC3b

CR2

C3d part of iC3b = C3dg > C3d > C3b

CR3

iC3b

CR4

iC3b, C3dg (?)

monocytes, macrophages, B cells, red cells, etc. B cells, lymphnodefollicular dendritic cells Monocytes, macrophages, neutrophils, etc. Monocytes, macrophages, neutrophils, etc.

Only part of the cells will be lysed whereas others remain intact but become agglutinable by anti-C4b and anti-C3b. Other complement-binding antibodies, such as anti-Jk”, only lead to coating of the red cells with C4b and C3b but do not cause direct lysis. Red cells coated with C4b and C3b adhere to complement receptors on phagocytic cells. The various complement receptors are listed in Table 1. Because C3b is rapidly coverted to iC3b, adherence to CR3 and CR4, which have a high affinity for iC3b, is important. The question arises whether adherence of E-C3b or E-iC3b to complement receptors on macrophages leads to destruction of the red cell. The data concerning phagocytosis of E-C3b or E-iC3b by nonactivated macrophages in uifro are contradictory but there is general agreement that in uitro E-C3b or E-iC3b are phagocytosed by activated macrophages. In uiuo, some of the E-C4bC3b injected into C6-deficient rabbits were phagocytosed and some were cytotoxically damaged (Brown et af.,1970). Severe in uiuo erythrophagocytosis may occur in post-infectious cold agglutinin disease (Dacie, 1962; Friedman & Dracker, 1991). In these patients the phagocytic cells are probably in a state of activation due to the infection. Thus there seems to be no doubt that adherence to complement recptors is a second mechanism of red cell destruction due to the activation of complement. If, in uiuo, red cells react with complement-binding antibodies without being lysed directly, many of the cells, after having adhered to complement receptors, will be detached from these receptors due ~o inactivation of C3b. They will return in the circulation coated with C3dg or C3d and if they were not cytotoxically damaged by the macrophages, these cells will not only survive but will actually be protected against further damage after interaction with complement-binding antibodies (Evans et af.,1968; Engelfriet ef af., 1972). The reason is that the presence of haemolytically inactive C3dg and C3d prevents the uptake of C3b. In patients with complement binding

autoantibodies there is a dual cell population. Part of the red cells have not reacted with the complementbinding antibodies and part of the red cells are in the state E-C3dgC3d. If incompatible red cells are injected into a patient with complement-binding alloantibodies, part of the red cells will be rapidly destroyed but another part of the red cells will become coated with C3dg and C3d and will have a normal survival. This explains the biphasic curve obtained in survival studies in patients with complement-binding antibodies (Mollison, 1962). In some circumstances it is important to be able to stop complement activation in uiuo, e.g. in patients with warm autohaemolysins which react with nonenzyme-treated red cells and in whom haemolysis is complement-dependent and severe, or in patients with severe haemolysis due to biphasic haemolysins. Theoretically C1-esterase inhibitor, which inhibits the esterase activity of activated C1, should have an inhibiting effect on the further activation of complement along the classical pathway. No data on the in uiuo effect of this inhibitor in patients with complement-dependent red cell destruction are available, but in uitro we observed no clear effect of CI-esterase inhibitor on the lysis of red cells by warm autohaemolysins or haemolytic anti-A, when the sera containing these haemolysins were mixed with an equal part of C1-esterase inhibitor or with inactivated AB serum as a control. Another possibility would be treatment with heparin which is anti-complementary. However, in lytic tests in uitro a high concentration of heparin (i.e. 100 iu/ml) is needed to reduce the CH50 to zero (see Mollison, 1979). However, some inhibition of complement activation can probably be achieved in uiuo but no data are available. An interesting and probably important observation is that high doses of IvIg inhibit the uptake of C4b and C3b onto red cells. Serum from a patient treated with IvIg reduced C4b and C3b uptake onto sensitized

Immune destruction of red cells

homologous red cells in vitro to ahnost background values compared to serum from the same patient before treatment (Basta et al., 1991) and in an animal model it was shown that IvIg significantly increased the survival of red cells sensitized in the absence of complement with complement-binding IgM antibodies (Basta et al., 1989). A D H E R E N C E T O FC-RECEPTORS There is now ample evidence that non-complementbinding red cell alloantibodies, i.e. the vast majority of both allo- and autoantibodies, bring about destruction of red cells by causing them to adhere to Fc receptors on cells of the monocyte-macrophage system. The characteristics of the three Fc receptors, known at present, are shown in Table 2. Which of these receptors is involved in the adherence of red cells sensitized with IgG non-complementbinding antibodies in uiuo and the phagocytosis and cytotoxic 'lysis which may be induced by this adherence? Table 2. F, receptors and their characteristics

Fc receptors

Characteristics

FcRI

72 kDa protein Transmembrane fixation High-affinity for monomeric IgG IgGl= IgG3 > IgG4 > > > IgGZ EA-IgG3 > EA-IgG I Monocy tes/macrophages Induced on PMN by IFN-7

FcRII

40 kDa protein Transmembrane fixation N o affinity monomeric IgG IgG dimers: IgG3 > IgG 1 > > > > IgG4 IgG2: depends on allele Monocytes/macrophages, PMN, platelets, B cells

FcRlIl FcRIIIa

45-70 kDa protein Macrophages, NK cells Cultured monocytes Transmembrane fixation Medium affinity for monomeric IgG: binds it at 4"C, but not at 37°C Affinity for TgG dimers?

Fc RI I I b

Neutrophils, PI-linked Does not bind monomeric IgG IgG dimers: IgG3 > IgG I NA polymorphism

3

It was shown by Klaassen ef al. (I 990) that in uitro FcR-I, -11, and -111 can be independently modulated from the surface of monocytes/macrophages by incubation with EA-IgG anti-D, which implies that EA-IgG adhere to all three Fc receptors. Further experiments, however, showed that only adherence of EA-IgG anti-D to the FcRI results in cytotoxic lysis of the adherent cells. In blocking experiments FcRI was blocked by mouse IgG2a and FcRII and -111 by monoclonal antibodies (mAb). Blocking of both FcRII and -111 had no effect on the lysis of EA-IgG anti-D by cultured monocytes, on which FcRIII is expressed, whereas blocking of FcRI completely inhibited such lysis (Klaassen et al., 1990; Kumpel & Hadley, 1990). If in these experiments red cells were used, sensitized with murine mAb against glycophorin A, blocking of FcRII, but not of FcRIII, also produced partial inhibition. The involvement of FcRII in cytotoxic lysis probably depends on the number of antibody molecules bound to the red cells, the number of antiglycophorin mAb bound being far greater than the number of antiD molecules. FcRI, therefore, is probably the only receptor which can induce lysis of red cells sensitized with human IgG allo- or autoantibodies except perhaps for IgG anti-A and -B. Whether this is also true for phagocytosis of EA-IgG has not been established. Phagocytosis was observed after adherence to FcRIII by Clarkson & Ory (1988). However, these experiments were done with red cells coated with mAb against FcRIII and the mechanism of adherence was therefore quite different. There remains the question to which receptors EAIgG first adhere in uiuo. Because FcRI has a high affinity for monomeric IgG, it is probably blocked in uivo, whereas FcRII and -111 are not. Possibly, therefore, the initial adherence of EA-IgG is to FcRII and -111, which could lead to stripping of monomeric IgG from FcRI upon which the red cells can adhere to this receptor. This would finally activate the effector mechanisms. A number of observations seem to favour this hypothesis: first, Clarkson et al. (1986a) have shown that in chimpanzees the half-life of EA-IgG is drastically increased after the injection of mAb against FcRIII. F(ab')2 fragments of the mAb also had such an effect although much less pronounced. They concluded that blocking of FcRIII was partly responsible for the increased survival. When the same mAb were given to a patient with therapy-resistant autoimmune thrombocytopenia, the platelet count rose steeply and the survival of EA-IgG increased (Clarkson el al. 1986b). Secondly, the affinities of monomeric IgG1 and IgG3 for FcRI are equal (Burton et al., 1988) whereas the

4

C. P . Engelfiier

affinity of IgG3 dimers for FcRIII was found to be greater than of IgG1 dimers (Huizinga et a)., 1989). This might be the reason why in uiuo the efficacy of IgG3 antibodies to destroy red cells is greater than that of IgGl antibodies. Other observations are, however, difficult to explain if the above theory is true: firstly, EA-IgG are mainly, and, if the number of antibodies is low, are exclusively sequestered in the spleen. FcRIII is expressed equally strongly on the Kupfer cells in the liver as on splenic macrophages (Clarkson et af., 1986b). If adherence to FcRII and -111 can take place uninhibited by plasma IgG, EA-IgG would be expected to be mainly sequestered in the liver as are E-C3b or E-iC3b. Secondly, in uitro the cytotoxic lysis of monocytes, on which FcRII is expressed, is completely inhibited by plasma or serum. Further experiments with cultured monocytes, on which all three receptors are expressed, may throw light on the question, which of the Fc-receptors is involved in the initial adherence of EA-IgG in uiuo? The answer is important for the selection of effector cells for in uitro assays to evaluate the in uiuo activity of non-complement-binding IgG antibodies. If the initial adherence is to FcRII and -ITI, cultured macrophages should be used in the presence of serum or plasma to imitate the in uiuo situation as well as possible. Another question with regard to such in vifro assays is whether cytotoxic lysis of EA-IgG should be measured, or phagocytosis or both? To answer this we must know whether the relative importance of these two effector mechanisms may vary in different antibody populations. Using monoclonal anti-Ds, Wiener et al. (1988) have shown that whereas IgGl anti-D mainly caused phagocytosis by monocytes in uitro, IgG3 anti-D mainly caused cytotoxic lysis. This implies that in mixtures of IgGl and IgG3 antibodies, the relative importance of these effects may indeed differ. Thus the best in uitro parameter for in uiuo activity would be the sum of cytotoxic lysis and phagocytosis. Others, e.g. Zupanska et al. (1990), however, found that the highest level of phagocytosis of EA-IgG anti-D (polyclonal) by monocytes was associated with IgG3 antibodies. Finally, another possibly important point is as follows: there are various isoforms of the FcRII encoded by three closely linked genes, FcRIIA, -B and -C. Of the FcRIIA gene there are two alleles which encode for products which differ greatly in their affinity for murine IgG1 (Tax et al., 1983). It has now been shown (Warmerdam et al., 1991) that the product of the FcRIIA allele with a low affinity formurine IgG 1 has a high affinity for dimers of IgG2 whereas the FcRTIA with a high affinity for murine TgG1 does not bind dimers of human IgG2. Although the biological

significance of this finding has not yet been established, it is possible that IgG2 antibodies may be biologically active in subjects with an FcRIIA with a high affinity for IgG2. IgG2 red cell antibodies might cause red celldestruction in such subjects and this also may be important for the selection of effector cells for in uitro assays in certain circumstances. Another important question is how the adherence of EA-IgG to Fc-receptors can be influenced in uiuo. The following factors have been shown to inhibit cytotoxic lysis of EA-IgG anti-D in yitro. (a) Blocking of FcRI by serum or plasma IgG; (b) Blocking of Fc receptors by monoclonal antibodies against the Fc receptors themselves; (c) Blocking of Fc receptors by IgG antibodies against Fc receptor-independent antigens, the so-called Kurlander phenomenon (Kurlander, 1983). In this case the antibodies, after they have reacted with their antigens on the monocyte/macrophage surface, react with the Fc-receptor by the Fcpart of their molecule. It has been shown that in uitro both HLA class I and class I1 antibodies block Fc receptor-dependent functions in this manner (Neppert et al., 1985). Furthermore corticosteroids in high concentration have been shown to inhibit the release of lysosomal enzymes by monocytes after adherence of EA-IgG and therefore to decrease cytotoxic lysis, which may explain the immediate in uiuo effect of corticosteroids in AIHA. In uiuo the following factors have been shown to influence Fc-receptors dependent destruction of EA-IgG. 1 High dose IvIg. Although the exact mechanisms

responsible for the effect of this treatment have not been conclusively elucidated, it is generally believed that the blocking of Fc-receptors is an important effect. 2 As mentioned blocking of FcRIII by monoclonal antibodies causes an increase in the survival of EA-IgG. 3 Could blocking of the Fc receptors by Fc receptorindependent antibodies occur in uiuo? It has been suggested that maternal HLA antibodies may diminish the destruction of D-positive red cells in the fetus and new-born by maternal anti-D by blocking the Fc receptors on macrophages (Neppert et al., 1988). The following observations in our laboratory (M. C. Dooren et al., unpublished observations) show that maternal alloantibodies reactive with the monocytes/macrophages of the fetus and new-born may protect against lysis of rea cells by maternal anti-D. When investigating the predictive value for the severity of HDN of an antibody-dependent-cellular cytotoxicity (ADCC) assay using standard red cells sensitized

Immune deslruction of red cells with maternal anti-D and monocytes as effector cells, the following correlations were found between the results of the assay and the severity of HDN. If less than 10% of the anti-D sensitized cells were lysed, there was no HDN; if 10-30% were lysed the HDN was only light; when more than 80% were lysed the HDN was severe. or fatal with a few exceptions (see below); when 30-80% of cells were lysed the correlation was less clear, the H D N could vary from mild to severe (Engelfriet & Ouwehand, 1990). In some cases, where more than 80% of red cells were lysed, the H DN was far less serious than expected or even absent. To investigate whether, in these cases, maternal IgG monocyte-reactive alloantibodies had prevented the destruction of the red cells, the maternal sera were tested in an inhibition ADCC test. In this test the effector monocytes are preincubated with the maternal serum, then washed before being used in the ADCC test. In a majority of cases in which the HDN was much less severe than expected from the ADCC activity of the maternal anti-D, the maternal serum blocked the Fc-receptor function of paternal, but not of autologous maternal monocytes. IgG antibodies reactive with the paternal but not the maternal monocytes were detected in these sera using a monocyte immunofluorescence test (Kuijpers el al., 1991). The results show that IgG alloantibodies capable of blocking monocyte Fc-receptor function were present in these sera (M. C. Dooren et af.,unpublished observati on s). As mentioned above, HLA class I antibodies are capable of blocking monocyte Fc-receptor function in uitro. However, because HLA class I antibodies are largely, if not completely, absorbed by fetal cells in the placenta, it seems unlikely that HLA class I antibodies are responsible for blocking Fc-receptors on monocytes/macrophages in vivo in the fetus or infant. To investigate the possible role of HLA class I antibodies in the protection against HDN, the maternal sera that blocked monocyte Fc-receptor function in uitro were absorbed with platelets, which did not affect their blocking capacity. Furthermore HLA class I antibodies were detectable as frequently in the serum of mothers whose children suffered from severe HDN as in the serum of mothers whose infants had unexpectedly mild HDN. Thus, HLA class I antibodies are not responsible for the protection against HDN (M. C. Dooren et al., unpublished observations). Further studies are needed to define the specificity of the blocking antibodies, which, because they are detectable in the monocyte fluorescence test, in which monocytes are used on which FcRI is not present. cannot be directed against a polymorphism on the FcRI itself. Non-HLA class I Fc-receptor blocking

5

antibodies were not detectable in sera from mothers whose infant’s H D N was as severe as expected from the ADCC activity of the maternal anti-D. Fc-receptor blocking antibodies were not detectable in all cases of unexpectedly mild HDN. There must be other factors which influence the severity of HDN in the face of highly active maternal anti-D. The inhibited placental passage of IgG and immature Fc-receptor function are possible mechanisms. Thus, there are several possibly therapeutic measures by which Fcreceptor-induced destruction of EA-IgG can be influenced in uiuo. In the case of HDN, and possibly also in alloimmune thrombocytopenia and neutropenia of the new-born, a ‘natural’ protective mechanism may diminish the severity of the disease. REFERENCES Basta, M., Fries, L.F. & Frank, M.M. (1991) High doses of intravenous Ig inhibit in vitro uptake of C4 fragments onto sensitized erythrocytes. Blood, 77, 376-380. Basta, M., Langlois, P.F., Marques, M., Frank, M.M. & Fries, L.F. ( I 989) High-dose intravenous immunoglobulin modifies complement-mediated in vivo clearance. Blood, 74,326-333. Brown, D.L., Lachmann, P.J. & Dacie, J.V. (1970) The in vivo behaviour of complement coated red cells. Studies in C6-deficient, C3-depleted and normal. Clinicaf and Experimental Immunology, 7,40 1-42I . Burton, D.R., Jefferis, R., Partridge, L.J. & Woof, J.M. (1988)Molecular recognition of antibody (IgG) by cellular Fc receptor (FcRI). Molecular Immunology, 25, I 1751181.

Clarkson, S.B., Kimberly, R.P., Valinsky, J.E., Witmer, M.D., Bussell, J.B., Nachman, R.L. & Unkeless, J.C. (l986a) Blockade ofclearance of immune complexes by an anti-FcR monoclonal antibody. Journal of Experimental Medicine, 164, 474-490. Clarkson, S.B., Bussel, J.B., Kimberley, R.P., Valinsky, J.E., Nachman, R.L. & Unkeless, J.C. (1986b) Treatment of refractory immune thrombocytopenic purpura with an anti-Fc-receptorantibody. New England Journal of Medicine, 314, 1236- 1239. Clarkson, S.B. & Ory, P.A. (1988) CD16 developmentally regulated IgG Fc receptors on cultured human rnonocytes. Journal of Experimental Medicine, 167,408-420. Dacie, J.V. ( 1 962) The Haemolytic Anaemias, Parts I and 11, 2nd edn. Grune and Stratton, New York. Engelfriet, C.P., von dem Borne A.E.G.Kr., Beckers, D., . Reynierse, E. & van Loghem, J.J. (1972) Autoimmune haemolytic anaemias V. Studies on the resistance against complement haemolysis of red cells of patients with chronic cold agglutinin disease. Clinical and E.rperimental Inlmuttology, 2, 255-000. EngelTriet, C.P. &Ouwehand, W.H. (1990)ADCC and other cellular bio-assays for predicting the clinical significance of

6

C. P . Engelfiiel

red cell alloantibodies. In: Blood Tran.$usion the Impact of New Technologies (ed. Contreras, M.). Bailliere's Clinical Haematology, London. Evans, R.S., Turner, E., Bingham, M. & Woods, R. (1968) Chronic hemolytic anemia due to cold agglutinins. 11. The role of C' in red cell destruction. Journal of Clinical Inoestigation, 47, 691-699. Friedman, H.D. & Dracker, R.A. (1991) Intravascular Erythrophagocytosis. Journal of the American Medical Association, 265, 1082. Fujita, T., Inoue, T., Ogawa, K., Iida, K. & Tanuria, N. (1987) The mechanism of action of decay-accelerating factor (DAF). D A F inhibits the assembly of C3 convertases by dissociating C2a and Bb. Journal of Experimental Medicine, 166, 1221-1228. Huizinga, T.W.J., Kerst, M., Nuyens, J.H., Vlug, A., von dem Borne, A.E.G. Kr., Roos, D. & Tetteroo, P.A.T. (1989) Binding characteristics of dimeric IgG subclass complexes to human nuetrophils. Journal of Immunology, 142,2359-2364. Klaassen, R.J.L.. Ouwehand, W.H., Huizinga, T.W.J., Engelfriet, C.P. & von dem Borne, E.G. Kr. (1990) The Fcreceptor I11 of cultured human monocytes. Journal of Immurrology, 144, 599-606. Kuijpers, R.W.A.M., Dooren, M.C., von dem Borne, A.E.G.Kr. & Ouwehand, W.H. (1991) Detection of human monocyte-reactive alloantibodies by flow cytometry following selective down modulation of Fc receptor I. Blood, 78, 2150-2156. Kumpel, B.M. & Hadley, A.G. (1990) Functional interactions of red cells sensitized by IgGl and IgG3 human monoclonal anti-D with enzyme-modified human rnonocytes and FcR-bearing cell lines. Molecular Immunology, 27,247-256. Kurlander, R.J. (1983) Blockade of Fc-receptor-mediated binding to U937 cells by murine monoclonal antibodies directed against a variety of surface antigens. Journal of Immunology, 131, 140-147. Mollison, P.L. (1 979) Blood Transfusionin Clinical Medicine, Blackwell Scientific Publications Ltd, Oxford. Mollison, P.L. (1962) Destruction of incompatible red cells in vioo in relation to antibody characteristics. In: MecAanisms of Cell and Tissue Damage Produced by Immune Reactions. 2nd International Symposium on Immunopathology, Brook Lodge (MI U.S.A.), p. 267. Schwabe, Basel. Neppert, J., Marguard, F. & Muller-Eckhardt, C. (1985)

Murine monoclonal antibodies and human alloantisera specific for HLA inhibit monocyte phagocytosis of anti-D sensitized red blood cells. European Journal of Immunology, 15, 559-563. Neppert. J., Mueller-Eckhardt, G. & Heine, 0. (1988) Reduced immune phagocytosis of monocytes from neonates whose mothers produce HLA antibodies. Vox Sanguinis, 54, 177-1 80. Rollins, S.A. & Sims, P.J. (1990) The complement-inhibitory activity of CD59 resides in its capacity to block incorporation of C9 into membrane C5b-9'. Journalof Immunolog.y, 144,3478-3483. Schonermark, S.E.W., Rauterberg, M.L., Shin, S. et (11. (1986) Homologous species restriction in lysis of human erythrocytes: a membrane-derived protein with C8 binding capacity functions as an inhibitor. Journal of Immunology, 136, 1772- 1776. Sugita, Y., Nakano, Y. & Tomita, M. (1988) Isolation from human erythrocytes of a new membrane protein which inhibits the formation of complement transmembrane channels. Journal of Biochemistry, Japan, 104, 633-641. Tax, W.J.M.. Willems, H.W., Reekers,P.P.M., Capel, P.J.A. & Koene, R.A.P. (1983) Polymorphism in mitogeniceffect of IgGl monoclonal antibodies against T3 antigen on human T cells. Nature, 304,445. Warmerdam, P.A.M., Van de Winkel, J.G.J., Vlug, A., Westerdaal, N.A.C. & Capel, P.J.A. (1991) A single amino acid in the second Ig-like domain of the human Fc gamma receptor I1 is critical for human IgG2 binding. Journal qf Immunology, 147, 1338- 1343. Wiener, E., Jullifer, V.M. &Scott, H.C.F. (1988) Differences between the activities of human monoclonal IgGl and IgG3 subclasses of anti-D (Rh) antibody in this abilities to mediate effector functions in monocytes. Immunolozy, 65, 159- 163. Zalman, L.S.L.M., Wood, L.M., Frank, M.K. & MuellerEckhardt, H.J. (1986) Deficiency of the homologous restriction factor in paroxysmal nocturnal hemoglobinuria. Journal of E.rperimenta1 Medicine, 165, 572-577. Zupanska, B., Brojer, E., McIntosh, J., Seyfried, H. & Howell, P. (1 990) Correlation of monocyte-monolayer assay results, number of erythrocyte-bound IgG molecules, and IgG subclass composition in the study of red cell alloantibodies other than D. Vox Sanguinis, 58,'276280.