Antinuclear Antibodies in Rabbit Antisera* by Eugene V

View metadata, citation and similar papers at core.ac.uk brought to you by CORE

provided by PubMed Central

ANTINUCLEAR ANTIBODIES IN RABBIT ANTISERA* BY EUGENE V. BARNETr,~,§ M.D., AND JOHN H. VAUGHAN,~ M.D. (From the Rochester General and Strong Memorial Hospitals, University of Rochester School of Medicine and Dentistry, Rochester, New York) Pl~r~ 71 (Received for publication 8 November 1965)

Antibodies to nuclear constituents have been found in the sera of man (1), mouse (2), dog (3), and chicken (4). Their presence is usually associated with clinical disease. In recent years evidence has been presented that experimental animals may respond to immunization with production of antibody reactive with single strand desoxyfibonucleic acid (DNA). Immunization with native or single strand DNA, however, does not result in antibody to DNA (5). Meischer et al. (6) were successful in inducing antibodies to DNA in rabbits and guinea pigs immunized repeately with Brucella and calf thymus nuclear protein. Others (7) have not had the same success. Levine et al. (8) induced complement-fixing antibody to single strand DNA in rabbits immunized with 0 X 174, and others with purines (9), pyrimidines, (10) or nucleotides (11) conjugated to serum proteins. Plescia et al. (12) obtained complement-fixing antibodies to DNA in rabbits immunized with an electrostatic precipitate of DNA and methylated bovine serum albumin. Christian et al. (5) detected complement-fixing antibodies to single strand DNA in rabbits repeatedly immunized with heat-killed Bacillus subtilis and Escherickla coll. The present study describes the production in rabbits of antibody to nuclear constituents following immunization with whole human serum or human serum fractions in complete Freund's adjuvant. Methods Antigens Used for lmmunization.--Rabbit DNA was prepared from liver from freshly killed normal adult full grown rabbits. The extraction of nuclear proteins, histones, and DNA was performed according to the method of Dounce et al. (13, 14) and was prepared by Mr. Vincent Agnello and Dr. Alexander Dounce, Department of Biochemistry, University of Rochester, School of Medicine and Dentistry. DNA was prepared from human liver obtained at autopsy within 2~,~ hr of death. Preparations of calf thymus DNA were also obtained from

* Pre~nted in part before the American Association Immunologists 14 April 1965. Sup- ported by Grants AMO8153 and AMO2443, National Institutes of Health. Dr. Barnett is a Senior Investigator of the Arthritis Foundation. Dr. Vanghan is a re- cipient of a Research Career Award, National Institutes of Health. § Present address: Department of Medicine for the Health Sciences, University of Cali- fornia, Los Angeles. 733 734 ANTINUCLEAR ANTIBODIES

Dr. Dounce, as well as from the Worthington Biochemical Corporation, Freehold, New Jersey. E. coli DNA was obtained from Worthington Biochemical Corporation. Single strand DNA was prepared by boiling the native DNA for 15 rain and then rapidly cooling the preparation in an ice bath. DNA concentration was estimated by absorption at 260 m/z in a Hitashi- Perkin-Elmer spectrophotometer. To prepare the anticomplement sera an immune precipitate consisting of egg albumin and antiegg albumin was formed at 4°C in fresh pooled human group 0 serum. After 48 hr, the precipitate was washed then resuspended. Generally the precipitates contained about 10% of the original weights in added human serum proteins. On injection into rabbits they induced antisera that gave in addition to several antibodies against human alpha and gamma proteins, strong antibodies against B1C-A proteins as detected in immunoelectrophoresis against whole human serum (15). Autologous gamma globulin was obtained by prebleeding the rabbits and precipitating the gamma globulin in half saturated ammonium sulfate. Precipitates were estimated by absorption at 277 m#. Immunization of Rabbits.--Fuli grown New Zealand white rabbits were immunized with antigens as shown in Table I. Three groups of 6 rabbits each were immunized with whole human serum in complete Freund's adjuvant. Six rabbits were immunized with a pool of whole human serum obtained from the clinical chemistry laboratory of Rochester General Hospital. The serum pool was made from blood specimens obtained from patients with a variety of disorders, but none had known collagen vascular diseases or malignancies. The sera were obtained within 2 hr of blood clotting. Emulsions were made in a 1 : 1 ratio with complete Freund's adjuvant (Difco Laboratories, Inc., Detroit). Each rabbit received 1 ml of whole human serum per immunization divided among 47 foot pads. Rabbits were immunized once each week for 4 injections and bled 1 week later. They received 2 subsequent immunizations at 4 week intervals and were bled 1 week after each booster injection. Six rabbits were immunized with an emulsion containing 2.5 ml of pooled normal human serum and 10 ml of complete Freund's adjuvant. Each rabbit received 1 ml of this emulsion subcutaneously weekly for 3 weeks and were boosted monthly thereafter. Rabbit serum was collected 1 week after each booster injection for 4 bleedings. A third group of 6 rabbits was immunized with 1 ml serum kom a single normal human donor mixed with an equal volume of complete Freund's adjuvant. The rabbits were immunized weekly for 4 weeks intramuscularly and bled 1 week later. Six rabbits were immunized with 25 nag of Cohn fraction III (E. R. Squibb and Sons, New York), in complete Freund's adjuvant, 4 with fraction IV-l, and 4 with fraction IV-4. These 14 rabbits were immunized weekly for 4 weeks and then bled 1 week following the 4th injection. They received a booster injection 1 month later and were again bled the following week. Sera were obtained from 21 rabbits that had been immunized repeatedly with Cohn fraction II in complete Freund's adjuvant for up to 6 months. Sera were obtained from 2 rabbits that had been immunized repeatedly for over 1 year with Colin fraction I in complete Freund's adjuvant and from 4 rabbits similarly immunized with Colin fraction V. To obtain anti-human complement sera, 6 rabbits were immunized daily with 2 mg of an egg albumin antiegg albumin precipitate formed in human serum, by the intravenous route on 4 consecutive days eachweek, for 3 weeks. The rabbits were then boosted 1 month later with 2 mg of immune precipitate intraperitoneally followed the next day by 2 mg of immune precipitate intravenously. This procedure was repeated 1 month later and the rabbits were bled 1 week following the final booster immunization. Five rabbits were immunized with 0.4 mg of rabbit single strand DNA in complete Freund's adjuvant (Difco) twice monthly for 4 injections. Bleedings were obtained prior to each injec- EUGENE V. BARNETT AND JOHN H. VAUGHAN 735 tion and 2 weeks following the last injection. Five rabbits were immunized with an emulsion containing 1 ml of human single strand DNA (0.5 mg/ml). The rabbits were bled prior to each immunization and 1 month following the last immunization. Six rabbits were immunized with an emulsion contalnlng 1.5 ml of complete Freund's adjuvant, 1 ml of phosphate-buffered saline pH 7, and 0.5 ml of autologous gamma globulin (concentration 20 mg protein per ml). Five rabbits were immunized with a mixture containing 0.5 ml of autologous gamma globulin (20 mg protein per ml) plus 1 ml of human single strand DNA (concentration 0.5 mg per ml) in complete Freund's adjuvant. Four rabbits were immunized with 50% emulsion of Freund's complete adjuvant in phosphate-buffered saline pH 7.3 ml total divided into 4 foot pads. All rabbits were bled prior to each immunization and 1 month following the last immunization. Serological Te~ts.---Quantitative complement fixation tests were performed by the method of Wasserman and Levine (16). The tests with rabbit serum 514 were performed in the laboratory of Dr. Lawrence Levine using native and single strand Bacillus na#o DNA supplied by Dr. Levine. Thirty per cent complement fixation or greater was considered significant and indicative of a complement-fixing antigen-antibody reaction. Immunofluorescence tests were done as previously described (1) using human, rabbit, or chicken blood smears fixed on glass slides at 37°C in 95% ethanol. Cryostat frozen sections of human liver, thyroid, mouse liver, and rat liver were also used as sources of nuclei. The goat anti-rabbit gamma globulin fluorescein conjugate used was prepared by the method of Riggs et ai. (17) and shown to give only a single llne with the rabbit serum in Ouchterlony double diffusion or in immunoclectrophoresis against whole rabbit serum. Identical immunofluorescence results were obtained with fluorescein-conjugated goat anti-rabbit gamma globulin obtained from Microbiological Associates, Bethesda, Maryland. Agglutination tests for rheumatoidlike factors were carried out in disposable plastic trays with sheep ceils sensitized with rabbit amboceptor (18). DNAse, RNAse, and trypsin pretreatment of human white blood cell nuclei on smears of peripheral blood (19) was used to determine the effects of these enzymes on the nuclear antigens involved in the immunofluorescence. DNAse, RNAse, and an enzyme from dogfish liver shown by Levine and Van Vunakis (20) to digest preferentially single strand DNA was used to digest soluble antigens used in the quantitative complement fixation test. Susceptibility of the antinuclear antibodies to inactivation by suLChydryl reagents was tested by incubation of the rabbit antisera in 0.1 M mereaptoethanol for 24 hr at room temperature (21). The specimens were then tested directly without dialysis or alkylation for the presence of antinuclear antibody by the immunofluorescencetechnique.

RESULTS As shown in Table I, immunization with whole human serum resulted in the development of antinuclear antibody reactive with human leukocyte nuclei in 15 of 18 rabbits. The immunofluorescence pattern (Fig. 1) was solid when un- diluted serum was used. Sera diluted from 1:4 to 1:16 gave speckled or ring patterns. No nuclear fluorescence was seen when human thyroid, human, mouse, or rat liver, chicken erythrocytes, or chicken leukocytes were used as sources of nuclei. Of 14 rabbits immunized with Cohn fractions HI or IV, 4 were found to have detectable antinuclear antibodies. Sera from animals injected with complete Freund's adjuvant alone or with mixtures of autologous gamma globulins in human single strand DNA, autologous gamma globulins alone, single strand 736 ANTIN-~CI~A~ ANTIBOD~S

o~ °~

~+ e~ o.~ ~z EUGENE V. BARNETT AND JOHN H. VAUGHAN 737

DNA alone, single strand rabbit DNA alone, human complement, or human Cohn fractions I, II, or V, contained no detectable antinuclear antibodies. Sera from 8 rabbits immunized with whole human serum were treated with 0.1 x~ mercapoethanol for 24 hr at room temperature (Table II). In only 1 rabbit serum, No. 513, was the antinuclear antibody activity destroyed by the sulfhydryl treatment. Serum from rabbit 163 was fractionated by sucrose gradi-

TABLE II Results of Iraraunofl~orescence Tests for Antinudear Ant@odies in Rabbit Antisera Trea~ witk 0.1 at Me~aptoetkanol

Control SH gx

NRS R159 + + R163 +* + R509 + ++ R512 + + R513 + R514 + + R515 + + R516 + +

+, antinuclear antibodies detected by immunofluorescence. --, no antinuclear antibodies detected by immunofluorescence. * Antinudear antibody activity found to sediment with 7S globulins on sucrose gradient ultracentrifugation.

TABLE III Results of Iraraunofluores~ence Tests on Human Leukocyte Nuclei TreoJed u,ith Various Enzymes

Control DNAse l~/Ase Trypsin

616" 1/6 6/6 4/6 * Number positive/number of sera tested.

ent ultracentrifugation and the antinuclear antibody was found to sediment with the 7S globulins. Antinuclear antibody tests were repeated on human white cell nuclei treated with various enzymes (Table HI). Five of the 6 rabbit antisera failed to react with nuclei which had been incubated with DNase. Two of the 6 rabbit sera failed to react with human white cell nuclei which had been digested with trypsin. RNAse treatment of the nuclei did not result in any lessening of the posi- tivity of tests with any of the 6 rabbit antisera. All sera were tested in a routine complement fixation test (22). Anticomple- 738 ANTINUCLEAR ANTrBODIES mentarity was frequent. However, complement fixing antibodies to one or more nuclear antigens could be demonstrated in 8 sera from rabbits immunized with whole human serum. Three of these antisera had complement-fixing antibodies in sufficiently high titer to be tested in the quantitative complement fixation test of Wasserman and Levine (16). Antiserum 514 diluted 1:200 was shown to give significant complement fixation with human denatured (single strand) DNA and Bacillus na#o denatured DNA (Text-fig. 1). Maximum complement fixation was obtained with 0.03 #g of the human DNA while 0.3 ]zg of the Bacillus natto DNA was required for maximum fixation. Only 40% fixation was obtained with 0.1/zg of native human DNA and there was no significant fixation with native Bacillus natto DNA.

80|1 ~ HUMANDNA ~,,AB. nattoDN& 6ot [~L ~"°'u"~ .." I,o.o,u,.I ss##~ ''S

HUMAN DNA ,--,,-~I[[I .,~f/".//\ ~. \ (nohvel ~,~ - .... -m~...... ~ mm I~ (notive) ..... i.~ o'.z ols ~9 DNA TExT-FIG. 1. Complement fixation by rabbit antiserum 514 diluted 1:200 and nuclear antigens.

Eighty per cent complement fixation was obtained with 0.03 and 0.1/zg of denatured rabbit DNA (Text-fig. 2). Rabbit antiserum 160 diluted 1: 300 gave 70 % complement fixation with 0.03 #g of denatured human DNA (Text-fig. 3). Mter digestion with DNAse from dogfish liver the DNA showed no significant complement-fixing ability. Com- plement fixation by rabbit antiserum 160 was also seen with human histone at 0.1 and 0.3/zg antigen concentrations. This antiserum gave 80% complement fixation with 0.1 /zg of purine-6-oyl-bovine serum albumin (Text-fig. 4). No complement fixation was obtained with bovine serum albumin alone. Seventy per cent complement fixation was found with 0.03/~g of denatured calf thymus DNA. Rabbit antiserum 163 diluted 1:300 (Text-fig. 5) gave significant complement fixation with denatured human DNA but did not react with denatured rabbit DNA. At a dilution of 1:100, however, rabbit antiserum 163 (Text-fig. 6) EUGENE V. BARNETT AND JOHN H. VAUGHAN 739 was shown to give significant complement fixation both with denatured human DNA and to a lesser degree with denatured rabbit DNA. Absorption of rabbit antisera 163, 160, and 514 with whole human serum or with Cohn fractions III, IV-l, or IV-4, or with single strand calf thymus DNA

IO0

~_. 6o

01 0.2 0.3 gg DNA TExT-FIG. 2. Complement ~tion by rabbit antiserum 514 diluted 1:200 and de4~tured (single strand) and native rabbit DNA.

°°1 80!~ ~ HUMAN

0.I 0.2 0.3 ~g ANTIGEN TF~cT-Fm. 3. Complement fz~tion by rabbit antiserum 160 diluted 1:300 and human histone, denatured human DNA and b.mau DNA, denatured and digested by DNABe from dogfish liver. 740 ,oo]ANTIN-13CLEAR ANTZBODrRS

8O e.PURINE-6-0yI-BSA

40 !

20 ~ CALFTHYMUS DNA b/'- ~ (dena~ed) ..o- ~ , ",~ 0.I 02 03 IZg ANTIGEN ~-Fm. 4. Complement fixation by rabbit antiserum 160 diluted 1:300 and denatured calf thymus DNA, pufine-6-oylBSA and BSA.

100-

80-

60- ~ H~ DNA ~ (flenofured)

20- RABBITDNA "-r- -" C (de.lured) o'.l o',2 63 ~g DNA 'I~_,xT-Fzo.5. Complement fixation by rabbit antiserum 163 diluted 1:300 with denatured bllman DNA but not with denatured rabbit DNA. resulted in increased anticomplementariness of the sera so that specific complement-fixing activity with DNA could no longer be demonstrated. The absorbed rabbit antisera were also tested in immunofluorescent tests on human leukocyte nuclei. Antinuclear antibodies were not detectable following the absorption with whole human serum, Cohn fractions, III, IV-l, and IV-4. Following ab- EUGENE V. BARNETT AND JOHNH. VAUGHAN 741 sorption with DNA, the rabbit antisera contained antinuclear antibodies in un- diminished titer. No antinuclear antibody was detected in 45 preimmunization bleedings in either the immunofluorescence of complement fixation tests. Rheumatoid factorlike activity was detected in 44 of 45 prelmmunization bleedings by agglutination of sheep cells coated with rabbit amboceptor. The titer of the rheumatoid factorlike activity in the 44 preimmunization sera ranged from 1:16 to 1: 2048.

°°l

40 ~ ~BeI.I~A

20 ¸

o'E 0'2 0'3 IJ-q DNA

TzxT-Fzo. 6. Complement fixation by rabbit antiserum 163 diluted l: 100 with denatured human and rabbit DNA.

Surprisingly these titers rose little (less than 4-fold) regardless of the number of immunizations or the type of antigen used. The single rabbit that had no detectable agglutinin for amboceptor-coated sheep ceils prior to {mmunlzation did not acquire agglutinins during multiple immuniT~.tions with whole human serum. Antinuclear antibodies developed, however, to a titer of 1:16 in the immunofluorescence tests. Twelve of the 18 rabbits immunized with whole human serum including rabbit 514 were sacrificed when antinuclear antibody as detected by immunofluorescence, was present in their sera. Specimens of all organs were fixed in formalin, sectioned, stained with hematoxylin and eosin, and examined micro- scopically. No lesions in any way suggestive of nephritis, amyloid, or vasculitis were found.

DISCUSSION Data have been presented to demonstrate that antinuclear antibodies appear in the sera of a majority of rabbits immunized repeatedly with whole human 742 ANTINUCLEAR ANTIBODIES serum in complete Freund's adjuvant. Antinuclear antibodies, as detected by immunofluorescence tests on human leukocyte nuclei, were found also in sera from rabbits immunized with Cohn factions III, IV-l, and IV-4. Only human leukocyte nuclei gave positive fluorescence tests with these antisera. Nuclei from other human tissues, rabbit nuclei, chicken leukocyte, and erythrocyte nuclei all gave negative results. Some hint as to the nuclear antigens to which antibodies were made was given by studies with enzyme treated cells. In 4 of 6 cases the leukocyte nuclei were no longer reactive following incubation with DNAse. In 2 of 6 cases trypsin also destroyed the nuclear antigens reactivity. These observations suggest that the nuclear antigen involved may be DNA or closely associated with DNA. In quantitative complement fixation tests 3 of the antisera were shown to have antibodies against DNA. There is no evidence from the present data that the antibodies against DNA detected by complement fixation were also detected in the immunofluorescence tests. Indeed, it would appear that the antiDNA antibodies themselves were not detected in the immunofluorescence tests. The results obtained on human white cell nuclei treated with various enzymes (Table III) could as well be explained if the antigens were nucleoproteins or a I)NA associated protein rather than DNA. The absorption experiment results suggested that the antinuclear antibodies in immunofluorescence tests were directed against antigens found in human serum and human serum fractions, but not in single strand calf thymus DNA. Furthermore, only human leukocyte nuclei gave positive results in immunofluorescence tests, while homologous or heterologous antigens could be used in the complement fixation tests. This further suggests that the different serological tests were detecting antibodies of different specificity. In every case the serological reactivity in complement fixation tests was greater against denatured than native DNA. The reactivity was greatest with denatured human DNA but excellent fixation was also found with denatured Bacillus natto DNA. Lesser fixation was found with homologous; i.e., rabbit, single strand DNA than with human single strand DNA. The reactivity of the DNA was markedly reduced by incubation with pancreatic DNAse and with DNAse obtained from dogfish liver shown to attack preferentially single strand DNA. Complement-fixing antibody against purine~6-oyl BSA was also detected in one of the antisera. These results are like those that have been found in lupus sera containing antiDNA antibodies (23, 24). Unlike the results found in lupus sera, is the suggestion here of somewhat greater reactivity of these rabbit antisera for heterologous than homologous DNA preparations, e.g. Text- fig. 5. The antinuclear antibody in the rabbit sera was detected by a goat anti-rabbit gamma globulin fluorescein conjugate. In one of 8 sera tested the antibody was completelyinactivated by mercaptoethanol. In rabbit antiserum 163, in which the EUGENE V. BARNETT AND JOIIN H. VAIYGHAN 743 antinuclear antibody was resistant to treatment with mercaptoethanol, the antibody was found to sediment with 7S globulins. No antinuclear antibodies were induced by immunization with DNA or by DNA mixed with gamma globulin. The stimulus for the induction of antinuclear antibodies by immunization with whole human serum is probably nuclear antigen present in serum and complexed to serum proteins. Electrostatic precipitates of DNA and methylated albumin (12) and purines (9), pyrimidines (10) and nucleotides (11) conjugated to protein have been shown to be antigenic in the rabbit. The immunofluorescence demonstrating positive tests only with human leukocyte nuclei suggests that the nuclear antigen present in human serum was derived from leukocytes. It is possible that leukocyte nuclear antigens may be spilled into the serum only during coagulation and that the plasma separated from the cells immediately after venepuncture would be free of these antigens. Nevertheless, the degree of trauma experienced by leukocytes during coagulation must be slight and probably this sort of trauma occurs intravascu- larly during normal life. The presence of antigenic nuclear material in human sera suggested by the present study is furthermore, in accord with data by Tan et al. (25) demonstrating immunochemically identifiable DNA in serum in cer- tain diseases. The positive reactions with diphenylamine (26) obtained with ex- tracts of human serum have also been interpreted to indicate the presence of serum DNA. The exact mechanism of serum transport for DNA, polynucleo- tides, or other antigenic nuclear materials is at present unknown. The binding of C14-1abeled T5 bacteriophage DNA to as globulins (27) together with the induction of antinuclear antibodies in the present study by immunization with Cohn fractions III, IV-I, or IV-4 as well as other studies (28) suggest that a specific globulin may act as a carrier protein for serum nuclear constituents. The antigenic stimulus for the induction of antinuclear antibodies in man is unknown. The specificity of some anfinuclear antibodies in rheumatoid arthritis for leukocyte nuclei (29, 1) may indicate that leukocytic nuclear material may be "antoantigenic" in some cases. Anti-gamma globulin factors similar to the rheumatoid factors found in human sera were found in all but one of the rabbit anfisera tested, but they were already present in the preimmunizafion bleedings. The titers did not increase much following immunization nor were the fiters correlated in any way with the development of antinuclear antibodies. This report outlines an additional method for induction of antinuclear antibodies including antiDNA in rabbits. The antiDNA antibodies in hyperimmunized rabbits reported by Christian, DeSimone, and Abruzzo (5), were detected only by complement fixation tests. Antinuclear antibodies reported here were detected first by immunofluorescence tests on human leukocyte nuclei. The complement fixation tests demonstrated that these rabbit anfisera contained antibody to DNA as well as to other nuclear antigens. There is no evidence from 744 ANTI_NUCLEAR ANTIBODIES

the present data that the complement-fixing antibodies against DNA were also detected by the immunofluorescence tests. Since the specificity of the antinuclear antibodies in the rabbit anfisera studied here appears to be directed pri- marily at human nuclear antigens, as determined by both immunofluorescence and complement fixation tests, it seems un]~I~elythat they arose by an autolm- mune mechanism such as that proposed by Christian et al. (5). These workers interpreted that the antinuclear antibodies were directed against autologous rabbit DNA released or otherwise made antigenic during the immunizat]on process. Since the bulk of the anfiDNA activity in their study was removed by absorption with E. toll, they also considered that antibodies were formed specific for bacterial DNA but cross-reactive with other DNA. In neither their rabbits immunized with bacteria nor the rabbits immunized with whole human serum in the present study was there a correlation between the titer of rheuma- toidllke and antinuclear antibodies. The biological significance of the induction of antinuclear antibodies in rabbits is unlmown. There were no pathological changes in the rabbits immunized with whole human sera that resembled in any way those found in systemic lupus erythematosus or rheumatoid arthritis. The induction of antiDNA and other ant]nuclear antibodies in the present study without the induction of disease that can be attributed to these antibodies is like the experience of others (5, 6, 9). This finding must certainly mold our thinking about the possible pathogenetic significance of similar antibodies found in human disease. There is increasing evidence that in many cases antinuclear antibodies follow in the course of the disease (1) and are not by themselves cytotoxic (30, 31). Nonetheless, antiDNA antibodies may contribute to the pathogenesis of systemic lupus erythematosus. It has been suggested (25) that circulating complexes of native DNA and antibodies to native DNA may contribute to the nephritis and vasculitis in the absence of a direct cytotoxic effect of the antibodies alone. Since the immunized rabbits in the present experiment may not have had such circulating complexes, the absence of pathological changes in no way excludes the role of complexes in the pathogenesis. Work in progress in our laboratory and others will bear directly on the role of DNA antiDNA complexes in pathogenesis.

SIYM'~4.R¥ Antinuclear antibodies were detected by immunofluorescence in most sera from rabbits immunized with whole human serum emulsified in Freund's complete adjuvant. Four of 14 sera from rabbits immunized with Cohn fractions III, IV-I, or IV-4 also gave positive nuclear fluorescence with human leukocyte nuclei. Other human and animal nuclei gave negative results with these rabbit antisera. Three rabbit anti-whole human sera had complement-fixing antibody against DNA in sufficient titer for study in quantitative complement fixation tests. Antibody with greatest reactivity with human single strand DNA was detected in the 3 rabbit antisera. Reactivity with rabbit, calf thymus, and B. EUGENE V. BARNETT AND ~OHN H. VAUGHAN 745 natto DNA was also detected. In each case reactivity was greater with single strand than with native DNA. Antibodies against human histone and purine-6- oly BSA were also detected. No correlation was found between the titers of rheumatoidlike factors and antinuclear antibodies present in the rabbit antisera. The induction of antinuclear antibodies in these rabbits was not associated with disease attributable to the antibodies. The induction of antinuclear antibody by immunization with whole human serum was interpreted as indi- cating the presence of antigenic nuclear material in whole human serum. BIBLIOGP~APHY 1. Barnett, E. V., North A. F., Jr., Condemi, J. J., Jacox, R. F., and Vaughan, J. H., Antinudear factors in systemic lupus erythematosus and rheumatoid arthritis, Ann. Int. Med., 1965, 63, 100. 2. Norins, L. C., and Holmes, M. C., Antinuclear factor in mice, J. Immunol., 1965, 93, 148. 3. Lewis, R. M., Schwartz, R., and Henry, W. B., Jr., Canine systemic lupus erythematosus, Blood, 1965, 9.5, 143. 4. Bardawil, W. A., Gallins, N., and Gavalle, R., Antinuclear globulins in fowl, Fed. Proc., 1965, 24, 620. 5. Christian, C. L., DeSimone, A. R., and Abruzzo, J. L., Anti-DNA antibodies in hyperimmunized rabbits, J. Exp. Med., 1965, ].9.1., 309. 6. Miescher, P., Cooper, N. S., and Benacerraf, B., Experimental production of antinuclear antibodies, J. Immunol., 1960, 85, 27. 7. Dubois, E. L., and Katz, Y. J., An attempt to produce systemic lupus erythematosus and rheumatoid arthritis by crude desoxyribonucleic acid and joint antigens, Arthritis and Rheum.at., 1960, 3, 403. 8. Levine, L., Murakami, W. T., Van Vunakis, H., and Grossman, L., Specific antibodies to thermally denatured desoxyribonucleic acid of phage T4, Proc. Nat. Acad. So., 1960, 46, 1038. 9. Butler, V. P., Jr., Beiser, S. M., Erlanger, B. F., Tanenbaum, S. W., Cohen, S., and Bendich, A., Purine-specific antibodies which react with desoxyribonucleic acid (DNA), Proc. Nat. Acad. So., 1962, 48, 1597. 10. Tanenbaum, S., and Beiser, S. M., Pyrimidine-specific antibodies which react with desoxyribonucleic acid (DNA), Prec. Nat. Acad. $6., 1963, 49, 662. 11. Erlanger, B. F., and Beiser, S. M., Antibodies specific for ribonucleosides and their reaction with DNA, Proc. ]Vat. Acad. So., 1964, 59., 68. 12. Plescia, O. J., Braun, W., and Palczuk, N. C., Production of antibodies to denatured desoxyribonudeic acid (DNA), Proc. Nat. Acad. S¢., 1964, 59., 279. 13. Dounce, A. L., in The Nucleic Acids, (E. ChargaffandJ. N. Davidson, editors), New York, Academic Press, 1955, 93. 14. Kay, E. R. M., Simons, N. S., and ])ounce, A. L., An improved preparation of sodium desoxyribonudeate, J. Am. Chem. Soc., 1952, "/4, 1724. 15. Leddy, J. P., Hill, R. W., Swischer, S. N., and Vaughan, J. H., Observations on the immunochemical nature of red cell autosensitization, Third International Symposium of Immunopathology, Basel, S. Karger, 1963, 318. 16. Wasserman, E., and Levine, L., Quantitative micro-complement fixation and its 746 ANTINUCLEAR ANTIBODIES

use in the study of antigenic structure by specific antigen-antibody inhibition, J. Immunol., 1961, 87, 290. 17. Riggs, J. L., Seiwald, A. J., Burckhalter, J. W., Downs, C. M., and Metcalf, T. G., Isothiocyanate compounds as fluorescent labeling agents for immune serum, Am. J. Pathol., 1958, 84, 1081. 18. Rose, H. M., Ragen, C., Pearce, E., and Lipman, M., Differential agglutination of normal and sensitized sheep erythrocytes by sera of patients with rheumatoid arthritis, Proc. Exp. Biol., 1948, 65, 1. 19. Bardawil, W. A., Toy, R. L., Galins, N., and Bayles, T. B., Disseminated LE, scleroderma, and dermaloimyosifis in manifestation of sensitization to DNA- probl. I. An immunohistochemical approach, Am. ]. Path., 1958, 34, 607. 20. Levine, L., and Van Vunakis, H., Desoxyribonucleases of mustelus canis liver, Biol. Bull., 1963, 125, 384. 21. Barnett, E. V., Condemi, J. J., Leddy, J. P., and Vaughan, J. H., Gammas, gammalA, and gammalM anfinuclear factors in human sera, J. Clin. Inv., 1964, 43, 1104. 22. Barnett, E. V., Dumonde, D. C., and Glynn, L. E. Induction of autoimmunity to adrenal gland, Immunology 1963, 4, 382. 23. Stollar, D., Levine, L., and Marmur, J., Antibodies to denatured desoxyribonucleic acid in lupus erythematosus serum. II. Characterization of antibodies in several sera, Biochem. et Biophysica Acta, 1962, 61, 7. 24. Stollar, D., and Levine, L., Antibodies to denatured desoxyribonucleic acid in lupus erythematosus serum. IV. Evidence of purine determinants in DNA, Arch. Biochem. and Biophysics, 1953, 101, 417. 25. Tan, E. M., Schur, P. H., and Kunkel, H. G., DNA in the serum of patients with systemic lupus erythematosus (SLE), J. Clin. Inv., 1965, 44, 1104. 26. Gray, J. D., The relationship between rheumatoid "factor" and serum desoxyribonucleic acid, J. Immunol., 1964, 92, 827. 27. Buffer, V. P., Jr., Tanenbaum, S. W., and Beiser, S. M., A study of the cross- reactivity of antipurine-6-oyl serum with desoxyribonucleic acid (DNA), J. Exp. Med., 1965, 121, 19. 28. Atchley, W. A., An immunoelectrophoretic analysis of antigens reacting with lupus serum, Arthritis and Rheumat., 1961, 5, 471. 29. Faber, V., Elling, P., Norup, G., Mansa, B., and Nissen, N., An antinuclear factor specific for leukocytes, Lancet, 1954, 9., 344. 30. Ward, J. R., Cloud, R. S., and Turner, L. M., Non-cytotoxicity of "nuclear antibodies" from lupus erythematosus sera in tissue culture, Ann. Rheumat. Dis., 1964, 23, 381. ~1. Marmont, A. M., The transfusion of active LE plasma into noninpus recipients with a note on the LE like cell, Ann. New York Acad. Sc., 1965, 124, 838.

EXPLANATION OF PLATE 71 FIG. 1. Fluorescence of human leukocyte nuclei by rabbit antiserum 514 and goat anti-rabbit %globulin fluorescein conjugate. THE JOURNAL OF EXPERIMENTAL MEDICINE VOL. 123 PLATE 71

(Barnett and Vaughan: Antinuclear antibodies)