Eur.J. Immunol. 1991.21: 1787-1792
Misako Matsumoto, Yuji SugitaO and Tsukasa Seya Department of Immonology, Center for Adult Diseases, Osaka, Department of Physiological Chemistry, School of Pharmaceutical Scienceso, Shows University Tokyo
Complement cytotoxicity on myeloid cells
1787
Alternative complement pathway-mediated myeloid cell cytotoxicity: repertoire of membrane factors participating in regulation of C3 deposition and cytolysis* Most human nucleated cells and cell lines possess C3 step regulators, decayaccelerating factor (DAF; CD55) and membrane cofactor protein (MCP; 0 4 6 ) and an inhibitor of membrane attack complex (MAC) formation (p18; CD59). Unless DAF and MCP were simultaneously blocked by their antibodies, Mg2+-EGTA-humanserum treatment did not induce C3 deposition on most nucleated cells. Furthermore, 40 ng/ml) of C5a irrespective of the Ab treatments. Under the same conditions, p39 produced > 200 ng/ml of C5a, and anti-DAFor anti-MCP appeared to potentiate the C5 activation slightly.
IS0
c
0
I,
K562
m 150
"937
I
i
The possibility still remains that the generated C5a was trapped by cell surface molecules including C5a receptors, thereby being undetected in the SN. We next analyzed the deposited C5 on these myeloid cells by anti-C5 and FCM (Fig. 4, upper panel). HLr60, when placed in Mg2+EGTA-NHS, accepted a little C5 on their membrane after the treatment with anti-DAF and/or anti-Ma. By FCM with anti-C5, however, HL-60treated with C5a desArg did not induce any positive shift (not shown). Therefore, depositionof C5b occurs by suppressingthe activity of DAF or MCI! Similar C5 deposition was observed in U-937 cells which were treated with anti-DAF and Mg2+-EGTA-NHS (C9-DS was used in this case). It is interesting to note in U-937 that no C5 deposition was observed following treatment with anti-MCF! Although not shown in the figure, p39 allowed the deposition of C5 similar to C3.
P39 I
Fluorescence Intensity
, ,
3.4 MAC formation on myeloid cells
-no Ab . . . . anti-^^^ _- anti-MCP - - - anti-DAF,MCP
By FCM using Ab against human C8 and C9, the degreesof C8 and C9 deposition were also assessed. Both C8 and C9 remained on the cell surface and accumulated more on I%-60 by anti-DAF and/or anti-MCP treatment (Fig. 4, lower panel). Under the same experimental conditions, U-937 was partly lysed into undetectable debris. Using the C9-deficient serum, C8 deposition was assessed again with respect to U-937 (Fig. 4, lower panel). C8 was successfully deposited on the anti-DAF-treated U-937 but not on the anti-MCP-treated one, which is consistent with the results of C3 and C5 deposition. C8 and C9 were also deposited on p39 (not shown).
'./
LI
,
Figure 2. C3 fragments deposited on myeloid cell lines by treatment with Ab against DAF and/or MCP. K-562, HL60,U-937 and p39 were preincubated with polyclonal anti-DAF * * * .), antiMCP (........), both anti-DAFand anti-MCP (---) or buffer alone (-).These cells were then incubated with MgZ+-EGTA-NHSor Mg2+-EGTA-C9DS.The deposited C3 was detected with mAb to human C3c and FITC-labeled goat anti-mouse IgG followed by FCM. Arrow in the panel of p39 indicates the position of the peak fluorescence intensity in EDTA-NHS-treated p39. (a
HL60
u937
L Q)
P
zE 200
1
- no Ab Control
HL60 Cell Lines
. . . . _ _ anti-DAF
P39
Figure 3. Determination of C5a generated by the antibody-treated cells and serum. HL60 and p39 were pretreated with anti-DAF, anti-MCP, anti-DAF and anti-MCP, or buffer alone, and then incubated with MgZ+-EGTA-NHS.The reaction mixtures were centrifuged and C5a generated in the SN was measured with a C5a radioimmunoassaykit (Amersham). As controls, EDTA-NHS and Mg2+-EGTA-NHScontaining no cells were incubated with the buffer, and C5a generation was determined as above. Mean values of duplicate experiments were plotted. Assuming that the concentration of c 5 in NHs is 100 p g / d , 100n g / d of C5a reflects -4% c 5 cleavage. Similar results were obtained with U-937 (not shown in the figure).
anti-MCP
- _ _ anti-DAF, MCP
Fluorescence Intensity Figure 4. Detection of C5, C8 and C9 on HL-60 and U-937 that were treated with Ab. HL60 and U-937 were treated with anti-DAF . .) anti-MCP (..-.....) or anti-DAF, MCP (---) followed by Mgz+-EGTA-NHSor Mg2+-EGTA-C9Dsas described in Fig. 2. C5b, C8 and C9 deposited and expressedon the cells were detected by treating the cells with goat antisera to the respective C proteins followed by FITC-labeled swine anti-goat IgG. C5, C8 and C9 were also detected in ~ 3 (data 9 not shown). (a
+
Complement cytotoxicity on myeloid cells
Eur. J. Immunol. 1991.21: 1787-1792
3.5 Cytolysis of myeloid cells via the alternative pathway
U-937, HL-60 and p39 were labeled with W r and then treated with combinations of Ab to DAF, MCP and p18. C-mediated cytotoxicitywas tested with these cells. HL60 was killed by C if its surface molecules, DAF, MCP and p18, were simultaneouslyblocked (Fig. 5). The killing efficiency was 60% at most. Actually, no lysis took place unless p18 was blocked. U-937 was destructed by the treatment with anti-DAF and -MCP by 50%. Blocking of p18 was unnecessary for the U-937 cytolysis (Fig. 1). About 25% of U-937 underwent cell damage by the treatment only with anti-DAF,while no damage was induced by anti-MCP alone (Fig. 5). Despite the extensive C3/C5 activation and deposition on p39, it was considerably resistant to C-mediated lysis even after the treatment with the Ab against DAF, MCP and p18 (Fig. 5).
HL60
u937 Cell Lines
P39
Figure 5. Effects of various antibodies against C regulatory proteins on alternative pathway-mediated cytotoxicity. HL-60, U-937 and p39 were labeled with W r and preincubated with anti-DAF (W), anti-MCP (W), anti-DAF anti-MCP (m),and anti-DAF anti-MCP anti-pl8 (B). The cells were washed thoroughly and incubated with Mg2+-EGTA-NHS.The 51Crreleased was measured in a y-counter. Mean values of triplicate experiments were shown.
+
+
+
Little lysis was observed in these cell lines if the polyclonal Ab against DAF and MCP were substituted with mAb. Little lysis (< 20%) was also observed on the other myeloid cell lines HEL, THPl and K-562 (not shown).
4 Discussion
1791
polyclonal anti-P.&croglobulin and anti-pl8 as a control to assess the effect of Ab itself. Far less or virtually no C3 deposition was observed with the control Ab, though the antigenic sites were two- to fivefold more in most cell lines tested than those of anti-DAF/anti-MCP Moreover, the amount of anti-DAF and anti-MCP deposited on 5 x lo5 cells is of the ng order [20], probably too small to induce an Ab-dependent alternative pathway activation. Therefore, the C3 deposition observed herein is mostly reflects the blocking of DAF or MCP. SuccessfulFCM visualization of C3 deposition by blocking DAF or MCP on HL-60 suggests that even in human cell lines there are C3-accepting molecules on which nascent C3b covalently anchors and amplifies the alternative pathway. DAF and MCP virtually regulate the C3 activation occurring on the accepting molecules. In U-937 cells, anti-DAF promotes C3 deposition while anti-MCP does not. This suggests that U-937 accepts C3b in a DAFdependenthim-independent manner. U-937, therefore, possesses a unique acceptor for C3b which is resistant to MCP but sensitive to DAF. Some B cell lines allow the deposition of C3b despite their expression of DAF and MCP [20,26-281 , and some T cell lines, though lacking DAF, are sufficiently protected from C3 targeting by MCP [21]. These and our present results enable us to classify human cells into four categories: (a) C3b deposited on them being resistant to both DAF and MCP, (b) those inhibited predominantly by DAF, (c) those that are inactivated predominantly by MCP (together with factor I), and (d) those that are susceptible to both. p39 and most CR2+ EBV-infected B cell lines are representatives of the first category. U-937 belongs to the second. DAFTcell lines, TALL and CEM, probably being protected sufficiently by MCP [21], are examples for the third category. K-562 (Fig. 2) and most human cell lines fall in the fourth. Some cell lines regulating C3 deposition other than by DAF and MCP, suggests that the membrane regulatory proteins are not all that determine homologous C3 deposition: properties of putative C3 acceptors and other factors (such as a membrane molecule capable of regulating the accessibility of plasma factor H to the membrane convertases) may critically influence the C3 deposition [43, 441.
Morgan et al. [30] demonstrated that Ab-sensitized U-937 cells can be damaged by the classical C pathway. Our data Human myeloid cell lines U-937 and HL-60 have been support their findings at the molecular level and further shown to become relatively susceptible to C by treatment clarified that U-937 is also susceptible to the alternative with their Ab [30]. The purpose of this study was to pathway. U-937 is fairly insensitive to C3 attack, but once elucidate the relationship between the known C regulatory DAF protective activity is circumvented, C3b can be proteins and susceptibility to homologous C of these human deposited and continued into successful formation of MAC cell lines. To compare C susceptibility among the myeloid and cell damage because of the absence of p18. Molecular cell lines, we used the same lots of polyclonal Ab against mechanisms of C susceptibilityof HL60 remain unclear.We DAF and MCP which had been characterized as inhibitors currently maintain that HL-60 possesses accepting molefor DAF and MCP but themselves induced no cytolysis [21]. cules for C3b whose activity cannot be suppressed by either mAb against DAF or MCP acted similarly but not as DAF or MCP and, thereby, easily induces extensive C3 effectively as the polyclonal Ab in enhancement of C3 deposition by treatment with either anti-DAFor anti-MCl? deposition via the alternative pathway [21]. In addition, Furthermore, it was lysed effectively by the additional g of antiviral polyclonal blocking of p18, contrasting with the other myeloids tested. virus-infected cells bound Ab, activate the alternative pathway [42]. Alternative It is, therefore, likely that the low potency of C3 and C9 pathway-activating effect of the polyclonal Ab may have step regulation engages C-mediated cell damage, which operated jointly with DAFMCP blocking effect, in the partly explain high C susceptibility of the Ab-sensitized efficient C3 deposition. For this reason, we employed HL-60 [30].
-
1792
M. Matsumoto,Y Sugita and T. Seya
The C3 activation mechanism of p39 is unknown. It must possess an activator, which is similar in function to but different in antigenicity from CR2, on its surface. Judging from analysis of the deposited C3 on SDS-PAGE and FCM (Matsumoto and Seya, unpublished data), an active form of C3b still remainsbound to p39 even after 90-min incubation with Mg2+-EGTA-NHS.C3b deposited on p39 is relatively resistant to proteolytic inactivation by factor I since the deposited C3b is not instantly converted to C3bi. These properties of the deposited C3b probably reflect those of the putative acceptor molecules on p39. An important finding is that the extensive C3 deposition in p39 is not in closely conjunction with effective C9 deposition or cell damage; 60% of the cells are damaged by C. Again, other mechanisms [l] or molecules (including HRF) [9, 10, 451 may additionally serve to protect host cells from homologous C-mediated cytolysis. We are grateful to Drs. Kumar (Washington Univ., St. Louis) and M. Tomita (Showa Univ., Tokyo) for critical reading, and to Drs. Iida (New York Univ. NY) and Hatanaka (Osaka Medical Colledge, Takatsuki) for providing their reagents. We thank JCRB for providing the human myeloid cell lines. We gratefully acknowledge general support by Drs. Akedo (Centerfor Adult Diseases, Osaka), Inoue (Kyowa Hakko Co., Tokyo) and Nagase (Mochida Co., Tokyo). Thanks are also due to Ms i? Hara for technical assistance and to Ms A . It0 for excellent secretarial assistance. Received December 10, 1990; in revised form March 20, 1991.
5 References 1 Morgan, B. F!, Biochem. J. 1989. 264: 1. 2 Koski, C. L. Ramm, L. E., Hammer, C. H., Mayer, M. M. and Shin, M. L., Proc. Natl. Acad. Sci. USA 1983. 80: 3816. 3 R a m , L. E.,Whitlow, M. B., Koski, C. L., Shin, M. L. and Mayer, M. M., J. Immunol. 1983. 131: 1411. 4 Fearon, D. T., Proc. Natl. Acad. Sci. USA 1979. 76: 5867. 5 Nicholson-Weller, A., Burge, J., Fearon, D. T., Weller, I? F. and Austen, K. F., J. Immunol. 1982.129: 184. 6 Medof, M. E., Kinoshita,T. and Nussenzweig,V.,J. Exp. Med. 1984. 160: 1558. 7 Cole, J. L., Housley, Jr., G. A., Dykman,T. R., MacDermott, R. E! and Atkinson, J. P., Proc. Natl. Acad. Sci. USA 1985.82: 859. 8 Seya,T.,Turner, J. R. and Atkinson, J. I?, J. Exp. Med. 1986. 163: 837. 9 Zalman, L. S. ,Wood, L. M. and Miiller-Eberhard, H. J., Proc. Natl. Acad. Sci. USA 1986. 83: 6975. 10 Schiinermark, S., Rauterberg, E. W., Shin, M. L., Loke, S., Roelcke, D. and Hansch, G. M., J. Immunol. 1986. 136: 1772.
Eur. J. Immunol. 1991.21: 1787-1792 11 Sugita,Y Nakano,Y. and Tomita, M., J. Biochem. 1988. 104: 633. 12 Okada, N., Harada, R., Fujita, T. and Okada, H., Inr. Immunol. 1989. 1: 205. 13 Davies, A., Simmons, D. L., Hale, G., Hanison, R. A. ,Tighe, H., Lachmann, E! J. and Waldmann, H., J. Exp. Med. 1989. 170: 637. 14 Holguin, M. H., Fredrick, L. R., Bernshaw, N. J. ,Wileox,L. A. and Parker, C. J., J. Clin. Invest. 1989. 84: 7. 15 Fearon, D. T., J. Exp. Med. 1980. 152: 20. 16 Kinoshita,T., Medof, M. E., Silber, R. and Nussenzweig,V.,J. Exp. Med. 1985. 162: 75. 17 Medof, M. E.,Walter, E. I., Rutgers, J. L., Knowles, D. M. and Nussenzweig,V., J. Exp. Med. 1987.165: 848. 18 Seya, T., Ballard, L. L., Bora, N. S., Kumar, V., Cui, W. and Atkinson, J. I?, Eur. J. lmmunol. 1988.18: 1289. 19 McNearney,T., Ballard, L., Seya,T. and Atkinson, J. E!, J. Clin. Invest. 1989. 84: 538. 20 Seya,T., Hara,T., Matsumoto, M. and Akedo, H., J. Immunol. 1990. 145: 238. 21 Seya,T., Hara,T., Matsumoto, M., Sugita,Y.and Akedo, H., J. Exp. Med. 1990. 172: 1673. 22 Pangburn, M. K. and Miiller-Eberhard, H. J., Proc. Natl. Acad. Sci. USA 1978. 75: 2416. 23 Pangburn, M. K., Momson, D. C., Schreiber, R. D. and Miiller-Eberhard, H. J., J. Immunol. 1980. 124: 977. 24 Fearon, D. T. and Austen, K. F., Proc. Natl. Acad. Sci. USA 1977. 74: 1683. 25 Fearon, D. T. and Austen, K. F., J. Exp. Med. 1977. 146: 22. 26 Praz, F. and Lesavre, I?, J. Immunol. 1983. 131: 1396. 27 Ramos, 0.F., Sarmay, G., Klein, E.,Yefenof, E. and Gergely, J., Proc. Natl. Acad. Sci. USA 1985. 82: 5470. 28 Mold, C., Nemerow, G. R., Bradt, B. M. and Cooper, N. R., J. Immunol. 1988. 140: 1923. 29 Tedder, T. F., Clement, L. T. and Cooper, M. D., J. Immunol. 1984. 133: 678. 30 Morgan, B. P.,Imagawa, D. K., Dankert, J. R. and Ramm, L. E., J. Immunol. 1986.136: 3402. 31 Nagasawa, S. and Stroud, R. M., Immunochemistry 1977.14: 749. 32 Markwell, M. A. K. and Fox, C. E , Biochemistry 1978. 17: 4807. 33 Seya, T., Farries, T. C., Nickels, M. W. and Atkinson, J. P., J. Immunol. 1987.139: 1260. 34 Seya,T., Nagasawa, S. and Atkinson, J. F!, J. Immunol. 1990. 144: 2312. 35 Seya,T., Hara,T., Uenaka, A., Nakayama, E. and Akedo, H., Complement Inflammation 1990. 7: 78. 36 Fujita, T., Inoue, I., Ogawa, K., Iida, K. and Tamura, N., J. Exp. Med. 1987. 166: 1221. 37 Iida, K., Mitomo, K., Fujita,T. and Tamura, N., Immunology 1987. 62: 413. 38 Kabat, E. A. and Mayer, M. M., Experimental Immunochemk t r j Charles C. Thomas Publisher, Springfield. 1964, p. 133. 39 Fukumori, Y , Yoshimura, K., Ohnoki, S., Yamaguchi, H., Akagaki,Y and Inai, S., Int. Immunol. 1989. 1: 85. 40 Seya,T., Okada, M., Matsumoto, M., Hong, K., Kinoshita,T., Atkinson, J. I?, Mol. Immunol. 1991, in press. 41 Cheung, N.V. ,Walter, E. I., Smith-Mensah,W. H., Ratnoff, W. D.,Tykocinski, M. L. and Medof, M. E., J. Clin. Invest. 1988. 81: 1122. 42 Perrin, L. H., Joseph, B. S., Cooper, N. R. and Oldstone, M. B. A., J. Exp. Med. 1976. 143: 1027. 43 Fischer, E. and Kazatchkine, M. D., J. Immunol. 1983. 130: 2821. 44 Weisman, H. F., Bartow,T., Leppo, M. K., Marsh, Jr., H. C , Carson, G. R., Concino, M. F., Boyle, M. F!, Roux, K. H., Weisfeldt, M. L. and Fearon, D. T., Science 1990. 249: 146. 45 Watts, M. J., Dankert, J. R. and Morgan, B. F!, Biochem. J. 1990. 265: 471.
Misako Matsumoto, Yuji SugitaO and Tsukasa Seya Department of Immonology, Center for Adult Diseases, Osaka, Department of Physiological Chemistry, School of Pharmaceutical Scienceso, Shows University Tokyo
Complement cytotoxicity on myeloid cells
1787
Alternative complement pathway-mediated myeloid cell cytotoxicity: repertoire of membrane factors participating in regulation of C3 deposition and cytolysis* Most human nucleated cells and cell lines possess C3 step regulators, decayaccelerating factor (DAF; CD55) and membrane cofactor protein (MCP; 0 4 6 ) and an inhibitor of membrane attack complex (MAC) formation (p18; CD59). Unless DAF and MCP were simultaneously blocked by their antibodies, Mg2+-EGTA-humanserum treatment did not induce C3 deposition on most nucleated cells. Furthermore, 40 ng/ml) of C5a irrespective of the Ab treatments. Under the same conditions, p39 produced > 200 ng/ml of C5a, and anti-DAFor anti-MCP appeared to potentiate the C5 activation slightly.
IS0
c
0
I,
K562
m 150
"937
I
i
The possibility still remains that the generated C5a was trapped by cell surface molecules including C5a receptors, thereby being undetected in the SN. We next analyzed the deposited C5 on these myeloid cells by anti-C5 and FCM (Fig. 4, upper panel). HLr60, when placed in Mg2+EGTA-NHS, accepted a little C5 on their membrane after the treatment with anti-DAF and/or anti-Ma. By FCM with anti-C5, however, HL-60treated with C5a desArg did not induce any positive shift (not shown). Therefore, depositionof C5b occurs by suppressingthe activity of DAF or MCI! Similar C5 deposition was observed in U-937 cells which were treated with anti-DAF and Mg2+-EGTA-NHS (C9-DS was used in this case). It is interesting to note in U-937 that no C5 deposition was observed following treatment with anti-MCF! Although not shown in the figure, p39 allowed the deposition of C5 similar to C3.
P39 I
Fluorescence Intensity
, ,
3.4 MAC formation on myeloid cells
-no Ab . . . . anti-^^^ _- anti-MCP - - - anti-DAF,MCP
By FCM using Ab against human C8 and C9, the degreesof C8 and C9 deposition were also assessed. Both C8 and C9 remained on the cell surface and accumulated more on I%-60 by anti-DAF and/or anti-MCP treatment (Fig. 4, lower panel). Under the same experimental conditions, U-937 was partly lysed into undetectable debris. Using the C9-deficient serum, C8 deposition was assessed again with respect to U-937 (Fig. 4, lower panel). C8 was successfully deposited on the anti-DAF-treated U-937 but not on the anti-MCP-treated one, which is consistent with the results of C3 and C5 deposition. C8 and C9 were also deposited on p39 (not shown).
'./
LI
,
Figure 2. C3 fragments deposited on myeloid cell lines by treatment with Ab against DAF and/or MCP. K-562, HL60,U-937 and p39 were preincubated with polyclonal anti-DAF * * * .), antiMCP (........), both anti-DAFand anti-MCP (---) or buffer alone (-).These cells were then incubated with MgZ+-EGTA-NHSor Mg2+-EGTA-C9DS.The deposited C3 was detected with mAb to human C3c and FITC-labeled goat anti-mouse IgG followed by FCM. Arrow in the panel of p39 indicates the position of the peak fluorescence intensity in EDTA-NHS-treated p39. (a
HL60
u937
L Q)
P
zE 200
1
- no Ab Control
HL60 Cell Lines
. . . . _ _ anti-DAF
P39
Figure 3. Determination of C5a generated by the antibody-treated cells and serum. HL60 and p39 were pretreated with anti-DAF, anti-MCP, anti-DAF and anti-MCP, or buffer alone, and then incubated with MgZ+-EGTA-NHS.The reaction mixtures were centrifuged and C5a generated in the SN was measured with a C5a radioimmunoassaykit (Amersham). As controls, EDTA-NHS and Mg2+-EGTA-NHScontaining no cells were incubated with the buffer, and C5a generation was determined as above. Mean values of duplicate experiments were plotted. Assuming that the concentration of c 5 in NHs is 100 p g / d , 100n g / d of C5a reflects -4% c 5 cleavage. Similar results were obtained with U-937 (not shown in the figure).
anti-MCP
- _ _ anti-DAF, MCP
Fluorescence Intensity Figure 4. Detection of C5, C8 and C9 on HL-60 and U-937 that were treated with Ab. HL60 and U-937 were treated with anti-DAF . .) anti-MCP (..-.....) or anti-DAF, MCP (---) followed by Mgz+-EGTA-NHSor Mg2+-EGTA-C9Dsas described in Fig. 2. C5b, C8 and C9 deposited and expressedon the cells were detected by treating the cells with goat antisera to the respective C proteins followed by FITC-labeled swine anti-goat IgG. C5, C8 and C9 were also detected in ~ 3 (data 9 not shown). (a
+
Complement cytotoxicity on myeloid cells
Eur. J. Immunol. 1991.21: 1787-1792
3.5 Cytolysis of myeloid cells via the alternative pathway
U-937, HL-60 and p39 were labeled with W r and then treated with combinations of Ab to DAF, MCP and p18. C-mediated cytotoxicitywas tested with these cells. HL60 was killed by C if its surface molecules, DAF, MCP and p18, were simultaneouslyblocked (Fig. 5). The killing efficiency was 60% at most. Actually, no lysis took place unless p18 was blocked. U-937 was destructed by the treatment with anti-DAF and -MCP by 50%. Blocking of p18 was unnecessary for the U-937 cytolysis (Fig. 1). About 25% of U-937 underwent cell damage by the treatment only with anti-DAF,while no damage was induced by anti-MCP alone (Fig. 5). Despite the extensive C3/C5 activation and deposition on p39, it was considerably resistant to C-mediated lysis even after the treatment with the Ab against DAF, MCP and p18 (Fig. 5).
HL60
u937 Cell Lines
P39
Figure 5. Effects of various antibodies against C regulatory proteins on alternative pathway-mediated cytotoxicity. HL-60, U-937 and p39 were labeled with W r and preincubated with anti-DAF (W), anti-MCP (W), anti-DAF anti-MCP (m),and anti-DAF anti-MCP anti-pl8 (B). The cells were washed thoroughly and incubated with Mg2+-EGTA-NHS.The 51Crreleased was measured in a y-counter. Mean values of triplicate experiments were shown.
+
+
+
Little lysis was observed in these cell lines if the polyclonal Ab against DAF and MCP were substituted with mAb. Little lysis (< 20%) was also observed on the other myeloid cell lines HEL, THPl and K-562 (not shown).
4 Discussion
1791
polyclonal anti-P.&croglobulin and anti-pl8 as a control to assess the effect of Ab itself. Far less or virtually no C3 deposition was observed with the control Ab, though the antigenic sites were two- to fivefold more in most cell lines tested than those of anti-DAF/anti-MCP Moreover, the amount of anti-DAF and anti-MCP deposited on 5 x lo5 cells is of the ng order [20], probably too small to induce an Ab-dependent alternative pathway activation. Therefore, the C3 deposition observed herein is mostly reflects the blocking of DAF or MCP. SuccessfulFCM visualization of C3 deposition by blocking DAF or MCP on HL-60 suggests that even in human cell lines there are C3-accepting molecules on which nascent C3b covalently anchors and amplifies the alternative pathway. DAF and MCP virtually regulate the C3 activation occurring on the accepting molecules. In U-937 cells, anti-DAF promotes C3 deposition while anti-MCP does not. This suggests that U-937 accepts C3b in a DAFdependenthim-independent manner. U-937, therefore, possesses a unique acceptor for C3b which is resistant to MCP but sensitive to DAF. Some B cell lines allow the deposition of C3b despite their expression of DAF and MCP [20,26-281 , and some T cell lines, though lacking DAF, are sufficiently protected from C3 targeting by MCP [21]. These and our present results enable us to classify human cells into four categories: (a) C3b deposited on them being resistant to both DAF and MCP, (b) those inhibited predominantly by DAF, (c) those that are inactivated predominantly by MCP (together with factor I), and (d) those that are susceptible to both. p39 and most CR2+ EBV-infected B cell lines are representatives of the first category. U-937 belongs to the second. DAFTcell lines, TALL and CEM, probably being protected sufficiently by MCP [21], are examples for the third category. K-562 (Fig. 2) and most human cell lines fall in the fourth. Some cell lines regulating C3 deposition other than by DAF and MCP, suggests that the membrane regulatory proteins are not all that determine homologous C3 deposition: properties of putative C3 acceptors and other factors (such as a membrane molecule capable of regulating the accessibility of plasma factor H to the membrane convertases) may critically influence the C3 deposition [43, 441.
Morgan et al. [30] demonstrated that Ab-sensitized U-937 cells can be damaged by the classical C pathway. Our data Human myeloid cell lines U-937 and HL-60 have been support their findings at the molecular level and further shown to become relatively susceptible to C by treatment clarified that U-937 is also susceptible to the alternative with their Ab [30]. The purpose of this study was to pathway. U-937 is fairly insensitive to C3 attack, but once elucidate the relationship between the known C regulatory DAF protective activity is circumvented, C3b can be proteins and susceptibility to homologous C of these human deposited and continued into successful formation of MAC cell lines. To compare C susceptibility among the myeloid and cell damage because of the absence of p18. Molecular cell lines, we used the same lots of polyclonal Ab against mechanisms of C susceptibilityof HL60 remain unclear.We DAF and MCP which had been characterized as inhibitors currently maintain that HL-60 possesses accepting molefor DAF and MCP but themselves induced no cytolysis [21]. cules for C3b whose activity cannot be suppressed by either mAb against DAF or MCP acted similarly but not as DAF or MCP and, thereby, easily induces extensive C3 effectively as the polyclonal Ab in enhancement of C3 deposition by treatment with either anti-DAFor anti-MCl? deposition via the alternative pathway [21]. In addition, Furthermore, it was lysed effectively by the additional g of antiviral polyclonal blocking of p18, contrasting with the other myeloids tested. virus-infected cells bound Ab, activate the alternative pathway [42]. Alternative It is, therefore, likely that the low potency of C3 and C9 pathway-activating effect of the polyclonal Ab may have step regulation engages C-mediated cell damage, which operated jointly with DAFMCP blocking effect, in the partly explain high C susceptibility of the Ab-sensitized efficient C3 deposition. For this reason, we employed HL-60 [30].
-
1792
M. Matsumoto,Y Sugita and T. Seya
The C3 activation mechanism of p39 is unknown. It must possess an activator, which is similar in function to but different in antigenicity from CR2, on its surface. Judging from analysis of the deposited C3 on SDS-PAGE and FCM (Matsumoto and Seya, unpublished data), an active form of C3b still remainsbound to p39 even after 90-min incubation with Mg2+-EGTA-NHS.C3b deposited on p39 is relatively resistant to proteolytic inactivation by factor I since the deposited C3b is not instantly converted to C3bi. These properties of the deposited C3b probably reflect those of the putative acceptor molecules on p39. An important finding is that the extensive C3 deposition in p39 is not in closely conjunction with effective C9 deposition or cell damage; 60% of the cells are damaged by C. Again, other mechanisms [l] or molecules (including HRF) [9, 10, 451 may additionally serve to protect host cells from homologous C-mediated cytolysis. We are grateful to Drs. Kumar (Washington Univ., St. Louis) and M. Tomita (Showa Univ., Tokyo) for critical reading, and to Drs. Iida (New York Univ. NY) and Hatanaka (Osaka Medical Colledge, Takatsuki) for providing their reagents. We thank JCRB for providing the human myeloid cell lines. We gratefully acknowledge general support by Drs. Akedo (Centerfor Adult Diseases, Osaka), Inoue (Kyowa Hakko Co., Tokyo) and Nagase (Mochida Co., Tokyo). Thanks are also due to Ms i? Hara for technical assistance and to Ms A . It0 for excellent secretarial assistance. Received December 10, 1990; in revised form March 20, 1991.
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