Clin. exp. Immunol. (1975) 19, 347-354.
AN UNUSUAL MOUSE MYELOMA PROTEIN BINDING NATIVE DNA J. A. DIXON, S. SUGAI AND N. TALAL* Clinical Immunology Section, Veterans Administration Hospital, and Department of Medicine, University of California, San Francisco, California, U.S.A. (Received 16 July 1974)
SUMMARY
SP 104 is an IgA-K myeloma protein produced by a lymphoid tumour of CA F1 mice. It arose originally in mice injected intraperitoneally with cell-free extract of spleen from a dog with systemic lupus erythematosus. The monoclonal nature of the IgA was shown by characteristic appearance on immunoelectrophoresis, restriction to a single light chain type and ability to induce anti-idiotypic antiserum. This protein has antibody activity against native (double-stranded) DNA and its specificity is similar to the antibodies against DNA found in the sera of humans with SLE and NZB/NZW F1 mice. Its idiotype does not cross-react with idiotypes of other mouse myeloma proteins known to bind DNA.
INTRODUCTION Antibodies directed against nucleic acids are found in the sera of humans with systemic lupus erythematosus (SLE) as well as in older New Zealand Black/White (NZB/NZW F1) hybrid mice which manifest a lupus-like syndrome (Talal, Steinberg & Daley, 1971; Talal, 1973; Attias, Sylvester & Talal, 1973; Talal & Gallo, 1972; Steinberg, Pincus & Talal, 1969). These antibodies bind double-stranded DNA (ds-DNA), double-stranded RNA (ds-RNA) and single-stranded DNA (ss-DNA). The antigens inducing the formation of these antibodies are unknown, although inhibition studies suggest that the binding of RNA is most specific for naturally occurring ds-RNA (Talal et al., 1971; Talal, 1973; Attias et al., 1973). Functional studies of mineral oil-induced myelomas in BALB/C mice have established that a significant number of the monoclonal immunoglobulins produced by these tumours precipitate with nucleic acid bases and/or nitrophenyl derivatives (Schubert, Jobe & Cohn, 1968). Three of these proteins (S23, S176, J504) were studied in detail and shown to bind ds-DNA (Schubert, Roman & Cohn, 1970). Two of them (S23 and S176) showed greater *
Present address and correspondence: Dr Norman Talal, Clinical Immunology Section (151T), Veterans Administration Hospital, 4150 Clement Street, San Francisco, California 94121, U.S.A.
347
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J. A. Dixon, S. Sugai and N. Talal
specificity for phage RNA and ss-DNA than for native ds-DNA. The other (J504) was equally specific for phage-RNA, ds-DNA and ss-DNA. Schwartz and his co-workers have studied a colony of dogs with a clinical syndrome closely resembling human SLE (Lewis, Schwartz & Henry, 1965; Lewis et al., 1963). Lewis et al. found that normal CA F1 mice injected with cell-free filtrates from spleens of SLE colony dogs developed anti-nuclear antibodies and antibodies to ds-DNA (Lewis et al., 1973). These serological abnormalities could be transmitted to healthy syngeneic mice by intraperitoneal injections of cell-free filtrates of spleens from affected mice. Moreover, a small percentage (5.90%) of the recipient mice were found at autopsy to have lymphoid tumours containing numerous A and C-type virus particles. One tumour-bearing animal was found to have a monoclonal protein spike on serum protein electrophoresis. This tumour, SP104, was easily maintained by serial passage in normal CA F1 mice and was kindly provided to us by Drs Schwartz and Lewis. Preliminary studies performed in our laboratory indicated that the monoclonal protein was an IgA-K type that had antibody activity against ds-DNA and polydeoxythymidylicpolyriboadenylic acid (dT rA), a synthetic DNA-RNA hybrid (Talal & Gallo, 1972). These findings led to a more detailed investigation of the nucleic acid binding and idiotypic properties of this protein which we now report. MATERIALS AND METHODS Transplantation of tumours Tumour SP104 is maintained in the solid form by serial subcutaneous passage into 6-10 week-old female CA F1 mice (Jackson Laboratory, Bar Harbor, Maine). The injection of 5-10 x 106 tumour cells results in a palpable tumour in 4-5 days and death of the animal in 2 weeks. S194A, an IgA-K type BALB/C plasmacytoma was obtained from the Cell Distribution Center, Salk Institute, La Jolla, California. The myeloma protein produced by this tumour has been shown to bind the haptens DNP, TNP, 5-acetyluracil and purine (Schubert et al., 1968). The tumour was maintained by serial intraperitoneal passage in 6-10-week-old female BALB/C mice (Simonsen Laboratory, San Jose, California). Serum proteins
Electrophoresis and immunoelectrophoresis were performed in 0.500 agarose gel buffered with 0 05 veronal (pH 8.6). A constant current of 15 mA per 1 cm of agarose gel was maintained for 45 minutes. Immunoglobulins were precipitated with monospecific goat antisera that were furnished by Dr Richard Asofsky. The following antisera were used: anti-IgM (G135A), anti-IgA (G134A), anti-IgGyl (G155), anti-IgGy2, and anti-) (135A2A). A rabbit anti-K antiserum was furnished by Dr Herbert Morse. Proteins and immunoprecipitin lines were stained with Amido Black 10B. Double diffusion (Ouchterlony) studies were also performed in 0.500 agarose buffered with 0-05 veronal (pH 8-6). Isolation of IgA Serum was used as a source of SP104 and S194A monoclonal protein. The proteins were isolated from serum by precipitation with 5000 saturated ammonium sulphate at 4°C followed by Sephadex G-200 gel filtration in borate-buffered saline (1 M, pH 8 0). The isolated proteins were concentrated and dialysed against borate-buffered saline (0 15 M,
Mouse myeloma protein binding native DNA
349
pH 8.0). They were then checked for purity and immunoglobulin content by double immunodiffusion and immunoelectrophoresis using antisera to normal mouse serum, to specific mouse immunoglobulins and to K and i light chains. The purified immunoglobulins contained only IgA and precipitated with anti-K but not anti-A serum. Preparation of anti-idiotypic antisera The purified SPI04 IgA isolated from the pooled sera of tumour-bearing animals was emulsified in Freund's complete adjuvant and used to immunize rabbits. Each rabbit received three 1-ml injections (containing 1 mg of protein) every 2 weeks. The rabbits were bled 2 weeks after the third injection. The rabbit antiserum was absorbed with normal CA F1 serum and purified BALB/c S194A IgA-c protein.
Nucleic acid binding The binding of nucleic acids was studied using a cellulose ester filter radioimmunoassay (Talal, 1973; Talal & Gallo, 1972). When purified SP104 protein was tested, various concentrations in 100 pu were incubated at 560C for 30 min. When whole serum was tested, 10 pl were diluted with 85 pI of borate-buffered saline (0.15 M, pH 8.0) and incubated at 560C for 30 min. Fixed quantities of radiolabelled nucleic acid were then added and the solution incubated for 30 minutes at 370C and then at 40C for 30 min. The radioactive antigenantibody complexes were collected on cellulose ester filters (Millipore Corporation, Bedford, Massachusetts) and assayed for radioactivity in a liquid scintillation counter. The radiolabelled nucleic acids and their specific activities in 5 pl are as follows: [3H]DNA (KB human cell line) (Electronucleonics, Rockville, Maryland) 500 ct/min/0-20 mpg; [14C]DNA (E. coli) (Amersham/Searle) 500 ct/min/I 67 mug; ["4C]poly I -poly C (Miles Laboratories, Elkhart, Indiana) 500 ct/min/1 2 5 mpg; [3H]poly dT * poly rA (kindly supplied by Dr Robert Gallo, NIH, Bethesda, Maryland) 390 ct/min/63 mpg; and [3H]poly As poly U (Miles Laboratories, Elkhart, Indiana) 400 ct/min/0-42 mpg. Inhibition of DNA and dT rA binding was tested as previously described (Talal & Gallo, 1972). The myeloma protein or serum was incubated with 5 pl ofinhibitor (containing 4 or 5 pg) at 370C for 30 min before the addition of the labelled nucleic acid. The following inhibitors were studied: reovirus RNA, phage RNA, poly I -poly C (Miles Laboratories, Elkhart, Indiana), dT~rA, poly A~poly U (Miles Laboratories), ss-DNA (calf thymus) (Worthington, Freehold, New Jersey) ds-DNA (calf thymus-Worthington, New Jersey). ss-DNA was made by heating ds-DNA for 10 minutes at 1000C followed by rapid chilling in an ice bath. All inhibitors were dissolved in borate-buffered saline (0 15 M, pH 8 0). RESULTS Serum electrophoresis The sera of SP104 tumour-bearing mice were studied by agarose gel electrophoresis. The fl-globulin fraction was increased probably due to the presence of the monoclonal protein migrating in the f-globulin region (Fig. 1).
Immunoelectrophoresis Sera were further studied by immunoelectrophoresis using specific anti-mouse immunoglobulin antisera. Precipitation with a monospecific antiserum to mouse IgA revealed a
350
J. A. Dixon, S. Sugai and N. Talal Alb a
Bly
Normal CA F1 serum
SF
104
SF
104
Anti-A
Anti-K
Normal
CA F1 serum
Anti-IgA SP 104
FIG. 1. Agarose gel electrophoresis and immunoelectrophoresis of sera from a normal CA F1 and a CA F1 mouse bearing the SPl04 tumour. The electrophoresis shows an increased fl-globulin fraction. The immunoelectrophoresis was developed with monospecific antisera to mouse IgA and light chains and shows the presence of an IgA-K myeloma protein.
mouse
400
350 -0
i 300
/
E 250 I 200 150
-
_E U
100Q 50-
007
035
0'7
35
Protein concentration (mg/ml)
FIG. 2. Binding of various nucleic acids by CA F1 IgA-K as a function of concentration of the SPI04 IgA myeloma protein. (0) ds-DNA. (o) Poly dt poly rAo(o) Poly rA- poly rU. (-) Poly rI -poly rC.
Mouse myeloma protein binding native DNA
351
heavy dense precipitin line in the /3-y region with prominent bowing, a typical feature of monoclonal immunoglobulins (Fig. 1). This line was also reproduced when anti-K antiserum was used. There was no reaction with anti-2-antiserum. The SP104 protein was thus identified as an IgA-K myeloma protein. It showed some splitting into two lines anodally.
Sephadex gelfiltration On Sephadex G-200 gel filtration, the bulk of the SPI04 IgA protein eluted in the first peak indicative of some high molecular weight aggregation. This material was then used for studies of nucleic acid binding. Nucleic acid binding The purified SPI04 protein bound significant amounts of ds-DNA (Fig. 2). The radioactivity retained on the filter was proportional to the concentration of the protein. Maximum retention of radioactivity on the filter occurred at a concentration of 0 7 mg/ml. No decline in binding was observed at higher concentrations as has sometimes been noted in studies of human SLE sera (Attias et al., 1973; Kredich, Skyler & Fotte, 1973). Maximum binding by the protein represented 7000 of the total radiolabelled antigen added. TABLE 1. Inhibition of DNA binding by SP104, IgA-K myeloma proteins Percentage inhibition of ['4C1DNA binding by:* ds-DNA
ss-DNA
Reovirus RNA
76+10t
45+4
15±5
Phage Poly (rA) Poly (rI)- Poly (dt) RNA poly (rU) poly (rC) poly (rA) 7+7
7+4
4±4
1±1
* Average of five experiments. t Mean ± standard error.
Increasing amounts of dT rA were bound with increasing concentrations of the protein (Fig. 2). However, the maximum binding observed represented only 210% of the total antigen added. Two different synthetic ds-RNAs, poly I poly C and poly A * poly U, were not bound by the protein. Purified S194A protein did not bind significant amounts of any nucleic acid tested. -
Inhibition of DNA binding Antibodies to ds-DNA appear spontaneously in humans with SLE, dogs with SLE and NZB/NZW mice. Because of the unusual derivation of the SP104 tumour from cell-free filtrates of spleens from SLE colony dogs, we studied the specificity of the SPI04 protein in an inhibition assay (Table 1). The binding of ds-DNA was inhibited 76% by calf thymus ds-DNA, 4500 by ss-DNA and 15% by reovirus ds-RNA (Table 1). The other nucleic acids did not give significant inhibition. Binding of dT rA was inhibited by dT rA (500%) and poly A -poly U (50%). This specificity was compared with the specificities of the anti-DNA antibodies found in the sera of human SLE patients and NZB/NZW mice (Table 2). There was a marked similarity in specificity. Ds-DNA inhibited the binding of ['4C]DNA by human SLE sera by
J. A. Dixon, S. Sugai and N. Talal
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TABLE 2. Inhibition of DNA binding in systemic lupus erythematosus and NZB/NZW mice
Percentage inhibition of [14C]DNA binding by:
Binding
[14C]DNA Patient C.D. N.C. L.T. N.L. Mean+s.e. NZB/NZW 112-3A 146-3A 131 142-3A 78-3A
Mean+s.e.
(ct/min)
ds-DNA
ss-DNA
Reovirus RNA
Phage RNA
414 386 386 426
64 38 80 43 56± 10
48 13 96 34 48+ 18
11 0 47 8 16+10
1 0 27 4 8+6
63 96 92 95 96 88+6
93 97 96 98 96 96±1
24 37 35 18 27
24 18 14 10 13 16+2
239 328 296 330 232
28±3
Poly (rA) Poly (rI)- Poly (dt) poly(rU) poly (rC) poly (rA)
4±3
0 0 4 0 1+1
6±4
3 5 4 10 1 5+2
0 4 4 1 1 2+1
0 18 14 14 5 10±3
2 0 12 0
5 0 18 0
56%. ss-DNA inhibited binding by 48% and reovirus inhibited it by 16%. In NZB/NZW mice the patterns of inhibition were similar. However, both ds-DNA and ss-DNA inhibited ['4C]DNA binding to a much greater extent than in either humans or in the case of SPl04 protein. Idiotype characteristics The purified SPl04 protein was used to immunize rabbits in order to prepare anti-idiotypic antiserum. After absorption of the antiserum, typical idiotypic reactivity was observed
J604 tis SP104
FIG. 3. Double diffusion in agarose gel with absorbed antiserum against the SP104 protein (A). Outer wells contain sera from five different BALB/C plasmacytomas and purified SP104 protein.
Mouse myeloma protein binding native DNA
353
when tested against a panel of fifteen sera from BALB/C IgA myelomas (kindly supplied by Dr Roy Riblett) and the SP104 IgA. The anti-idiotypic antiserum reacted strongly with the SP104 IgA but not with S194A or with any other IgA myeloma tested (Fig. 3). It is of interest that two of the BALB/C proteins tested (S23 and S176) which did not cross-react with the SP104 idiotype have previously been shown to bind ds-DNA (Schubert et al., 1970). DISCUSSION SP104 is a tumour that arose in a CA F1 mouse which was injected intraperitoneally with a cell-free filtrate of dog spleen. The dog was bred from a colony of dogs with systemic lupus erythematosus (Lewis et al., 1965, 1973). Histologically, SP104 is composed of a mixture of poorly differentiated cells consisting of plasmablasts and reticulum cells. It is similar to oil-induced plasmacytomas in BALB/C mice in its diffuse mesenteric origin and in its content of A and C-type virus particles (Potter, 1972). It is also similar in producing an IgA myeloma protein, since 60 65% of BALB/C plasmacytomas secrete IgA (Potter, 1972). The purified SP104 IgA-K protein has unusual antibody activity. It binds ds-DNA and small amounts of dT * rA but has no activity against two different synthetic double-stranded RNA preparations. The inhibition of DNA binding by ds-DNA is much greater than by ss-DNA. A small amount of inhibition was observed with reovirus ds-RNA. The binding of ds-DNA is not unique to this monoclonal protein. Three other BALB/C IgA myeloma proteins bind ds-DNA (Schubert et al., 1970). None of these, however, showed specificities of binding similar to SP104. Two (S23 and S176) showed greater specificity for ss-DNA and phage RNA. The other (J504) was equally specific for phage RNA, ss-DNA and ds-DNA. Moreover, neither S23 nor S176 share a common idiotype with SP104. Sera from humans with SLE and NZB/NZW mice also contain antibodies to ds-DNA (Steinberg et al., 1969). In contrast to the BALB/C myelomas, these antibodies have the same specificity for ds-DNA as the SP104 protein. Their binding of radioactive ds-DNA is inhibited to a greater extent by ds-DNA than by ss-DNA. Binding is inhibited to a small extent by ds-RNA. These findings suggest but do not prove that the naturally occurring nucleic acid immunogen against which these antibodies are formed may be similar in the case of the SP104 protein, humans with SLE and NZB/NZW mice, and different in BALB/C oil-induced plasmacytomas. However, neither the source nor the nature of this immunogen is known, nor are the mechanisms regulating the immune response to nucleic acids. In NZB/NZW mouse disease, viruses may play a significant pathogenetic role in addition to genetic and immunological factors (Talal, 1970; Levy, 1973; Lerner et al., 1972). The viruses may act as a potent source of nucleic acid antigen or may, through their cytotoxic effects, be responsible for altering the immunogenicity of normal host DNA. The work of Lewis et al. (1973) suggests, but does not prove, the role of virus in the transmission of anti-DNA antibodies and lymphoid tumours from SLE colony dogs to normal CA F1 mice (Lewis et al., 1973). It is postulated that antigens present during the induction of oil-induced plasmacytomas in BALB/C mice may exert a selective effect upon the antibody activity of the monoclonal proteins that are eventually produced (Potter, 1972). Perhaps the same nucleic acid antigen (? viral) responsible for initiating the anti-DNA autoimmune response in animals that do not develop tumours could also exert a selective
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effect which is responsible for the specificity of the SP104 protein, a myeloma protein highly specific for ds-DNA. ACKNOWLEDG MENTS
We wish to acknowledge the excellent secretarial assistance of Mrs Nina Croomes and the technical assistance of Mr Edgar Adjahoe. The research was supported in part by grant number AM 16140 from the U.S. Public Health Service and in part by the Veterans Administration. We appreciate the generosity of Drs Robert Schwartz and Robert Lewis in providing us with the SP104 tumour. REFERENCES ATTIAS, M.R., SYLVESTER, R.A. & TALAL, N. (1973) Filter radioimmunoassay for antibodies to reovirus RNA in systemic lupus erythematosus. Arthr. and Rheum. 16, 719. KREDICH, N.M., SKYLER, J.S. & FOTTE, L.J. (1973) Antibodies to native DNA in systemic lupus erythematosus: a technique for rapid and quantitative determination. Arch. intern. Med. 131, 639. LERNER, R.A., JENSEN, F., KESSEL, S.J., DIXON, F.J., DES ROCHES, G. & FRANKE, U. (1972) Karotype, virologic and immunologic analysis of two continuous lymphocyte lines established from New Zealand black mice: possible relationship of chromosomal mosaicism to autoimmunity. Proc. nat. Acad. Sci. (Wash.), 69, 2965. LEVY, J.A. (1973) Zenotropic viruses: murine leukemia viruses associated with NIH Swiss, NZB, and other mouse strains. Science, 182, 1151. LEWIS, R.M., ANDRE-SCHWARTZ, J., HARRIS, G.S., HIRSCH, M.S., BLACK, P.H. & SCHWARTZ, R.S. (1973) Canine systemic lupus erythematosus: transmission of serologic abnormalities by cell-free filtrates. J. clin. Invest. 52, 1893. LEWIS, R.M., HENRY, W.B., JR, THORNTON, G.W. & GILMORE, C.E. (1963) A syndrome of autoimmune hemolytic anemia and thrombocytopenia in the dog. Sci. Proc. J. Amer. vet. Med. Assoc. supplement 1, 140. LEWIS, R.M., SCHWARTZ, R.S. & HENRY, W.B., JR (1965) Canine systemic lupus erythematosus. Blood, 25, 143. POTTER, M. (1972) Immunoglobulin-producing tumors and myeloma proteins of mice. Physiol. Rev. 52, 631. SCHUBERT, D., JOBE, A. & COHN, M. (1968) Mouse myelomas producing precipitating antibody to nucleic acid bases and/or nitrophenyl derivatives. Nature (Lond.), 220, 882. SCHUBERT, D., ROMAN, A. & COHN, M. (1970) Anti-nucleic specificities of mouse myeloma immunoglobulins. Nature (Lond.), 225, 154. STEINBERG, A.D., PINCUS, T. & TALAL, N. (1969) DNA-binding assay for detection of anti-DNA antibodies in NZB/NZW F1 mice. J. Immunol. 102, 788. TALAL, N. (1970) Immunologic and viral factors in the pathogenesis of systemic lupus erythematosus. Arthr. and Rheum. 13, 887. TALAL, N. (1973) Antibodies binding 3H-reovirus RNA in systemic lupus erythematosus. Clin. Immunol. Immunopathol. 1, 230. TALAL, N. & GALLO, R.C. (1972) Antibodies to a DNA-RNA hybrid in systemic lupus erythematosus measured by a cellulose ester filter radioimmunoassay. Nature: New Biology, 240, 240. TALAL, M., STEINBERG, A.D. & DALEY, G. (1971) Inhibition of antibodies binding polyinosinic-polycytidylic acid in human and mouse lupus sera by viral and synthetic ribonucleic acids. J. clin. Invest. 50, 1248.
AN UNUSUAL MOUSE MYELOMA PROTEIN BINDING NATIVE DNA J. A. DIXON, S. SUGAI AND N. TALAL* Clinical Immunology Section, Veterans Administration Hospital, and Department of Medicine, University of California, San Francisco, California, U.S.A. (Received 16 July 1974)
SUMMARY
SP 104 is an IgA-K myeloma protein produced by a lymphoid tumour of CA F1 mice. It arose originally in mice injected intraperitoneally with cell-free extract of spleen from a dog with systemic lupus erythematosus. The monoclonal nature of the IgA was shown by characteristic appearance on immunoelectrophoresis, restriction to a single light chain type and ability to induce anti-idiotypic antiserum. This protein has antibody activity against native (double-stranded) DNA and its specificity is similar to the antibodies against DNA found in the sera of humans with SLE and NZB/NZW F1 mice. Its idiotype does not cross-react with idiotypes of other mouse myeloma proteins known to bind DNA.
INTRODUCTION Antibodies directed against nucleic acids are found in the sera of humans with systemic lupus erythematosus (SLE) as well as in older New Zealand Black/White (NZB/NZW F1) hybrid mice which manifest a lupus-like syndrome (Talal, Steinberg & Daley, 1971; Talal, 1973; Attias, Sylvester & Talal, 1973; Talal & Gallo, 1972; Steinberg, Pincus & Talal, 1969). These antibodies bind double-stranded DNA (ds-DNA), double-stranded RNA (ds-RNA) and single-stranded DNA (ss-DNA). The antigens inducing the formation of these antibodies are unknown, although inhibition studies suggest that the binding of RNA is most specific for naturally occurring ds-RNA (Talal et al., 1971; Talal, 1973; Attias et al., 1973). Functional studies of mineral oil-induced myelomas in BALB/C mice have established that a significant number of the monoclonal immunoglobulins produced by these tumours precipitate with nucleic acid bases and/or nitrophenyl derivatives (Schubert, Jobe & Cohn, 1968). Three of these proteins (S23, S176, J504) were studied in detail and shown to bind ds-DNA (Schubert, Roman & Cohn, 1970). Two of them (S23 and S176) showed greater *
Present address and correspondence: Dr Norman Talal, Clinical Immunology Section (151T), Veterans Administration Hospital, 4150 Clement Street, San Francisco, California 94121, U.S.A.
347
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J. A. Dixon, S. Sugai and N. Talal
specificity for phage RNA and ss-DNA than for native ds-DNA. The other (J504) was equally specific for phage-RNA, ds-DNA and ss-DNA. Schwartz and his co-workers have studied a colony of dogs with a clinical syndrome closely resembling human SLE (Lewis, Schwartz & Henry, 1965; Lewis et al., 1963). Lewis et al. found that normal CA F1 mice injected with cell-free filtrates from spleens of SLE colony dogs developed anti-nuclear antibodies and antibodies to ds-DNA (Lewis et al., 1973). These serological abnormalities could be transmitted to healthy syngeneic mice by intraperitoneal injections of cell-free filtrates of spleens from affected mice. Moreover, a small percentage (5.90%) of the recipient mice were found at autopsy to have lymphoid tumours containing numerous A and C-type virus particles. One tumour-bearing animal was found to have a monoclonal protein spike on serum protein electrophoresis. This tumour, SP104, was easily maintained by serial passage in normal CA F1 mice and was kindly provided to us by Drs Schwartz and Lewis. Preliminary studies performed in our laboratory indicated that the monoclonal protein was an IgA-K type that had antibody activity against ds-DNA and polydeoxythymidylicpolyriboadenylic acid (dT rA), a synthetic DNA-RNA hybrid (Talal & Gallo, 1972). These findings led to a more detailed investigation of the nucleic acid binding and idiotypic properties of this protein which we now report. MATERIALS AND METHODS Transplantation of tumours Tumour SP104 is maintained in the solid form by serial subcutaneous passage into 6-10 week-old female CA F1 mice (Jackson Laboratory, Bar Harbor, Maine). The injection of 5-10 x 106 tumour cells results in a palpable tumour in 4-5 days and death of the animal in 2 weeks. S194A, an IgA-K type BALB/C plasmacytoma was obtained from the Cell Distribution Center, Salk Institute, La Jolla, California. The myeloma protein produced by this tumour has been shown to bind the haptens DNP, TNP, 5-acetyluracil and purine (Schubert et al., 1968). The tumour was maintained by serial intraperitoneal passage in 6-10-week-old female BALB/C mice (Simonsen Laboratory, San Jose, California). Serum proteins
Electrophoresis and immunoelectrophoresis were performed in 0.500 agarose gel buffered with 0 05 veronal (pH 8.6). A constant current of 15 mA per 1 cm of agarose gel was maintained for 45 minutes. Immunoglobulins were precipitated with monospecific goat antisera that were furnished by Dr Richard Asofsky. The following antisera were used: anti-IgM (G135A), anti-IgA (G134A), anti-IgGyl (G155), anti-IgGy2, and anti-) (135A2A). A rabbit anti-K antiserum was furnished by Dr Herbert Morse. Proteins and immunoprecipitin lines were stained with Amido Black 10B. Double diffusion (Ouchterlony) studies were also performed in 0.500 agarose buffered with 0-05 veronal (pH 8-6). Isolation of IgA Serum was used as a source of SP104 and S194A monoclonal protein. The proteins were isolated from serum by precipitation with 5000 saturated ammonium sulphate at 4°C followed by Sephadex G-200 gel filtration in borate-buffered saline (1 M, pH 8 0). The isolated proteins were concentrated and dialysed against borate-buffered saline (0 15 M,
Mouse myeloma protein binding native DNA
349
pH 8.0). They were then checked for purity and immunoglobulin content by double immunodiffusion and immunoelectrophoresis using antisera to normal mouse serum, to specific mouse immunoglobulins and to K and i light chains. The purified immunoglobulins contained only IgA and precipitated with anti-K but not anti-A serum. Preparation of anti-idiotypic antisera The purified SPI04 IgA isolated from the pooled sera of tumour-bearing animals was emulsified in Freund's complete adjuvant and used to immunize rabbits. Each rabbit received three 1-ml injections (containing 1 mg of protein) every 2 weeks. The rabbits were bled 2 weeks after the third injection. The rabbit antiserum was absorbed with normal CA F1 serum and purified BALB/c S194A IgA-c protein.
Nucleic acid binding The binding of nucleic acids was studied using a cellulose ester filter radioimmunoassay (Talal, 1973; Talal & Gallo, 1972). When purified SP104 protein was tested, various concentrations in 100 pu were incubated at 560C for 30 min. When whole serum was tested, 10 pl were diluted with 85 pI of borate-buffered saline (0.15 M, pH 8.0) and incubated at 560C for 30 min. Fixed quantities of radiolabelled nucleic acid were then added and the solution incubated for 30 minutes at 370C and then at 40C for 30 min. The radioactive antigenantibody complexes were collected on cellulose ester filters (Millipore Corporation, Bedford, Massachusetts) and assayed for radioactivity in a liquid scintillation counter. The radiolabelled nucleic acids and their specific activities in 5 pl are as follows: [3H]DNA (KB human cell line) (Electronucleonics, Rockville, Maryland) 500 ct/min/0-20 mpg; [14C]DNA (E. coli) (Amersham/Searle) 500 ct/min/I 67 mug; ["4C]poly I -poly C (Miles Laboratories, Elkhart, Indiana) 500 ct/min/1 2 5 mpg; [3H]poly dT * poly rA (kindly supplied by Dr Robert Gallo, NIH, Bethesda, Maryland) 390 ct/min/63 mpg; and [3H]poly As poly U (Miles Laboratories, Elkhart, Indiana) 400 ct/min/0-42 mpg. Inhibition of DNA and dT rA binding was tested as previously described (Talal & Gallo, 1972). The myeloma protein or serum was incubated with 5 pl ofinhibitor (containing 4 or 5 pg) at 370C for 30 min before the addition of the labelled nucleic acid. The following inhibitors were studied: reovirus RNA, phage RNA, poly I -poly C (Miles Laboratories, Elkhart, Indiana), dT~rA, poly A~poly U (Miles Laboratories), ss-DNA (calf thymus) (Worthington, Freehold, New Jersey) ds-DNA (calf thymus-Worthington, New Jersey). ss-DNA was made by heating ds-DNA for 10 minutes at 1000C followed by rapid chilling in an ice bath. All inhibitors were dissolved in borate-buffered saline (0 15 M, pH 8 0). RESULTS Serum electrophoresis The sera of SP104 tumour-bearing mice were studied by agarose gel electrophoresis. The fl-globulin fraction was increased probably due to the presence of the monoclonal protein migrating in the f-globulin region (Fig. 1).
Immunoelectrophoresis Sera were further studied by immunoelectrophoresis using specific anti-mouse immunoglobulin antisera. Precipitation with a monospecific antiserum to mouse IgA revealed a
350
J. A. Dixon, S. Sugai and N. Talal Alb a
Bly
Normal CA F1 serum
SF
104
SF
104
Anti-A
Anti-K
Normal
CA F1 serum
Anti-IgA SP 104
FIG. 1. Agarose gel electrophoresis and immunoelectrophoresis of sera from a normal CA F1 and a CA F1 mouse bearing the SPl04 tumour. The electrophoresis shows an increased fl-globulin fraction. The immunoelectrophoresis was developed with monospecific antisera to mouse IgA and light chains and shows the presence of an IgA-K myeloma protein.
mouse
400
350 -0
i 300
/
E 250 I 200 150
-
_E U
100Q 50-
007
035
0'7
35
Protein concentration (mg/ml)
FIG. 2. Binding of various nucleic acids by CA F1 IgA-K as a function of concentration of the SPI04 IgA myeloma protein. (0) ds-DNA. (o) Poly dt poly rAo(o) Poly rA- poly rU. (-) Poly rI -poly rC.
Mouse myeloma protein binding native DNA
351
heavy dense precipitin line in the /3-y region with prominent bowing, a typical feature of monoclonal immunoglobulins (Fig. 1). This line was also reproduced when anti-K antiserum was used. There was no reaction with anti-2-antiserum. The SP104 protein was thus identified as an IgA-K myeloma protein. It showed some splitting into two lines anodally.
Sephadex gelfiltration On Sephadex G-200 gel filtration, the bulk of the SPI04 IgA protein eluted in the first peak indicative of some high molecular weight aggregation. This material was then used for studies of nucleic acid binding. Nucleic acid binding The purified SPI04 protein bound significant amounts of ds-DNA (Fig. 2). The radioactivity retained on the filter was proportional to the concentration of the protein. Maximum retention of radioactivity on the filter occurred at a concentration of 0 7 mg/ml. No decline in binding was observed at higher concentrations as has sometimes been noted in studies of human SLE sera (Attias et al., 1973; Kredich, Skyler & Fotte, 1973). Maximum binding by the protein represented 7000 of the total radiolabelled antigen added. TABLE 1. Inhibition of DNA binding by SP104, IgA-K myeloma proteins Percentage inhibition of ['4C1DNA binding by:* ds-DNA
ss-DNA
Reovirus RNA
76+10t
45+4
15±5
Phage Poly (rA) Poly (rI)- Poly (dt) RNA poly (rU) poly (rC) poly (rA) 7+7
7+4
4±4
1±1
* Average of five experiments. t Mean ± standard error.
Increasing amounts of dT rA were bound with increasing concentrations of the protein (Fig. 2). However, the maximum binding observed represented only 210% of the total antigen added. Two different synthetic ds-RNAs, poly I poly C and poly A * poly U, were not bound by the protein. Purified S194A protein did not bind significant amounts of any nucleic acid tested. -
Inhibition of DNA binding Antibodies to ds-DNA appear spontaneously in humans with SLE, dogs with SLE and NZB/NZW mice. Because of the unusual derivation of the SP104 tumour from cell-free filtrates of spleens from SLE colony dogs, we studied the specificity of the SPI04 protein in an inhibition assay (Table 1). The binding of ds-DNA was inhibited 76% by calf thymus ds-DNA, 4500 by ss-DNA and 15% by reovirus ds-RNA (Table 1). The other nucleic acids did not give significant inhibition. Binding of dT rA was inhibited by dT rA (500%) and poly A -poly U (50%). This specificity was compared with the specificities of the anti-DNA antibodies found in the sera of human SLE patients and NZB/NZW mice (Table 2). There was a marked similarity in specificity. Ds-DNA inhibited the binding of ['4C]DNA by human SLE sera by
J. A. Dixon, S. Sugai and N. Talal
352
TABLE 2. Inhibition of DNA binding in systemic lupus erythematosus and NZB/NZW mice
Percentage inhibition of [14C]DNA binding by:
Binding
[14C]DNA Patient C.D. N.C. L.T. N.L. Mean+s.e. NZB/NZW 112-3A 146-3A 131 142-3A 78-3A
Mean+s.e.
(ct/min)
ds-DNA
ss-DNA
Reovirus RNA
Phage RNA
414 386 386 426
64 38 80 43 56± 10
48 13 96 34 48+ 18
11 0 47 8 16+10
1 0 27 4 8+6
63 96 92 95 96 88+6
93 97 96 98 96 96±1
24 37 35 18 27
24 18 14 10 13 16+2
239 328 296 330 232
28±3
Poly (rA) Poly (rI)- Poly (dt) poly(rU) poly (rC) poly (rA)
4±3
0 0 4 0 1+1
6±4
3 5 4 10 1 5+2
0 4 4 1 1 2+1
0 18 14 14 5 10±3
2 0 12 0
5 0 18 0
56%. ss-DNA inhibited binding by 48% and reovirus inhibited it by 16%. In NZB/NZW mice the patterns of inhibition were similar. However, both ds-DNA and ss-DNA inhibited ['4C]DNA binding to a much greater extent than in either humans or in the case of SPl04 protein. Idiotype characteristics The purified SPl04 protein was used to immunize rabbits in order to prepare anti-idiotypic antiserum. After absorption of the antiserum, typical idiotypic reactivity was observed
J604 tis SP104
FIG. 3. Double diffusion in agarose gel with absorbed antiserum against the SP104 protein (A). Outer wells contain sera from five different BALB/C plasmacytomas and purified SP104 protein.
Mouse myeloma protein binding native DNA
353
when tested against a panel of fifteen sera from BALB/C IgA myelomas (kindly supplied by Dr Roy Riblett) and the SP104 IgA. The anti-idiotypic antiserum reacted strongly with the SP104 IgA but not with S194A or with any other IgA myeloma tested (Fig. 3). It is of interest that two of the BALB/C proteins tested (S23 and S176) which did not cross-react with the SP104 idiotype have previously been shown to bind ds-DNA (Schubert et al., 1970). DISCUSSION SP104 is a tumour that arose in a CA F1 mouse which was injected intraperitoneally with a cell-free filtrate of dog spleen. The dog was bred from a colony of dogs with systemic lupus erythematosus (Lewis et al., 1965, 1973). Histologically, SP104 is composed of a mixture of poorly differentiated cells consisting of plasmablasts and reticulum cells. It is similar to oil-induced plasmacytomas in BALB/C mice in its diffuse mesenteric origin and in its content of A and C-type virus particles (Potter, 1972). It is also similar in producing an IgA myeloma protein, since 60 65% of BALB/C plasmacytomas secrete IgA (Potter, 1972). The purified SP104 IgA-K protein has unusual antibody activity. It binds ds-DNA and small amounts of dT * rA but has no activity against two different synthetic double-stranded RNA preparations. The inhibition of DNA binding by ds-DNA is much greater than by ss-DNA. A small amount of inhibition was observed with reovirus ds-RNA. The binding of ds-DNA is not unique to this monoclonal protein. Three other BALB/C IgA myeloma proteins bind ds-DNA (Schubert et al., 1970). None of these, however, showed specificities of binding similar to SP104. Two (S23 and S176) showed greater specificity for ss-DNA and phage RNA. The other (J504) was equally specific for phage RNA, ss-DNA and ds-DNA. Moreover, neither S23 nor S176 share a common idiotype with SP104. Sera from humans with SLE and NZB/NZW mice also contain antibodies to ds-DNA (Steinberg et al., 1969). In contrast to the BALB/C myelomas, these antibodies have the same specificity for ds-DNA as the SP104 protein. Their binding of radioactive ds-DNA is inhibited to a greater extent by ds-DNA than by ss-DNA. Binding is inhibited to a small extent by ds-RNA. These findings suggest but do not prove that the naturally occurring nucleic acid immunogen against which these antibodies are formed may be similar in the case of the SP104 protein, humans with SLE and NZB/NZW mice, and different in BALB/C oil-induced plasmacytomas. However, neither the source nor the nature of this immunogen is known, nor are the mechanisms regulating the immune response to nucleic acids. In NZB/NZW mouse disease, viruses may play a significant pathogenetic role in addition to genetic and immunological factors (Talal, 1970; Levy, 1973; Lerner et al., 1972). The viruses may act as a potent source of nucleic acid antigen or may, through their cytotoxic effects, be responsible for altering the immunogenicity of normal host DNA. The work of Lewis et al. (1973) suggests, but does not prove, the role of virus in the transmission of anti-DNA antibodies and lymphoid tumours from SLE colony dogs to normal CA F1 mice (Lewis et al., 1973). It is postulated that antigens present during the induction of oil-induced plasmacytomas in BALB/C mice may exert a selective effect upon the antibody activity of the monoclonal proteins that are eventually produced (Potter, 1972). Perhaps the same nucleic acid antigen (? viral) responsible for initiating the anti-DNA autoimmune response in animals that do not develop tumours could also exert a selective
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effect which is responsible for the specificity of the SP104 protein, a myeloma protein highly specific for ds-DNA. ACKNOWLEDG MENTS
We wish to acknowledge the excellent secretarial assistance of Mrs Nina Croomes and the technical assistance of Mr Edgar Adjahoe. The research was supported in part by grant number AM 16140 from the U.S. Public Health Service and in part by the Veterans Administration. We appreciate the generosity of Drs Robert Schwartz and Robert Lewis in providing us with the SP104 tumour. REFERENCES ATTIAS, M.R., SYLVESTER, R.A. & TALAL, N. (1973) Filter radioimmunoassay for antibodies to reovirus RNA in systemic lupus erythematosus. Arthr. and Rheum. 16, 719. KREDICH, N.M., SKYLER, J.S. & FOTTE, L.J. (1973) Antibodies to native DNA in systemic lupus erythematosus: a technique for rapid and quantitative determination. Arch. intern. Med. 131, 639. LERNER, R.A., JENSEN, F., KESSEL, S.J., DIXON, F.J., DES ROCHES, G. & FRANKE, U. (1972) Karotype, virologic and immunologic analysis of two continuous lymphocyte lines established from New Zealand black mice: possible relationship of chromosomal mosaicism to autoimmunity. Proc. nat. Acad. Sci. (Wash.), 69, 2965. LEVY, J.A. (1973) Zenotropic viruses: murine leukemia viruses associated with NIH Swiss, NZB, and other mouse strains. Science, 182, 1151. LEWIS, R.M., ANDRE-SCHWARTZ, J., HARRIS, G.S., HIRSCH, M.S., BLACK, P.H. & SCHWARTZ, R.S. (1973) Canine systemic lupus erythematosus: transmission of serologic abnormalities by cell-free filtrates. J. clin. Invest. 52, 1893. LEWIS, R.M., HENRY, W.B., JR, THORNTON, G.W. & GILMORE, C.E. (1963) A syndrome of autoimmune hemolytic anemia and thrombocytopenia in the dog. Sci. Proc. J. Amer. vet. Med. Assoc. supplement 1, 140. LEWIS, R.M., SCHWARTZ, R.S. & HENRY, W.B., JR (1965) Canine systemic lupus erythematosus. Blood, 25, 143. POTTER, M. (1972) Immunoglobulin-producing tumors and myeloma proteins of mice. Physiol. Rev. 52, 631. SCHUBERT, D., JOBE, A. & COHN, M. (1968) Mouse myelomas producing precipitating antibody to nucleic acid bases and/or nitrophenyl derivatives. Nature (Lond.), 220, 882. SCHUBERT, D., ROMAN, A. & COHN, M. (1970) Anti-nucleic specificities of mouse myeloma immunoglobulins. Nature (Lond.), 225, 154. STEINBERG, A.D., PINCUS, T. & TALAL, N. (1969) DNA-binding assay for detection of anti-DNA antibodies in NZB/NZW F1 mice. J. Immunol. 102, 788. TALAL, N. (1970) Immunologic and viral factors in the pathogenesis of systemic lupus erythematosus. Arthr. and Rheum. 13, 887. TALAL, N. (1973) Antibodies binding 3H-reovirus RNA in systemic lupus erythematosus. Clin. Immunol. Immunopathol. 1, 230. TALAL, N. & GALLO, R.C. (1972) Antibodies to a DNA-RNA hybrid in systemic lupus erythematosus measured by a cellulose ester filter radioimmunoassay. Nature: New Biology, 240, 240. TALAL, M., STEINBERG, A.D. & DALEY, G. (1971) Inhibition of antibodies binding polyinosinic-polycytidylic acid in human and mouse lupus sera by viral and synthetic ribonucleic acids. J. clin. Invest. 50, 1248.
