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Deficiency of complement decay-accelerating factor (DAF, CD55) in non-Hodgkin's lymphoma
Immunolog)' Letters, 29 (1991) 205-210 Elsevier IMLET 01631
Deficiency of complement decay-accelerating factor (DAF, CD55) in non-Hodgkin's lymphoma H i r o k o F u k u d a 2, Tsukasa Seya ~, T o m o k o H a r a 1, M i s a k o M a t s u m o t o 1, Taroh K i n o s h i t a 3 a n d Toru M a s a o k a 2 IDepartment o f bnmunology and 2Department o f Hematology Center for Adult Diseases, Osaka and 3Research Institute for Microbial Diseases, Osaka University Medical School, Suita Osaka, Japan (Received 5 February 1991; revision received 13 March 1991; accepted 18 March 1991)
1. Summary
2. Introduction
We have assessed levels of surface-expressed complement regulatory proteins, decay-accelerating factor (DAF) and membrane cofactor protein (MCP) on cells from patients with hematological malignancies. Neither malignant ceils nor unaffected nucleated blood cells from the patients lacked MCP. On the other hand, complete deficiency of DAF was found in 2/10 of non-Hodgkin's lymphoma (NHL), while none of the 38 patients with acute nonlymphocytic leukemia (ANLL) (14 cases), chronic myelogenous leukemia (CML) (6 cases), acute lymphocytic leukemia (ALL) (12 cases) and chronic lymphocytic leukemia (CLL) (6 cases) lacked DAE The two patients with DAF-negative NHL had no history of paroxysmal nocturnal hemoglobinuria (PNH), and their peripheral blood cells were DAF-positive. One DAFnegative N H L exhibited T cell markers and the other those of B cell. In both cases, treatment of the DAFnegative lymphoma cells with antibody against MCP (M177) followed by MgZ+-EGTA-serum resulted in efficient deposition of homologous C3. These results infer that some NHL specifically lack DAF and, through treatment with M177, are targeted by homologous C3.
Human nucleated blood cells and cell lines are generally resistant to homologous complement. This insusceptibility to complement has been explained in part by the presence of membrane complement regulatory proteins which protect host cells from autologous complement attack (reviewed in [1]). They mainly block activation of the C3 or C9 step. The C3 step regulators are decay-accelerating factor (DAF, CD55) [2], membrane cofactor protein (MCP, CD46) [3], and C3b/C4b receptor (CRI, CD35) [4]; the former two appear to be major factors engaged in host cell protection from homologous C3 attack [5, 6]. The C9 step regulators so far identified are p18 (CD59) [7-10] and homologous restriction factor (HRF) [11, 12]. They are found on most human cells and cell lines but not on foreign material, and this in part causes the difference of susceptibility to complement between "self" and "non-self" cells. During the course of our study, we found that, even on human cell lines, homologous C3 tends to be depositable, once DAF and MCP are functionally impaired [6]. For instance, DAF-negative tumor cell lines permitted deposition of homologous C3b/C3bi if MCP on these ceils was blocked with antibodies and the cells were then treated with human serum (source of complement). Normal DAFpositive cells are barely damaged, even by anti-MCP treatment [6]. These C3b/C3bi-bearing cells will become target for both complement immunocytolysis [5] and host effector cells with C3 receptors [13]. In this work, we examined levels of DAF and MCP
Key words: Deposition of complement C3; Decay-accelerating factor (DAF, CD55); Membrane cofactor protein (MCP, CD46); Non-Hodgkin's lymphoma Correspondence to: T. Seya, M. D., Ph.D., Department of Immunology, Center for Adult Diseases, Osaka, Higashinari-ku, Osaka 537, Japan. 0165 2478 / 91 / $ 3.50 ,77, 1991 Elsevier Science Publishers B.V.
205
Dickinson, Mountain View, CA (Leul, Leul2, Leul6, anti-CALLA, HLA-DR), Ortho Diagnostic Inc., Raritan, NJ (OKT4, OKT8, OKTll, MI3), and Coulter Immunology (MY4, MY7, MY9). All of the listed antibodies were purified materials.
on cells of hematological malignancies, and found that certain kinds of NHL lack DAE 3. Materials and Methods
3.1. Cells and antibodies 3.2. Measurement of levels of DAF and MCP Cells from patients were prepared from heparinsupplemented blood, bone marrow or lymph nodes by the Ficoll-Conray sedimentation method. If the samples contained tissue debris, they were suspended in MEMI99/5% FCS and dispersed mechanically with pipettes. The cell suspension was then poured over the Ficoll layer and centrifuged. The purity of the tumor cells was usually < 80%. The cells were suspended in RPMI/10% FCS and used within 5 h. Cells from normal donors were also prepared from blood supplemented with 10 mM of EDTA by the Polyprep method [14]. Antibodies against DAF [15] and MCP [16] were prepared as previously reported. IAI0 (IgG2a) and M177 (IgG1) were used for assessment of DAF and MCP, respectively. A monoclonal antibody to CR1, 39B, was prepared in our laboratory [16]. For blocking studies, IC6 (IgGl), which blocks DAF activity, was purchased from Wako Pure Chemicals, Japan [17]. Antibody against CD45 (IgG1) was from Coulter Immunology, Hialeah, FL. Surface markers for myeloid cells and lymphocytes were from Becton
Flow cytometry analysis was performed for assessment of surface-expressed DAF and MCP as described earlier [18]. About l x 10 6 cells were incubated with 10-25/zg of IA10 or M177. After two washes, the cells were reacted with FITC-labeled goat-anti mouse IgG (Cappel, Malvern, PA). The cells were then fixed with paraformaldebyde and analyzed within 7 days. 3.3. Morphological and histological analysis Morphological analysis was performed with phase-shift microscopy (Nikon, Japan). Surface antigens on the sample cells were analyzed for diagnosis by a combination of fluorescent staining and phase-shift fluorescent microscopy [19]. For selective visual detection of FITC fluorescence, a vertical illuminator, containing filter sets suitable to the microscope, was used. To facilitate diagnosis, we employed MayGruenwald-Giemsa stain, esterase stain, acid phos-
TABLE 1 DAF and MCP in patients with hematological malignancies. Diagnosis
Number of patients
Source of cells a
MCP
DAF
ANLL
18
PB BM
8 6
0,"14
0,.'14
CML
6
PB BM
3 3
0/6
0., 6
NHL
10
PB LN
2 8
0"10
2 l0
ALL
12
PB 10 BM 2
0./12
0/12
CLL
6
PB
0/'6
0/'6
6
a PB, peripheral blood; BM, bone marrow; LN, lymph node.
206
phatase stain, periodate-acid-Schiff (PAS) stain, TDT analysis and NAP score analysis (reviewed in [20]). Diagnosis was performed according to the classifications of MacGlave et al. [211.
TABLE 2
3.4. Assessment o f C3 deposition
MT 5.3 KF 0
Cell surface antigens on the two DAF-negative NHLs. CRI a D A F a MCPa
Ceils (5×105) were pretreated with M177 for 60 min at 37 °C. Non-immune mouse IgG and antibodies to CD45 and DAF (1C6) were used as controis. After two washes, the cells were incubated with 50/zl of Mg z*-EGTA-serum and 150 #1 of Mg 2+EGTA-GVB for 90 min at 37 °C. For controls, the same batch of cells was incubated with 50/A of EDTA-serum and 150 ttl of EDTA-GVB. C3 fragments deposited on the cell surface were analyzed with rabbit anti-human C3c (Behringer) followed by FITC-labeled goat anti-rabbit IgG (Cappel) and flow cytometry [6]. Mean fluorescence and its shift were calculated by the attached computer.
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MCP was present in all ANLL, CML, NHL, ALL and CLL cells tested (Table 1). DAF, although found on all of the tested A N L L , CML, ALL and CLL, was negative in two NHL cases (Table 1). One patient (KF) displayed T cell markers, whereas another
RBC
...........
NHL
0 85
4.1. D A F and M C P on tumor cells
e.,
'-
0 79
KF
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4. Results
KF
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a Mean fluorescence shift in flow cytometry (see Fig. 1); b % positive cells under a fluorescent phase-shift microscope.
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--MCP Fig. 1. Assessment by flow cytometry of CRI, DAF, and M C P on malignant and unaffected cells from two patients with DAF-negative NHL. N H L cells and unaffected peripheral blood cells from two N H L patients, KF and MT, were treated with antibodies against CRI (39B), DAF (IAI0) and M C P (M177), and FITC-labeled second antibody. Flow cytometric profiles of the N H L cells (NHL) and red blood cells (RBC) from the two patients are shown in the left-hand and center panels. Unaffected lymphocytes (LYM) and polymorphonuclear leukocytes (PMN) were DAF-positive (right-hand panels). Unaffected lymphocytes (LYM) from KF and polymorphonuclear leukocytes (PMN) from MT, though not shown in the figure, were also DAF- and MCP-positive.
207
(MT) displayed B cell markers (Table 2). The latter expressed CR1. They were morphologically and histologically l y m p h o m a cells. Surface markers we examined are shown in Table 2. The absence of DAF in these two N H L samples was further confirmed using the protein A-rosette assa3, which is more sensitive, being capable of detecting < 50 copies of antigen/cell (not shown). Unaffected peripheral blood cells of these two patients, MT and KF, were prepared and levels of DAF and MCP tested next. Their granulocytes and lymphocytes possessed DAF and MCP in amounts similar to those from normal donors (Fig. 1). The flow cytometry profile of the lymphocyte DAF of MT was not unimodal, displaying 3 major peaks with a broad range, which was similar to those of normal donors and consistent with a previous report [18]. Their erythrocytes also expressed DAF and CR1 within the normal range (Fig. 1), and both the sugarwater test and the H A M test were negative (data not shown). 4.2. C3 deposition test N H L cells from MT and KF were pretreated with M177, 1C6 or anti-CD45, and then incubated with Mg2+-EGTA-serum (Fig. 2). C3 was deposited efficiently on the M177-treated N H L cells, although M177-untreated cells were still resistant to C3 target-
~~ --
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.
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.
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Fluorescence Intensity
Fig. 2. C3 deposition on the DAF-negative NHL cells pretreated with M177. NHL cells from KF were pretreated with anti-DAF (IC6) (left-hand panel) or anti-MCP (M177) (right panel), and then incubated with Mg 2+-EGTA-serum ( ....... ) or EDTA-serum ( - - ) . The NHL cells untreated with antibody or treated with anti-CD45 (not shown) were also incubated with Mg 2+ EGTAserum ( . . . . ) as controls. Deposited C3 fragments were detected with rabbit anti-human C3c and FITC-labeled second antibody and analysed by flow cytometry. Similar, but less, C3 deposition was observed with M177-treated NHL ceils from MT (not shown).
208
ing. Hardly any cell lysis was observed, however, in either case (not shown). No other monoclonal antibodies, anti-DAF or anti-CD45, induced C3 deposition on these cells (Fig. 2). 5. Discussion
This communication documents that some species of N H L lack DAE P N H [22, 23] and Cromerrelated Ag deficiency [24] have been reported to be states involving the loss of DAF from the blood cell surface. Two N H L patients reported here, however, had no history of P N H or Cromer-related Ag deftciency. Furthermore, the unaffected blood cells from the patients with DAF-negative-NHL expressed normal amounts of DAF and were protected satisfactorily from C3 deposition. Hence, DAF-negative N H L should be independent of P N H or Cromerrelated Ag deficiency. Although some kinds of tumor cell line appear to be deficient in DAF [25], the presence of DAF on their progenitor cells is still unverified. On the other hand, normal blood cells are essentially DAFpositive, including NK cells, which possess only a small amount of DAF [18, 26]. Indeed, in our quantitative analysis of DAF, 2 of the 8 lymphoid cell lines lacked DAF, while no myeloid cell lines lacked it [16]. The presence of DAF on all myeloid cells and cell lines, but not on all N H L and lymphoid cell lines, may imply that DAF disappears specifically from some lymphoid cells during malignant processing. In h u m a n nucleated cells, DAF and MCP jointly protect host cells from alternative pathway-mediated C3-attack [5, 6]. CR1 serves on human erythrocytes (having no MCP) as an inhibitor of the classical pathway [27]. In most human tumor cells and cell lines, CRI becomes negative concomitantly with an increase in MCP [16]. Our findings that the tumor cells lacking DAF are still insensitive to C3, and that DAF-negative/MCP-positive cells are converted to C3-bearing targets after treatment with M177 and Mg2+-EGTA-serum, suggest that MCP is a major factor for protection against complement attack in some tumor cells. Therefore, treatment with antiMCP may be effective in the specific elimination of DAFonegative-NHL. The pathogenesis of P N H has not yet been sufficiently elucidated. P N H is an acquired disease: phosphatidyl inositol (PI)-anchored proteins, in-
c l u d i n g DAF, p18 a n d H R E d i s a p p e a r f r o m the b l o o d cell s u r f a c e in a s s o c i a t i o n w i t h cell m a t u r a t i o n [28, 29]. Loss o f DAF, as well as p18 a n d H R F , o n the cells is closely c o r r e l a t e d with disease a c t i v i t y in P N H [30]. F o r this r e a s o n , we also m e a s u r e d the p18 level o n cells f r o m 20 p a t i e n t s , i n c l u d i n g KF, by flow c y t o m e t r y , p18 was f o u n d o n b o t h a f f e c t e d a n d u n a f f e c t e d cells in K F as well as the o t h e r p a t i e n t s . T h e r e f o r e , DAF, b u t n o t o t h e r P l - l i n k e d p r o t e i n s , specifically d i s a p p e a r s s e c o n d a r y to a m a l i g n a n t t r a n s f o r m a t i o n o f the l y m p h o c y t e s . We favor the interpretation that genomic disorders yielding NHL m a y s o m e t i m e s be related to loss o f D A E while t h o s e c a u s i n g o t h e r l e u k e m i a s are not. F u r t h e r s t u d y inc l u d i n g g e n o m i c a n d m e s s a g e a n a l y s e s o f D A F will be n e e d e d as a first step to test this hypothesis.
Acknowledgements We are g r a t e f u l to Drs. T e s h i m a , T o m i n a g a , Y a m a g a m i , U e d a , H i r a o k a a n d S h i b a t a ( C e n t e r for A d u l t Diseases, O s a k a ) for p r o v i d i n g the p a t i e n t s ' s a m p l e s . We also t h a n k Dr. A k e d o ( C e n t e r for A d u l t Diseases, O s a k a ) for critical d i s c u s s i o n . T h a n k s are also d u e to Ms. Ito for excellent secretarial assistance. T h i s w o r k was s u p p o r t e d in p a r t by Kyowa H a k k o Co., a n d M o c h i d a Co., J a p a n , a n d by g r a n t s (to TS) f r o m the Cell Science Research F o u n d a t i o n a n d t h e Kowa Research F o u n d a t i o n .
References [1] Atkinson, J. P. and Farries, T. C. (1988) lmmunol. Today 8, 212. [2] Nicholson-Wetler, A., Burge, J., Fearon, D. T., Weller, P. F. and Austen, K. F. (1982) J. lmmunol. 129, 184. [3] Seya, T., Turner, J. R. and Atkinson, J. P, (1986) J. Exp. Med. 163,837. [4] Fearon, D. T. (1979) Proc. Natl. Acad. Sci. USA 76, 5867. [5] Medof, M. E., Kinoshita, T., Silber, R. and Nussenzweig, V. (1985) Proc. Natl. Acad. Sci. USA 82, 2980. [6] Seya, T., Hara, T., Matsumoto, M., Sugita, Y. and Akedo, H. (1990) J. Exp. Med. 172, 1673.
[7] Sugita, Y., Nakano, Y. and Tomita, M. (1988) J. Biochem. 104, 633. [8] Davies, A., Simmons, D. L., Hale, G., Harrison, R.A., Tighe, H., Lachmann, P. J. and Waldmann, H. (1989)J. Exp. Med. 170, 637. [91 Okada, N., Harada, R., Fujita, T. and Okada, H. (1989) Int. Immunol. l, 205. [10] Holguin, M. H., Fredrick, L. R., Bernshaw, N. J., Wilcox, L, A. and Parker, C. J. (1989) J. Clin. Invest. 84, 7. [11] Zalman, L. S., Wood, L. M., Frank, M. M. and MullerEberhard, H. J. (1987) J. Exp. Med. 165, 572. [12] H~insch, G. M., Sch6nermark, S. and Roelcke, D. (1987) J. Clin. Invest. 80, 7. [131 Ramos, O. E, Kai, C., "~'~fenof,E. and Klein, E. (19881J. lmmunol. 140, 1239. [14] Seya, T., Ballard, L., Bora, N., Kumar, V., Cui, W. and Atkinson, J. P. (1988) Eur. J. Immunol. 18, 1289. [15] Kinoshita, T., Medof, M. E., Silber, R. and Nussenzweig, V. (1985) J. Exp. Med. 162, 75. [16] Seya, T., Hara, T., Matsumoto, M. and Akedo, H. (1990) J. Immunol. 145, 238. [17] Fujita, T., Inoue, T., Ogawa, K., Iida, K. and Yamura, N. (1987) J. Exp. Med. 166, 1221. [18] Nicholson-Weller, A., Russian, D.A. and Austen, K. E (1986) J. Immunol. 137, 1275. [19] Bessis, M. (1976) Blood Smears Reinterpreted. SpringerVerlag, New York. [20] Shibata, A. (1985) Clin. Lab. Haematol. 7, 55. [21] McGlave, P. B., Johnson, G. J. and Spiers, A. S. (1988) Manual of Clinical Hematology. Mazza, J. J. ed. p. 193. [22] Pangburn, M. K., Schreiber, R. D. and Muller-Eberhard, H. J. (1983) Proc. Natl. Acad. Sci. USA 80, 5430. [231 Nicholson-Weller, A., March, J.P., Rosenfeld, S. 1. and Austen, K. E (1983) Proc. Natl. Acad. Sci. USA 80, 5066. [24] Telen, M. J., Hall, S. E., Green, A. M., Moulds, J. J. and Rosse, W. E (1988) J. Exp. Med. 167, 1993. [25] Cheung, N. V., Walter, E. I., Smith-Mensah, W. H., Ratnoff, W. D., Tykocinski, M. k. and Medof, M. E. (1988) J. Clin. Invest. 81, 1122. [261 Ueda, E., Kinoshita, T., Nojima, J., lnoue, K. and Kitani, T. (1989) J. lmmunol. 143, 1274. [27] Kinoshita, T., Medof, M. E., Hong, K. and Nussenzweig, V. (1986) J. Exp. Med. 164, 1377. [28] Ueda, E., Kinoshita, T., Terasawa, T., Shichishima, T., ~hwata, Y., Inoue, K, and Kitani, Y. (1990) Blood 75, 762. [29] Van der Schoot, C. E., Huizinga, T. W. J., Van 't VeerKorthof, E. T., Wijmans, R., Pinkster, J. and von dem Borne, A. E. G. K. (1990) Blood 76, 1853. [30] Rosse, W. F. and Parker, C. J. (1984) Clin. Hematol. 14, 105.
209
Deficiency of complement decay-accelerating factor (DAF, CD55) in non-Hodgkin's lymphoma H i r o k o F u k u d a 2, Tsukasa Seya ~, T o m o k o H a r a 1, M i s a k o M a t s u m o t o 1, Taroh K i n o s h i t a 3 a n d Toru M a s a o k a 2 IDepartment o f bnmunology and 2Department o f Hematology Center for Adult Diseases, Osaka and 3Research Institute for Microbial Diseases, Osaka University Medical School, Suita Osaka, Japan (Received 5 February 1991; revision received 13 March 1991; accepted 18 March 1991)
1. Summary
2. Introduction
We have assessed levels of surface-expressed complement regulatory proteins, decay-accelerating factor (DAF) and membrane cofactor protein (MCP) on cells from patients with hematological malignancies. Neither malignant ceils nor unaffected nucleated blood cells from the patients lacked MCP. On the other hand, complete deficiency of DAF was found in 2/10 of non-Hodgkin's lymphoma (NHL), while none of the 38 patients with acute nonlymphocytic leukemia (ANLL) (14 cases), chronic myelogenous leukemia (CML) (6 cases), acute lymphocytic leukemia (ALL) (12 cases) and chronic lymphocytic leukemia (CLL) (6 cases) lacked DAE The two patients with DAF-negative NHL had no history of paroxysmal nocturnal hemoglobinuria (PNH), and their peripheral blood cells were DAF-positive. One DAFnegative N H L exhibited T cell markers and the other those of B cell. In both cases, treatment of the DAFnegative lymphoma cells with antibody against MCP (M177) followed by MgZ+-EGTA-serum resulted in efficient deposition of homologous C3. These results infer that some NHL specifically lack DAF and, through treatment with M177, are targeted by homologous C3.
Human nucleated blood cells and cell lines are generally resistant to homologous complement. This insusceptibility to complement has been explained in part by the presence of membrane complement regulatory proteins which protect host cells from autologous complement attack (reviewed in [1]). They mainly block activation of the C3 or C9 step. The C3 step regulators are decay-accelerating factor (DAF, CD55) [2], membrane cofactor protein (MCP, CD46) [3], and C3b/C4b receptor (CRI, CD35) [4]; the former two appear to be major factors engaged in host cell protection from homologous C3 attack [5, 6]. The C9 step regulators so far identified are p18 (CD59) [7-10] and homologous restriction factor (HRF) [11, 12]. They are found on most human cells and cell lines but not on foreign material, and this in part causes the difference of susceptibility to complement between "self" and "non-self" cells. During the course of our study, we found that, even on human cell lines, homologous C3 tends to be depositable, once DAF and MCP are functionally impaired [6]. For instance, DAF-negative tumor cell lines permitted deposition of homologous C3b/C3bi if MCP on these ceils was blocked with antibodies and the cells were then treated with human serum (source of complement). Normal DAFpositive cells are barely damaged, even by anti-MCP treatment [6]. These C3b/C3bi-bearing cells will become target for both complement immunocytolysis [5] and host effector cells with C3 receptors [13]. In this work, we examined levels of DAF and MCP
Key words: Deposition of complement C3; Decay-accelerating factor (DAF, CD55); Membrane cofactor protein (MCP, CD46); Non-Hodgkin's lymphoma Correspondence to: T. Seya, M. D., Ph.D., Department of Immunology, Center for Adult Diseases, Osaka, Higashinari-ku, Osaka 537, Japan. 0165 2478 / 91 / $ 3.50 ,77, 1991 Elsevier Science Publishers B.V.
205
Dickinson, Mountain View, CA (Leul, Leul2, Leul6, anti-CALLA, HLA-DR), Ortho Diagnostic Inc., Raritan, NJ (OKT4, OKT8, OKTll, MI3), and Coulter Immunology (MY4, MY7, MY9). All of the listed antibodies were purified materials.
on cells of hematological malignancies, and found that certain kinds of NHL lack DAE 3. Materials and Methods
3.1. Cells and antibodies 3.2. Measurement of levels of DAF and MCP Cells from patients were prepared from heparinsupplemented blood, bone marrow or lymph nodes by the Ficoll-Conray sedimentation method. If the samples contained tissue debris, they were suspended in MEMI99/5% FCS and dispersed mechanically with pipettes. The cell suspension was then poured over the Ficoll layer and centrifuged. The purity of the tumor cells was usually < 80%. The cells were suspended in RPMI/10% FCS and used within 5 h. Cells from normal donors were also prepared from blood supplemented with 10 mM of EDTA by the Polyprep method [14]. Antibodies against DAF [15] and MCP [16] were prepared as previously reported. IAI0 (IgG2a) and M177 (IgG1) were used for assessment of DAF and MCP, respectively. A monoclonal antibody to CR1, 39B, was prepared in our laboratory [16]. For blocking studies, IC6 (IgGl), which blocks DAF activity, was purchased from Wako Pure Chemicals, Japan [17]. Antibody against CD45 (IgG1) was from Coulter Immunology, Hialeah, FL. Surface markers for myeloid cells and lymphocytes were from Becton
Flow cytometry analysis was performed for assessment of surface-expressed DAF and MCP as described earlier [18]. About l x 10 6 cells were incubated with 10-25/zg of IA10 or M177. After two washes, the cells were reacted with FITC-labeled goat-anti mouse IgG (Cappel, Malvern, PA). The cells were then fixed with paraformaldebyde and analyzed within 7 days. 3.3. Morphological and histological analysis Morphological analysis was performed with phase-shift microscopy (Nikon, Japan). Surface antigens on the sample cells were analyzed for diagnosis by a combination of fluorescent staining and phase-shift fluorescent microscopy [19]. For selective visual detection of FITC fluorescence, a vertical illuminator, containing filter sets suitable to the microscope, was used. To facilitate diagnosis, we employed MayGruenwald-Giemsa stain, esterase stain, acid phos-
TABLE 1 DAF and MCP in patients with hematological malignancies. Diagnosis
Number of patients
Source of cells a
MCP
DAF
ANLL
18
PB BM
8 6
0,"14
0,.'14
CML
6
PB BM
3 3
0/6
0., 6
NHL
10
PB LN
2 8
0"10
2 l0
ALL
12
PB 10 BM 2
0./12
0/12
CLL
6
PB
0/'6
0/'6
6
a PB, peripheral blood; BM, bone marrow; LN, lymph node.
206
phatase stain, periodate-acid-Schiff (PAS) stain, TDT analysis and NAP score analysis (reviewed in [20]). Diagnosis was performed according to the classifications of MacGlave et al. [211.
TABLE 2
3.4. Assessment o f C3 deposition
MT 5.3 KF 0
Cell surface antigens on the two DAF-negative NHLs. CRI a D A F a MCPa
Ceils (5×105) were pretreated with M177 for 60 min at 37 °C. Non-immune mouse IgG and antibodies to CD45 and DAF (1C6) were used as controis. After two washes, the cells were incubated with 50/zl of Mg z*-EGTA-serum and 150 #1 of Mg 2+EGTA-GVB for 90 min at 37 °C. For controls, the same batch of cells was incubated with 50/A of EDTA-serum and 150 ttl of EDTA-GVB. C3 fragments deposited on the cell surface were analyzed with rabbit anti-human C3c (Behringer) followed by FITC-labeled goat anti-rabbit IgG (Cappel) and flow cytometry [6]. Mean fluorescence and its shift were calculated by the attached computer.
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MCP was present in all ANLL, CML, NHL, ALL and CLL cells tested (Table 1). DAF, although found on all of the tested A N L L , CML, ALL and CLL, was negative in two NHL cases (Table 1). One patient (KF) displayed T cell markers, whereas another
RBC
...........
NHL
0 85
4.1. D A F and M C P on tumor cells
e.,
'-
0 79
KF
I
:1 .1
0 96
4. Results
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--MCP Fig. 1. Assessment by flow cytometry of CRI, DAF, and M C P on malignant and unaffected cells from two patients with DAF-negative NHL. N H L cells and unaffected peripheral blood cells from two N H L patients, KF and MT, were treated with antibodies against CRI (39B), DAF (IAI0) and M C P (M177), and FITC-labeled second antibody. Flow cytometric profiles of the N H L cells (NHL) and red blood cells (RBC) from the two patients are shown in the left-hand and center panels. Unaffected lymphocytes (LYM) and polymorphonuclear leukocytes (PMN) were DAF-positive (right-hand panels). Unaffected lymphocytes (LYM) from KF and polymorphonuclear leukocytes (PMN) from MT, though not shown in the figure, were also DAF- and MCP-positive.
207
(MT) displayed B cell markers (Table 2). The latter expressed CR1. They were morphologically and histologically l y m p h o m a cells. Surface markers we examined are shown in Table 2. The absence of DAF in these two N H L samples was further confirmed using the protein A-rosette assa3, which is more sensitive, being capable of detecting < 50 copies of antigen/cell (not shown). Unaffected peripheral blood cells of these two patients, MT and KF, were prepared and levels of DAF and MCP tested next. Their granulocytes and lymphocytes possessed DAF and MCP in amounts similar to those from normal donors (Fig. 1). The flow cytometry profile of the lymphocyte DAF of MT was not unimodal, displaying 3 major peaks with a broad range, which was similar to those of normal donors and consistent with a previous report [18]. Their erythrocytes also expressed DAF and CR1 within the normal range (Fig. 1), and both the sugarwater test and the H A M test were negative (data not shown). 4.2. C3 deposition test N H L cells from MT and KF were pretreated with M177, 1C6 or anti-CD45, and then incubated with Mg2+-EGTA-serum (Fig. 2). C3 was deposited efficiently on the M177-treated N H L cells, although M177-untreated cells were still resistant to C3 target-
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Fig. 2. C3 deposition on the DAF-negative NHL cells pretreated with M177. NHL cells from KF were pretreated with anti-DAF (IC6) (left-hand panel) or anti-MCP (M177) (right panel), and then incubated with Mg 2+-EGTA-serum ( ....... ) or EDTA-serum ( - - ) . The NHL cells untreated with antibody or treated with anti-CD45 (not shown) were also incubated with Mg 2+ EGTAserum ( . . . . ) as controls. Deposited C3 fragments were detected with rabbit anti-human C3c and FITC-labeled second antibody and analysed by flow cytometry. Similar, but less, C3 deposition was observed with M177-treated NHL ceils from MT (not shown).
208
ing. Hardly any cell lysis was observed, however, in either case (not shown). No other monoclonal antibodies, anti-DAF or anti-CD45, induced C3 deposition on these cells (Fig. 2). 5. Discussion
This communication documents that some species of N H L lack DAE P N H [22, 23] and Cromerrelated Ag deficiency [24] have been reported to be states involving the loss of DAF from the blood cell surface. Two N H L patients reported here, however, had no history of P N H or Cromer-related Ag deftciency. Furthermore, the unaffected blood cells from the patients with DAF-negative-NHL expressed normal amounts of DAF and were protected satisfactorily from C3 deposition. Hence, DAF-negative N H L should be independent of P N H or Cromerrelated Ag deficiency. Although some kinds of tumor cell line appear to be deficient in DAF [25], the presence of DAF on their progenitor cells is still unverified. On the other hand, normal blood cells are essentially DAFpositive, including NK cells, which possess only a small amount of DAF [18, 26]. Indeed, in our quantitative analysis of DAF, 2 of the 8 lymphoid cell lines lacked DAF, while no myeloid cell lines lacked it [16]. The presence of DAF on all myeloid cells and cell lines, but not on all N H L and lymphoid cell lines, may imply that DAF disappears specifically from some lymphoid cells during malignant processing. In h u m a n nucleated cells, DAF and MCP jointly protect host cells from alternative pathway-mediated C3-attack [5, 6]. CR1 serves on human erythrocytes (having no MCP) as an inhibitor of the classical pathway [27]. In most human tumor cells and cell lines, CRI becomes negative concomitantly with an increase in MCP [16]. Our findings that the tumor cells lacking DAF are still insensitive to C3, and that DAF-negative/MCP-positive cells are converted to C3-bearing targets after treatment with M177 and Mg2+-EGTA-serum, suggest that MCP is a major factor for protection against complement attack in some tumor cells. Therefore, treatment with antiMCP may be effective in the specific elimination of DAFonegative-NHL. The pathogenesis of P N H has not yet been sufficiently elucidated. P N H is an acquired disease: phosphatidyl inositol (PI)-anchored proteins, in-
c l u d i n g DAF, p18 a n d H R E d i s a p p e a r f r o m the b l o o d cell s u r f a c e in a s s o c i a t i o n w i t h cell m a t u r a t i o n [28, 29]. Loss o f DAF, as well as p18 a n d H R F , o n the cells is closely c o r r e l a t e d with disease a c t i v i t y in P N H [30]. F o r this r e a s o n , we also m e a s u r e d the p18 level o n cells f r o m 20 p a t i e n t s , i n c l u d i n g KF, by flow c y t o m e t r y , p18 was f o u n d o n b o t h a f f e c t e d a n d u n a f f e c t e d cells in K F as well as the o t h e r p a t i e n t s . T h e r e f o r e , DAF, b u t n o t o t h e r P l - l i n k e d p r o t e i n s , specifically d i s a p p e a r s s e c o n d a r y to a m a l i g n a n t t r a n s f o r m a t i o n o f the l y m p h o c y t e s . We favor the interpretation that genomic disorders yielding NHL m a y s o m e t i m e s be related to loss o f D A E while t h o s e c a u s i n g o t h e r l e u k e m i a s are not. F u r t h e r s t u d y inc l u d i n g g e n o m i c a n d m e s s a g e a n a l y s e s o f D A F will be n e e d e d as a first step to test this hypothesis.
Acknowledgements We are g r a t e f u l to Drs. T e s h i m a , T o m i n a g a , Y a m a g a m i , U e d a , H i r a o k a a n d S h i b a t a ( C e n t e r for A d u l t Diseases, O s a k a ) for p r o v i d i n g the p a t i e n t s ' s a m p l e s . We also t h a n k Dr. A k e d o ( C e n t e r for A d u l t Diseases, O s a k a ) for critical d i s c u s s i o n . T h a n k s are also d u e to Ms. Ito for excellent secretarial assistance. T h i s w o r k was s u p p o r t e d in p a r t by Kyowa H a k k o Co., a n d M o c h i d a Co., J a p a n , a n d by g r a n t s (to TS) f r o m the Cell Science Research F o u n d a t i o n a n d t h e Kowa Research F o u n d a t i o n .
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