Immunology, 1975, 28, 1165.
Activated Guinea-pig C3 and the Immune Adherence Receptor (a Complement Receptor) on Cell Membranes* H. OKADA AND NORIKO OKADA National Cancer Center Research Institute, Tsukiji, Tokyo, and Central Laboratories, Sankyo Company, Hiromachi, Tokyo
(Received 29th January 1974; accepted for publication 16th November 1974) Summary. By treating C3 with purified C1, C4 and C2 in the fluid phase, haemolytically inactive C3 was prepared. This was shown to bind to human erythrocytes by use of radio-labelled (Fab')2 antibody to guinea-pig C3. The activated C3 preparation inhibited immune adherence between EAC43 and human erythrocytes. These findings indicate that the activated C3 attaches to the immune adherence receptor on human erythrocytes. In addition the fluid-phase activated C3 adhered to thymus cells and sheep erythrocytes, whereas EAC43 did not. Thus the immune adherence receptor may be present on so called immune adherencenegative cells, but in insufficient concentration to form rosettes with EAC43. INTRODUCTION The term immune adherence (IA) has been used by Nelson (1953) to describe the phenomenon where antigen-antibody-complement complexes adhere to human erythrocytes (HuE). In Ag-Ab-C1423 complexes, the C3 molecules do not require any other factor to exhibit IA reactivity to HuE (Nishioka and Linscott, 1963), although these complexes require another factor in addition to C3 for adherence to other complement receptors (Okada and Nishioka, 1973a, b; Ross, Polley, Rabellino and Grey, 1973; Eden, Miller and Nussenzweig, 1973; Okada and Okada, 1974). In a previous experiment, conjugation of purified guinea-pig C3 to tannic acid-treated sheep erythrocytes (TaSRBC) made the erythrocytes adherent to HuE (Okada, Kawachi and Nishioka, 1970). This finding suggested that only C3 molecules are required for IA reactivity. Since the tanned cells conjugated with purified guinea-pig C3 (TaSRBC-C3) adhered to HuE without any prior treatment of C3, it was felt that a proportion of the C3 molecules may have been activated nonspecifically during preparation, enabling them to react with IA receptors (Nelson, 1963) on the cell membrane (Okada and Nishioka, 1972). To investigate the reaction between activated C3 and the IA receptor on cell membranes, 125I-labelled (Fab')2 antibody to guinea-pig C3 was used. MATERIALS AND METHODS Complement components and intermediate cells Purified components of guinea-pig complement were prepared with minor modifica* Parts of this communication were presented at the 2nd Congress of Japanese Society for Immunology (Tokyo, 1972) and the 8th International Congress for Allergology (Tokyo, 1973). Correspondence: Dr H. Okada, Virology Division, National Cancer Center Research Institute, Tsukiji 5-chome,
Chuo-ku, Tokyo, Japan.
1165
H. Okada and Noriko Okada 1166 tions as described by Nelson, Jensen, Gigli and Tamura (1967). Human C4 was purified as described by Shimada, Mayumi, Sekine and Nishioka (1972). Complement-intermediate cells were prepared as described previously (Okada, Nishioka and Sindo, 1969; Okada, Kojima, Yoshida and Nishioka, 1972). The guinea-pig C3 preparation used in these experiments gave a single line in immunoelectrophoresis. The C3 purity was about 98 per cent when submitted to quantitative radio-immunoelectrophoresis (Okada et al., 1970). Diluents Glucose gelatin-veronal-buffered saline (gl-GVB2 +) and EDTA-gelatin-veronabuffered saline (EDTA-GVB) were prepared as described elsewhere (Nelson et al., 1967). Cell suspensions HuE, Type 0, Rh( +) and sheep erythrocytes (SRBC) were washed twice with EDTA-GVB and three times with an appropriate diluent. Mouse lymph node cells, spleen cells and thymus cells were obtained from C3H/He mice. Cells were washed three times with saline and suspended in EDTA-GVB. P3HR-1 and AdL-l were cultured cell lines from Burkitt's lymphoma and nasopharyngeal carcinoma respectively. The cells were washed four times before use.
Radio-labelled (Fab')2 fragments of antibody to guinea-pig C3 The IgG fraction of rabbit antiserum to guinea-pig C3 was treated with pepsin at 370 (Utsumi and Karush, 1965). The (Fab')2 thus prepared was labelled with 125I as described by McConahey and Dixon (1966). The labelled (Fab')2 anti-C3 was obtained by affinity chromatography on a Sepharose column conjugated with purified guinea-pig C3 (Cautrecasas, Wilcher and Anfinsen, 1968; Wofsy and Burr, 1969). Radio-labelled (Fab')2 with 75 per cent specificity to guinea-pig C3 was eluted with glycine-HCl buffer, pH 2-4. The radio-labelled (Fab')2 anti-guinea-pig C3 was used as 1251-labelled anti-C3.
Preparation of a standard curve of the radioactivity of '25I-labelled anti-C3 vs the number of C3 molecules on the cell membrane 0-5-ml samples of guinea-pig C3 at different concentrations in gl-GVB2 + were treated with an equal volume of 1 x 108/ml EAC142. The cells were washed three times and resuspended in 0-5 ml of EDTA-GVB. To the cell suspension, 0-1 ml of 125I-labelled anti-C3 (20,000 ct/min/ml) was added. After 60 minutes incubation at 300, the cells were washed three times with EDTA-GVB and the uptake of radioactivity on the cells was determined on an auto-y counter (Nuclear, Chicago). Since more than 50 per cent of the C3 molecules were fixed on EAC 142 at low C3 concentration (Okada and Nishioka, 1972), the standard curve was drawn with the assumption that C3 molecules fix on EAC142 at the rate of 50 per cent (Fig. 1). The non-linearity of uptake of C3 at an input of 100,000 molecules per cell may be due to the limiting number of C42 sites on the cell membrane.
C3 and the Immune Adherence Receptor
1167
4
o3 _
/~~~~~~~~~~~
/
C
C-)
£2
X
1000 330 4-1 12 37 110 Molecules of C3 on EAC1423 cells (xl01) FIG. 1. The relationship between the number of C3 molecules on the cell membrane and the radioactivity of 125I-labelled anti-C3 fixed on the cell membrane. In the calculation, 50 per cent of molecules in the reaction mixture were assumed to fix to EAC142.
RESULTS FIXATION OF C3 CONVERTASE-TREATED GUINEA-PIG C3 TO HuE
Guinea-pig C3 was treated with a mixture of Cl, C4 and C2 as follows. To 0 3 ml of a mixture of human C4 (7 x 1010 SFU) and guinea-pig C2 (5 x 1010 SFU) in gl-GVB2 +, 0-2 ml of varying concentrations of guinea-pig Cl were added, and incubated at 300 for 10 minutes. 0-1 ml of EDTA-GVB and 0-1 ml of guinea-pig C3 (0.1 mg/ml) were then added, and the incubation at 370 was continued for 1 hour. One millilitre of 1 x 108/ ml HuE were then added and further incubated for 1 hour at 370. The HuE were washed twice and suspended in 0-5 ml of EDTA-GVB. To each HuE suspension, 0 3 ml of 12511 labelled anti-C3 (circa 2000 ct/min) were added and the mixture was incubated at room temperature (about 200) for 1 hour. After washing three times with EDTA-GVB, the radioactive uptake by HuE was counted. The number of C3 molecules taken up by HuE was calculated using the standard curve shown in Fig. 1. At the optimum concentration range of C1, C3 molecules were efficiently taken up by HuE. C3 convertase prepared with 7 x 1010 SFU of C4, 5 x 1010 SFU of C2 and 4 x 108 SFU of Cl made over 25%Y of 4 x I0" molecules of C3 reactive to the C3 receptor on HuE (Table 1). This treatment destroyed 95 per cent of the haemolytic activity of C3. UPTAKE OF
C3 BY SEVERAL KINDS OF CELLS
Because it has been demonstrated that activated C3 fixes on HuE, its affinity to other cells was tested as follows: C3 was treated with an optimum ratio of C 1, C4 and C2 as mentioned above. Treated C3 or non-treated C3 (2 x 10'3 molecules in 0 5 ml) was mixed with 0-5 ml of cell suspension. After 30 minutes incubation at 370, the cells were washed twice and the number of C3 molecules fixed on the cell membrane was determined with 1251I-labelled anti-C3 as mentioned above. The number of C3 molecules specifically L
H. Okada and foriko Okada
1168
TABLE 1
RELATIONSHIP
OF AMOUNT OF
Cl
C4
(SFU)
(SFU)
4x 1010 4x 109 4x 108 4x 107
4x10O6 0
7x 7x 7x 7x 7x 7x
5x 5x 5x 5x 5x 5x
C3
USED AND THAT OF ERYTHROCYTES
C2 (SFU)
1010 1010 1010 1010 1010 10'0
FIXATION
Cl
OF TREATED
C3 fixed (molecules)
C3 (molecules)
10'0 1010 1010 1010 1010 1010
4x 4x 4x 4x
1013 1013 1013 1013
4xx I3
4x 1013
FIXED TO HUMAN
Per cell
Total 44x 8-6x 11 x 7 Ox 1 3x 8-Ox
TABLE 2 C3 TO SEVERAL KINDS
1011 1012
10'3 1010 101
1010
4400 86,000 110,000 700 1300 800
OF CELLS
C3 fixed (molecules)
Cells
C3H/He spleen C3H/He lymph node C3H/thymus HuE SRBC AdL-l P3HR-1
Number of cells 4-5 x 1 6x 2-9 x 2O x 10-x 88x 6 6x
101 107 107 108 109
106 106
Total
1-3 x 1 7x 2-1 x 5 7x 33x 6-1 x 2 6x
1012 1012 1012 1012
10'2 1012 1012
Per cell
29,000 110,000 72,000 29,000 3300 690,000 390,000
cell-bound were calculated by subtraction of the number of non-treated C3 fixed on control cells from that of treated C3 on test cells. As shown in Table 2, treated C3 was taken up by all cell types used, including IA receptor-negative cells such as SRBC, thymus cells and P3HR-1 which do not form rosettes with EAC43 cells (Nishioka, Tachibana, Hirayama, de The, Klein, Takada and Kawamura, 1971). INHIBITORY ACTIVITY OF ACTIVATED C3 ON IA REACTION BETWEEN HuE AND
EAC43 In order to examine the specific reaction of activated C3 with the IA receptor on the cell membrane, the inhibitory activity of activated C3 on the IA reaction between HuE and EAC43 was tested. Activated C3 was prepared by treating 4 x 1013 molecules of C3 with 4 x 108 SFU of Cl, 7 x 1010 SFU of C4 and 5 x 1010 SFU of C2 as described above. To 25-jl dilutions of activated C3, 25 pul of 1 x 108/ml HuE in EDTA-GVB and 25 p1 of 5 x IO7/ml EAC43 in EDTA-GVB were added. After 90 minutes incubation at 370, agglutination patterns due to IA were read. Activated C3 inhibited IA to a dilution of 1:8. In another experiment, 1 x 108/ml HuE in EDTA-GVB and an equal volume of activated C3 were mixed and incubated at 370 for 60 minutes. After washing twice with EDTA-GVB, the treated HuE (1 x 108/ml) were mixed with an equal volume of 5 x 107/ml EAC43. The rate of rosette formation was reduced from 61 to 4 per cent.
C3 and the Immune Adherence Receptor
1169
DISCUSSION C3 is known to be an essential factor for IA to HuE (Nishioka and Linscott, 1963). Since tanned erythrocytes (TaSRBC-C3) adhered to HuE, C3 molecules were expected to combine directly with IA receptors on HuE (Okada et al., 1970). C3 molecules on TaSRBC-C3 did not require any special treatment for their activation. Therefore, it was presumed that non-specifically activated C3 molecules were present in the C3 preparation used. This was confirmed by absorption of the C3 preparation with HuE prior to its conjugation to TaSRBC (Okada and Nishioka, 1972). These results also indicated that activated C3 molecules are able to combine with IA receptors on HuE. Recently, Bokisch and Theofilopoulos (1973) reported that not only C3b but also purified native C3 molecules fix on cultured cells from Burkitt lymphoma (Raji). However, our findings lead us to believe that the native C3 used probably contained non-specifically activated C3 molecules. In order to investigate fixation of activated C3 to HuE, guinea-pig C3 was treated with mixtures of C1, C4 and C2. Since purified guinea-pig C1 is in an activated state the mixture was expected to generate C42 or C3 convertase (Muller-Eberhard, Polley and Calcott, 1967) which in turn generated C3b. In our experiment, the concentrations of C4, C2 and C3 were fixed and that of Cl was varied. At optimum C1 concentration approximately 25 per cent of the C3 molecules were reactive to HuE (Table 1). The optimum concentration of C I should have been dependent on the activity of the Cl inactivator (Tamura and Nelson, 1967) contaminating the C4 preparation used. It is possible that C42 complexes were still active and generating nascent C3b which fix on the cell membrane at its C3d portion (Muller-Eberhard, Damasso and Calcott, 1966). However, the remaining haemolytic activity of C3 after incubation with C3 convertase was less than 5 per cent of its initial activity. In other words, 95 per cent of the C3 molecules were unable to become nascent C3b. Almost all C3 molecules could be regarded as decayed C3b that were unable to fix on the cell membrane at their C3d portion. Since 25 per cent of the C3 molecules fixed on HuE, this indicated that decayed C3b was able to combine with the IA receptor on HuE. This was confirmed by inhibition of the IA reaction between HuE and EAC43 with treated C3. Since decayed C3 are able to fix to the IA receptor on cell membranes, it may be possible that a portion of the deposition of C3 (fllc/filA globulin) on localized regions, such as in the nephritic kidney, could occur in a similar manner.
In order to detect IA receptor-positive cells, SRBC are usually employed as the anti-
genic particle in the indicator immune complement complex. A relatively strong bond is probably required to hold IA receptor cells and the indicator SRBC together. Therefore some cells which have IA receptors in low populations on cell membranes may be reas IA receptor-negative cells. To detect IA receptors on IA-negative cells, the cells were reacted with C3 convertase-treated C3 (decayed C3b). IA receptors were detected on IA receptor-negative cells including mouse thymus cells and P3HR-1, as well as on SRBC (Table 2). The number of C3 molecules combined with IA receptor-negative cells was unexpectedly high. The mean amount of C3 fixed on thymus cells was 65 per cent of that on an equal number of lymph node cells. If we calculate on the assumption
garded
that 20 per cent of lymph node cells are IA receptor-positive cells with EAC43 and that the IA receptor-negative lymph node cells (80 per cent) bind C3 to the same extent as thymus cells, then the positive cells bind only four times as much C3 as the negative cells. When x and y are taken as the amounts of receptors on the positive and negative L*
1170 H. Okada and Noriko Okada cells respectively, then the ratio x/y is calculated as follows: ly/(0-2x+0-8y) = 0.65, then x/y = 4. The number of IA receptors on some receptor-negative cells with EAC43 is not so small. IA receptors in these cases may diffuse and be unable to concentrate at some localized portion on the cell membrane to effect a type of cap formation (Edelman, Yahara and Wang, 1973) where EAC43 might bind. Lack of a concentrated IA receptor site on IA-negative cells may not afford a strong enough bond to bind EAC43, which is a relatively large particle. The number of C3 molecules fixed on the spleen cells were smaller than that on thymus cells, which is difficult to explain. One possible explanation may be fixation of mouse C3 on spleen cells through the preparation of spleen cell suspension, since mouse complement is easily activated non-specifically. IA-negative P3HR- 1 fixed ten times more the activated C3 than HuE which is an IA-positive cell. This fact may be due to the larger surface area of the cell membrane of P3HR-1, which is not only bigger in diameter than HuE but also has a lot of microvilli on the cell surface. ACKNOWLEDGMENTS This work was supported by research grants from the Ministry of Health and the Ministry of Education of the Japanese Government. The authors thank Dr K. Nishioka for valuable discussions and the excellent technical assistance of Miss K. Yamanushi. REFERENCES
BOKISCH, V. A. and THEOFILOPOULOS, A. N. (1973). 'Receptors for native C3 on human lymphoblastoid cell lines.'J. Immunol., 111, 300. CAUTRECASAS, P., WILCHEK, M. and ANFINSEN, C. B. (1968). 'Selective enzyme purification by affinity chromatography.' Proc. nat. Acad. Sci. (Wash.), 61, 636. EDELMAN, G. M., YAHARA, I. and WANG,J. L. (1973). 'Receptor mobility and receptor-cytoplasmic interaction in lymphocytes.' Proc. nat. Acad. Sci. (Wash.), 70, 1442. EDEN, A., MILLER, G. W. and NUSSENZWEIG, V. (1973). 'Human lymphocytes bear membrane receptors for C3b and C3d.'_J. cdin. Invest., 52, 3239. MCCONAHEY, P. F. and DIXON, F. J. (1966). 'A method of trace iodination of proteins for immunologic studies.' Int. Arch. Allergy, 29, 185. MULLER-EBERHARD, H. J., DAMASSO, A. P. and CALCOTT, M. A. (1966). 'The reaction mechanism of f6lc-globulin (C'3) in immune hemolysis.' 7. exp. Med., 123, 33. MULLER-EBERHARD, H. J., POLLEY, M. J. and CALCOTT, M. A. (1967). 'Formation and functional significance of a molecular complex derived from the second and the fourth component of human exp. Med., 125, 359. complement.'_J. NELSON, D. S. (1963). 'Immune adherence.' Advanc. Immunol., 3, 131. NELSON, R. A. (1953). 'The immune-adherence phenomenon. An immunologically specific reaction between microorganisms and erythrocytes leading to enhanced phagocytosis.' Science, 118, 733. NELSON, R. A., JENSEN, J., GIGLI, I. and TAMURA, N. (1967). 'Methods for the separation, purification and measurement of nine components of hemolytic complement in guinea pig serum.' Immunochemistry, 3, 111. NISHIOKA, K. and LINSCOTT, W. D. (1963). 'Compon-
ents of guinea pig complement. I. Separation of a serum fraction essential for immune hemolysis and immune adherence.' J. exp. Med., 118, 767. NISHIOKA, K., TACHIBANA, T., HIRAYAMA, T., THE, G. DE, KLEIN, G., TAKADA, M. and KAWAMURA, A., JR (1971). 'Immunological studies on the cell membrane receptors of cultured cells derived from nasopharyngeal cancer, Burkitt's lymphoma and infectious mononucleosis.' Recent Advances in Human Tumor Virology and Immunology (ed. by W. Nakahara, K. Nishioka, T. Hiyarama and Y. Ito), p. 401. University of Tokyo Press, Tokyo. OKADA, H., KAWACHI, S. and NISHIOKA, K. (1970). 'Immune adherence reactivity by C3 molecules without antibody and other factors of the complement system.' Biochem. biophys. Acta (Amst.), 208, 514. OKADA, H., KoJIMA, K., YOSHIDA, T. 0. and NiSHIOKA, K. (1972). 'Electrokinetic behavior of intermediate cells in immune hemolysis.'_J. Immunol., 108, 59. OKADA, H. and NISHIOKA, K. (1972). 'Studies on the mechanism of immune adherence and electric charge of intermediate cells.' Biological Activities of Complement (ed. by D. G. Ingram), p. 229. Karger, Basel. OKADA, H. and NiSHIOK.A, K. (1973a). 'Two C receptors on lymphoid cells.'_7. Immunol., 111, 309. OKADA, H. and NISHIOKA, K. (1973b). 'Complement receptors on cell membranes. I. Evidence for two complement receptors.' J. Immunol., 111, 1444. OKADA, H., NISHIOKA, K. and SINDO, T. (1966). 'The effects of Cu-chlorophyllin on the active site formation of each component of guinea-pig complement.' Immunology, 16, 473. OKADA, H. and OKADA, N. (1974). 'Complement receptors on cell membranes. II. Separation from mouse plasma of two essential factors for complement receptors on cell membrane.' jap. 7. exp. Aled., 44, 301. Ross, G. D., POLLEY, M. J., RABELLINO, E. M. and
C3 and the Immune Adherence Receptor GREY, H. M. (1973). 'Two different complement receptors on human lymphocytes. One specific for C3b and one specific for C3b inactivator-cleaved C3b.'_J. exp. Med., 138, 798. SHIMADA, K., MAYUMI, M., SEKINE, T. and NISHIOKA, K. (1972). 'An improved method for separation of the fourth component of complement and C4 inactivating substance.' Jap. J. exp. Aled., 42, 423. TAMURA, N. and NELSoN, R. A. (1967). 'Three
1171
naturally occurring inhibitors of components of complement in guinea pig and rabbit serum.' J. Immunol., 99, 582. UTSUMI, S. and KARUSH, F. (1965). 'Peptic fragmentation of rabbit yG immunoglobulin.' Biochemistrv, 4, 1766. WoFsy, L. and BURR, B. (1969). 'The use of affinity chromatography for specific purification of antibodies and antigens.'_7. Immunol., 103, 380.
Activated Guinea-pig C3 and the Immune Adherence Receptor (a Complement Receptor) on Cell Membranes* H. OKADA AND NORIKO OKADA National Cancer Center Research Institute, Tsukiji, Tokyo, and Central Laboratories, Sankyo Company, Hiromachi, Tokyo
(Received 29th January 1974; accepted for publication 16th November 1974) Summary. By treating C3 with purified C1, C4 and C2 in the fluid phase, haemolytically inactive C3 was prepared. This was shown to bind to human erythrocytes by use of radio-labelled (Fab')2 antibody to guinea-pig C3. The activated C3 preparation inhibited immune adherence between EAC43 and human erythrocytes. These findings indicate that the activated C3 attaches to the immune adherence receptor on human erythrocytes. In addition the fluid-phase activated C3 adhered to thymus cells and sheep erythrocytes, whereas EAC43 did not. Thus the immune adherence receptor may be present on so called immune adherencenegative cells, but in insufficient concentration to form rosettes with EAC43. INTRODUCTION The term immune adherence (IA) has been used by Nelson (1953) to describe the phenomenon where antigen-antibody-complement complexes adhere to human erythrocytes (HuE). In Ag-Ab-C1423 complexes, the C3 molecules do not require any other factor to exhibit IA reactivity to HuE (Nishioka and Linscott, 1963), although these complexes require another factor in addition to C3 for adherence to other complement receptors (Okada and Nishioka, 1973a, b; Ross, Polley, Rabellino and Grey, 1973; Eden, Miller and Nussenzweig, 1973; Okada and Okada, 1974). In a previous experiment, conjugation of purified guinea-pig C3 to tannic acid-treated sheep erythrocytes (TaSRBC) made the erythrocytes adherent to HuE (Okada, Kawachi and Nishioka, 1970). This finding suggested that only C3 molecules are required for IA reactivity. Since the tanned cells conjugated with purified guinea-pig C3 (TaSRBC-C3) adhered to HuE without any prior treatment of C3, it was felt that a proportion of the C3 molecules may have been activated nonspecifically during preparation, enabling them to react with IA receptors (Nelson, 1963) on the cell membrane (Okada and Nishioka, 1972). To investigate the reaction between activated C3 and the IA receptor on cell membranes, 125I-labelled (Fab')2 antibody to guinea-pig C3 was used. MATERIALS AND METHODS Complement components and intermediate cells Purified components of guinea-pig complement were prepared with minor modifica* Parts of this communication were presented at the 2nd Congress of Japanese Society for Immunology (Tokyo, 1972) and the 8th International Congress for Allergology (Tokyo, 1973). Correspondence: Dr H. Okada, Virology Division, National Cancer Center Research Institute, Tsukiji 5-chome,
Chuo-ku, Tokyo, Japan.
1165
H. Okada and Noriko Okada 1166 tions as described by Nelson, Jensen, Gigli and Tamura (1967). Human C4 was purified as described by Shimada, Mayumi, Sekine and Nishioka (1972). Complement-intermediate cells were prepared as described previously (Okada, Nishioka and Sindo, 1969; Okada, Kojima, Yoshida and Nishioka, 1972). The guinea-pig C3 preparation used in these experiments gave a single line in immunoelectrophoresis. The C3 purity was about 98 per cent when submitted to quantitative radio-immunoelectrophoresis (Okada et al., 1970). Diluents Glucose gelatin-veronal-buffered saline (gl-GVB2 +) and EDTA-gelatin-veronabuffered saline (EDTA-GVB) were prepared as described elsewhere (Nelson et al., 1967). Cell suspensions HuE, Type 0, Rh( +) and sheep erythrocytes (SRBC) were washed twice with EDTA-GVB and three times with an appropriate diluent. Mouse lymph node cells, spleen cells and thymus cells were obtained from C3H/He mice. Cells were washed three times with saline and suspended in EDTA-GVB. P3HR-1 and AdL-l were cultured cell lines from Burkitt's lymphoma and nasopharyngeal carcinoma respectively. The cells were washed four times before use.
Radio-labelled (Fab')2 fragments of antibody to guinea-pig C3 The IgG fraction of rabbit antiserum to guinea-pig C3 was treated with pepsin at 370 (Utsumi and Karush, 1965). The (Fab')2 thus prepared was labelled with 125I as described by McConahey and Dixon (1966). The labelled (Fab')2 anti-C3 was obtained by affinity chromatography on a Sepharose column conjugated with purified guinea-pig C3 (Cautrecasas, Wilcher and Anfinsen, 1968; Wofsy and Burr, 1969). Radio-labelled (Fab')2 with 75 per cent specificity to guinea-pig C3 was eluted with glycine-HCl buffer, pH 2-4. The radio-labelled (Fab')2 anti-guinea-pig C3 was used as 1251-labelled anti-C3.
Preparation of a standard curve of the radioactivity of '25I-labelled anti-C3 vs the number of C3 molecules on the cell membrane 0-5-ml samples of guinea-pig C3 at different concentrations in gl-GVB2 + were treated with an equal volume of 1 x 108/ml EAC142. The cells were washed three times and resuspended in 0-5 ml of EDTA-GVB. To the cell suspension, 0-1 ml of 125I-labelled anti-C3 (20,000 ct/min/ml) was added. After 60 minutes incubation at 300, the cells were washed three times with EDTA-GVB and the uptake of radioactivity on the cells was determined on an auto-y counter (Nuclear, Chicago). Since more than 50 per cent of the C3 molecules were fixed on EAC 142 at low C3 concentration (Okada and Nishioka, 1972), the standard curve was drawn with the assumption that C3 molecules fix on EAC142 at the rate of 50 per cent (Fig. 1). The non-linearity of uptake of C3 at an input of 100,000 molecules per cell may be due to the limiting number of C42 sites on the cell membrane.
C3 and the Immune Adherence Receptor
1167
4
o3 _
/~~~~~~~~~~~
/
C
C-)
£2
X
1000 330 4-1 12 37 110 Molecules of C3 on EAC1423 cells (xl01) FIG. 1. The relationship between the number of C3 molecules on the cell membrane and the radioactivity of 125I-labelled anti-C3 fixed on the cell membrane. In the calculation, 50 per cent of molecules in the reaction mixture were assumed to fix to EAC142.
RESULTS FIXATION OF C3 CONVERTASE-TREATED GUINEA-PIG C3 TO HuE
Guinea-pig C3 was treated with a mixture of Cl, C4 and C2 as follows. To 0 3 ml of a mixture of human C4 (7 x 1010 SFU) and guinea-pig C2 (5 x 1010 SFU) in gl-GVB2 +, 0-2 ml of varying concentrations of guinea-pig Cl were added, and incubated at 300 for 10 minutes. 0-1 ml of EDTA-GVB and 0-1 ml of guinea-pig C3 (0.1 mg/ml) were then added, and the incubation at 370 was continued for 1 hour. One millilitre of 1 x 108/ ml HuE were then added and further incubated for 1 hour at 370. The HuE were washed twice and suspended in 0-5 ml of EDTA-GVB. To each HuE suspension, 0 3 ml of 12511 labelled anti-C3 (circa 2000 ct/min) were added and the mixture was incubated at room temperature (about 200) for 1 hour. After washing three times with EDTA-GVB, the radioactive uptake by HuE was counted. The number of C3 molecules taken up by HuE was calculated using the standard curve shown in Fig. 1. At the optimum concentration range of C1, C3 molecules were efficiently taken up by HuE. C3 convertase prepared with 7 x 1010 SFU of C4, 5 x 1010 SFU of C2 and 4 x 108 SFU of Cl made over 25%Y of 4 x I0" molecules of C3 reactive to the C3 receptor on HuE (Table 1). This treatment destroyed 95 per cent of the haemolytic activity of C3. UPTAKE OF
C3 BY SEVERAL KINDS OF CELLS
Because it has been demonstrated that activated C3 fixes on HuE, its affinity to other cells was tested as follows: C3 was treated with an optimum ratio of C 1, C4 and C2 as mentioned above. Treated C3 or non-treated C3 (2 x 10'3 molecules in 0 5 ml) was mixed with 0-5 ml of cell suspension. After 30 minutes incubation at 370, the cells were washed twice and the number of C3 molecules fixed on the cell membrane was determined with 1251I-labelled anti-C3 as mentioned above. The number of C3 molecules specifically L
H. Okada and foriko Okada
1168
TABLE 1
RELATIONSHIP
OF AMOUNT OF
Cl
C4
(SFU)
(SFU)
4x 1010 4x 109 4x 108 4x 107
4x10O6 0
7x 7x 7x 7x 7x 7x
5x 5x 5x 5x 5x 5x
C3
USED AND THAT OF ERYTHROCYTES
C2 (SFU)
1010 1010 1010 1010 1010 10'0
FIXATION
Cl
OF TREATED
C3 fixed (molecules)
C3 (molecules)
10'0 1010 1010 1010 1010 1010
4x 4x 4x 4x
1013 1013 1013 1013
4xx I3
4x 1013
FIXED TO HUMAN
Per cell
Total 44x 8-6x 11 x 7 Ox 1 3x 8-Ox
TABLE 2 C3 TO SEVERAL KINDS
1011 1012
10'3 1010 101
1010
4400 86,000 110,000 700 1300 800
OF CELLS
C3 fixed (molecules)
Cells
C3H/He spleen C3H/He lymph node C3H/thymus HuE SRBC AdL-l P3HR-1
Number of cells 4-5 x 1 6x 2-9 x 2O x 10-x 88x 6 6x
101 107 107 108 109
106 106
Total
1-3 x 1 7x 2-1 x 5 7x 33x 6-1 x 2 6x
1012 1012 1012 1012
10'2 1012 1012
Per cell
29,000 110,000 72,000 29,000 3300 690,000 390,000
cell-bound were calculated by subtraction of the number of non-treated C3 fixed on control cells from that of treated C3 on test cells. As shown in Table 2, treated C3 was taken up by all cell types used, including IA receptor-negative cells such as SRBC, thymus cells and P3HR-1 which do not form rosettes with EAC43 cells (Nishioka, Tachibana, Hirayama, de The, Klein, Takada and Kawamura, 1971). INHIBITORY ACTIVITY OF ACTIVATED C3 ON IA REACTION BETWEEN HuE AND
EAC43 In order to examine the specific reaction of activated C3 with the IA receptor on the cell membrane, the inhibitory activity of activated C3 on the IA reaction between HuE and EAC43 was tested. Activated C3 was prepared by treating 4 x 1013 molecules of C3 with 4 x 108 SFU of Cl, 7 x 1010 SFU of C4 and 5 x 1010 SFU of C2 as described above. To 25-jl dilutions of activated C3, 25 pul of 1 x 108/ml HuE in EDTA-GVB and 25 p1 of 5 x IO7/ml EAC43 in EDTA-GVB were added. After 90 minutes incubation at 370, agglutination patterns due to IA were read. Activated C3 inhibited IA to a dilution of 1:8. In another experiment, 1 x 108/ml HuE in EDTA-GVB and an equal volume of activated C3 were mixed and incubated at 370 for 60 minutes. After washing twice with EDTA-GVB, the treated HuE (1 x 108/ml) were mixed with an equal volume of 5 x 107/ml EAC43. The rate of rosette formation was reduced from 61 to 4 per cent.
C3 and the Immune Adherence Receptor
1169
DISCUSSION C3 is known to be an essential factor for IA to HuE (Nishioka and Linscott, 1963). Since tanned erythrocytes (TaSRBC-C3) adhered to HuE, C3 molecules were expected to combine directly with IA receptors on HuE (Okada et al., 1970). C3 molecules on TaSRBC-C3 did not require any special treatment for their activation. Therefore, it was presumed that non-specifically activated C3 molecules were present in the C3 preparation used. This was confirmed by absorption of the C3 preparation with HuE prior to its conjugation to TaSRBC (Okada and Nishioka, 1972). These results also indicated that activated C3 molecules are able to combine with IA receptors on HuE. Recently, Bokisch and Theofilopoulos (1973) reported that not only C3b but also purified native C3 molecules fix on cultured cells from Burkitt lymphoma (Raji). However, our findings lead us to believe that the native C3 used probably contained non-specifically activated C3 molecules. In order to investigate fixation of activated C3 to HuE, guinea-pig C3 was treated with mixtures of C1, C4 and C2. Since purified guinea-pig C1 is in an activated state the mixture was expected to generate C42 or C3 convertase (Muller-Eberhard, Polley and Calcott, 1967) which in turn generated C3b. In our experiment, the concentrations of C4, C2 and C3 were fixed and that of Cl was varied. At optimum C1 concentration approximately 25 per cent of the C3 molecules were reactive to HuE (Table 1). The optimum concentration of C I should have been dependent on the activity of the Cl inactivator (Tamura and Nelson, 1967) contaminating the C4 preparation used. It is possible that C42 complexes were still active and generating nascent C3b which fix on the cell membrane at its C3d portion (Muller-Eberhard, Damasso and Calcott, 1966). However, the remaining haemolytic activity of C3 after incubation with C3 convertase was less than 5 per cent of its initial activity. In other words, 95 per cent of the C3 molecules were unable to become nascent C3b. Almost all C3 molecules could be regarded as decayed C3b that were unable to fix on the cell membrane at their C3d portion. Since 25 per cent of the C3 molecules fixed on HuE, this indicated that decayed C3b was able to combine with the IA receptor on HuE. This was confirmed by inhibition of the IA reaction between HuE and EAC43 with treated C3. Since decayed C3 are able to fix to the IA receptor on cell membranes, it may be possible that a portion of the deposition of C3 (fllc/filA globulin) on localized regions, such as in the nephritic kidney, could occur in a similar manner.
In order to detect IA receptor-positive cells, SRBC are usually employed as the anti-
genic particle in the indicator immune complement complex. A relatively strong bond is probably required to hold IA receptor cells and the indicator SRBC together. Therefore some cells which have IA receptors in low populations on cell membranes may be reas IA receptor-negative cells. To detect IA receptors on IA-negative cells, the cells were reacted with C3 convertase-treated C3 (decayed C3b). IA receptors were detected on IA receptor-negative cells including mouse thymus cells and P3HR-1, as well as on SRBC (Table 2). The number of C3 molecules combined with IA receptor-negative cells was unexpectedly high. The mean amount of C3 fixed on thymus cells was 65 per cent of that on an equal number of lymph node cells. If we calculate on the assumption
garded
that 20 per cent of lymph node cells are IA receptor-positive cells with EAC43 and that the IA receptor-negative lymph node cells (80 per cent) bind C3 to the same extent as thymus cells, then the positive cells bind only four times as much C3 as the negative cells. When x and y are taken as the amounts of receptors on the positive and negative L*
1170 H. Okada and Noriko Okada cells respectively, then the ratio x/y is calculated as follows: ly/(0-2x+0-8y) = 0.65, then x/y = 4. The number of IA receptors on some receptor-negative cells with EAC43 is not so small. IA receptors in these cases may diffuse and be unable to concentrate at some localized portion on the cell membrane to effect a type of cap formation (Edelman, Yahara and Wang, 1973) where EAC43 might bind. Lack of a concentrated IA receptor site on IA-negative cells may not afford a strong enough bond to bind EAC43, which is a relatively large particle. The number of C3 molecules fixed on the spleen cells were smaller than that on thymus cells, which is difficult to explain. One possible explanation may be fixation of mouse C3 on spleen cells through the preparation of spleen cell suspension, since mouse complement is easily activated non-specifically. IA-negative P3HR- 1 fixed ten times more the activated C3 than HuE which is an IA-positive cell. This fact may be due to the larger surface area of the cell membrane of P3HR-1, which is not only bigger in diameter than HuE but also has a lot of microvilli on the cell surface. ACKNOWLEDGMENTS This work was supported by research grants from the Ministry of Health and the Ministry of Education of the Japanese Government. The authors thank Dr K. Nishioka for valuable discussions and the excellent technical assistance of Miss K. Yamanushi. REFERENCES
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