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HYPERSENSITIVITY TO HISTONE INDUCED EXPERIMENTALLY IN RABBITS
888 bled periodically from the marginal ear assessed for antibody content by the indirect or layering " fluorescent antibody technique. Tissue substrates included autologous rabbit skin obtained by biopsy under local anxsthesia (procaine hydrochloride l"o); homologous rabbit liver and heterologous bovine aorta secured immediately after death; and heterologous human skin and placenta, obtained respectively at surgery and at delivery. Fresh frozen sections were stained with rabbit serum and then counterstained with anti-rabbit gamma-globulin goat-serum conjugate. Cross-inhibitions, as described elsewhere (Bardawil et al. 7), were performed between rabbit and human sera. Stained sections were examined under a Reichert ’Zetopan’ fluorescence microscope and photographed in colour upon Super Anscochrome’ daylight emulsion with a ’Leica IIIF’ 35-mm. camera withMicro Ibso ’ attachment. All the animals
Preliminary Communication
vein, and their
were
sera were
"
HYPERSENSITIVITY TO HISTONE INDUCED EXPERIMENTALLY IN RABBITS
*
AN abnormal gamma-globulin has been found in the of patients with acute disseminated lupus erythematosus (L.E.), scleroderma, dermatomyositis, and certain forms of rheumatoid arthritis in which L.E. cells are present This abnormal protein has been investigated histochemically by the fluorescent antibody technique2 supplemented by enzymatic digestions and inhibition procedures, and has been shown to possess an apparently specific reactive affinity for intranuclear desoxyribonucleoproteins in autologous, homologous, and heterologous tissues. A similar histochemical phenomenon in lupus patients has been described by Friou.34 The constancy of the abnormal gamma-globulin in patients with the listed disorders and the resemblance between the intranuclear binding of nucleoprotein by gamma-globulin and the findings in immune reactions suggest that hypersensitivity to desoxyribose-nucleicacid-proteins may play a significant role in the pathogenesis of some of the so-called diseases of collagen. To test this we have attempted to sensitise rabbits against heterologous nucleoprotein, in order to establish the immunological state supposed to exist in human patients, and perhaps even to create in animals an experimental disease state similar to, if not altogether identical with, that found in human systemic lupus erythematosis, scleroderma, and dermatosera
myositis.
’
Results Reactions Of the three animals in group I two died of concomitant infection the first week following injection. The surviving H-1 first showed the rabbit, Adj (table i), presence of antibodies to histone 49 days after the initial injection of antigen and about
Staining
7.
Bardawil, W. A., Toy, B. L., Hertig, A. 1958, p. 708.
T.
Amer J. Obstet. Gynec. April,
Methods
apportioned at random each lot containing both groups (table i), males and females. The antigen was commercial calf-thymus histone prepared by the method of Mirsky and Pollister,5 dissolved in 0-2 M hydrochloric acid, and adjusted to neutrality with 10 M sodium hydroxide. Rabbits in groupI received, in addition to the nucleoprotein antigen, an adjuvant based on that of Fischel et al.(table 11). Each dose contained the same constituents, but they were mixed in different proportions, and with the Fischel adjuvant a different antigen was used. Initial immunisation of the rabbits in group I was attempted by the intramuscular injection of two doses of the antigen-adjuvant combination described. Each rabbit in group II was given intravenous histone 50 mg. in aqueous solution, and those in group III were injected intramuscularly as their fellows in group I but received adjuvant alone. A month later the rabbits were given subcutaneous booster doses of histone 50 mg. Albino rabbits
were
to
*
Supported by Grant C-2451 from the U.S. Public Health Service, Bethesda, Maryland, U.S.A.
Toy, B. L., Galins, N., Bayles, Path. 1958, in the press. 2. Coons, A. H. Int. Rev. Cytol. 1956, 5, 1. 3. Friou, G. J., Finch, S. C., Detre, K. D. Fed. Proc. 1. Bardawil, W. A., T. B.
Amer. J.
1957, 16, 413. 4. Friou, G. J. Society of Clinical Investigation meetings, Atlantic City, N.J., 1957. 5. Mirsky, A. E., Pollister, A. W. J. gen. Physiol. 1946, 30, 117. 6. Fischel, E. E., Kabat, E. A., Stoerk, H. C., Bezer, A. E. J. Immunol. 1952, 69, 611.
Fig. 1-Autologous localisation of serum-gamma-globulin from rabbit Adj H-l. Note nuclear staining within superficial layers of epithelium. Fig. 2-Heterologous localisation of serum-gamma-globulin from rabbit Adj H-I. Human skin
as
tissue substrate. Note brilliant fluorescence of stained cellular nuclei.
889 TABLE I-EXPERIMENTAL GROUPS
15 days after the secondary dose. The antibody level was estimated crudely by the intensity of nuclear staining, and was observed to increase gradually over about 55 days, thereafter declining until staining became weak, irregular, or (as it often did) disappeared. A second booster injection, given to this rabbit about 10 weeks after the first, restored the capacity for intense intranuclear staining. Serum-antibodies in the hypersensitive rabbit are bound histochemically with intranuclear material in autologous, homologous, and heterologous tissues (figs. 1 and 2). In addition to staining bovine nuclei, from which the antigen had been derived, the serum-gamma-globulin of rabbit Adj H-1 reacted also with rabbit and human nucleoprotein. In group II rabbit H-3 died during the first week after injection. Rabbits H-1 and H-2 and the two controls in group III never produced antibodies measurable by the present technique. The participation of the rabbits’ own gammaglobulin was excluded by direct staining for this protein by techniques described elsewhere.7 Because a direct conjugate of serum-globulin from rabbit Adj H-1 was not available, auto-inhibition was not attempted. -
TABLE II-SENSITISING SOLUTION
.
Emulsifying
Modified from Fischel et al.6 by Duke Laboratories Inc.
agent made
identical with that of the so-called abnormal globulin or "L.E. factor " reported in human collagen disease. The heterophil reactivity observed is to be expected in view of the demonstration by Allfrey et al.that desoxyribose nucleic acid from various sources-including calf thymus and kidney, chick erythrocytes, paracentrotus and trout sperm, and wheat germ-can ,restore to nuclei their capacity for synthesis after it has been destroyed by desoxyribonuclease. Though nucleoproteins can mediate the most complex biological functions, they seem to remain sufficiently primitive to be immunologically non-
species-specific. If one of the so-called collagen diseases is shown to have definite basis in hypersensitivity against nucleoprotein, the experimentally noted lack of species specificity may be taken to imply that such allergy in the human patient may follow antigenic stimulation by viral, bacterial, or animal It has been found in this desoxyribonucleoprotein. that sera stain ordinary smears of laboratory positive the viridans streptococci, including group. Indeed, if a sensitisation non-specific against nucleoprotein is assumed, it is possible to construct a simple and orderly pathogenic theory to account for the classical morphological lesions found in the collagen diseases, embracing not only their character but also their topographical distribution in the a
’
body.
Demonstration of the intranuclear binding of nucleoprotein is but the first step in the establishment of nucleoprotein sensitivity as a factor in the pathogenesis of some of the collagen diseases. The cross-inhibition of serum from a patient with scleroderma provides additional suggestive evidence of a common process. The most arduous task of all, that of inducing clearcut clinical disease in animals artificially, has not yet been
accomplished. Summary
Serum from this rabbit has
successfully cross-inhibited staining from the gamma-globulin of a patient
a conjugate made with active scleroderma.
by
Collateral Tests At the apparent peak of nuclear staining intensity in Adj H-l, 35 days after the booster injection, a positive Arthus reaction (local oedema and necrosis) was obtained by the intradermal injection of histone 1-5 mg. An attempt to induce an Arthus reaction after the capacity for intranuclear staining had waned was fruitless, but an additional intradermal injection applied about a month after the ’second booster dose caused severe local haemorrhage and necrosis. Attempts to elicit similar responses from the rabbits in groups II and III failed. In attempted ring precipitations, rabbit and human sera were tested against neutral solutions of histone in multiple dilutions. The results were inconclusive because both test and control sera precipitated the antigen at practically all concentration levels, perhaps because of the presence of calcium and magnesium cations in the sera. L.E.-cell preparations made with serum from rabbit Adj H-1 did not show typical inclusion bodies. Blood-counts on the sensitised rabbit showed marked anxmia and leucopenia (lymphocytes 70%, segmented forms 29%, basophils 1%). Discussion
In the
rabbit (Adj H-l) hypersensitivity surviving induced histone, experimentally, led to the appearance of a circulating serum-antibody with a demonstrable reactive affinity for autologous, homologous, and heterologous intranuclear nucleoproteins. The histochemical behaviour of this antibody seems to be morphologically
to
test
The histochemical behaviour of sera from human patients with certain so-called collagen diseases has been simulated by samples from an animal sensitised against bovine histone. Evidence is presented that the observed effect depends on immunological reaction, and the relation to human disease is discussed. WADI A. BARDAWIL M.D.
Mexico
BENJAMIN Departments of Pathology, Harvard Medical School and St. Margaret’s Hospital, Boston, U.S.A. 8.
D.M.D.
L. TOY
Harvard
NORA GALINS Susquehanna.
B.A.
Allfrey, V., Mirsky, A. E., Osawa, S. The Nucleus and Protein Synthesis, in The Chemical Basis of Heredity (edited by W. D. McElroy and B. Glass); pp. 200 231, especially table 4, p. 221 (listing of species) and p. 218 ff. Baltimore, 1957.
" The fact that most students attend lectures has repercussions on their lecturers. Having a good attendance, teachers may assume that their students must learn a good deal from them, or else they would not attend. Accordingly, any lecturer who does not come into close contact with students in laboratory classes, or in other ways, can very well live in a pleasant dream-world about the effectiveness of his teaching. Perhaps the easiest and cheapest way to improve university teaching about one hundred per cent would be to make it compulsory for every lecturer to spend a good deal of time with his students immediately after each lecture to find out what they did, and did not, learn from it."-ARTHUR W. HAM, Canad. med. Ass. 1958, 78, 482.
Preliminary Communication
vein, and their
were
sera were
"
HYPERSENSITIVITY TO HISTONE INDUCED EXPERIMENTALLY IN RABBITS
*
AN abnormal gamma-globulin has been found in the of patients with acute disseminated lupus erythematosus (L.E.), scleroderma, dermatomyositis, and certain forms of rheumatoid arthritis in which L.E. cells are present This abnormal protein has been investigated histochemically by the fluorescent antibody technique2 supplemented by enzymatic digestions and inhibition procedures, and has been shown to possess an apparently specific reactive affinity for intranuclear desoxyribonucleoproteins in autologous, homologous, and heterologous tissues. A similar histochemical phenomenon in lupus patients has been described by Friou.34 The constancy of the abnormal gamma-globulin in patients with the listed disorders and the resemblance between the intranuclear binding of nucleoprotein by gamma-globulin and the findings in immune reactions suggest that hypersensitivity to desoxyribose-nucleicacid-proteins may play a significant role in the pathogenesis of some of the so-called diseases of collagen. To test this we have attempted to sensitise rabbits against heterologous nucleoprotein, in order to establish the immunological state supposed to exist in human patients, and perhaps even to create in animals an experimental disease state similar to, if not altogether identical with, that found in human systemic lupus erythematosis, scleroderma, and dermatosera
myositis.
’
Results Reactions Of the three animals in group I two died of concomitant infection the first week following injection. The surviving H-1 first showed the rabbit, Adj (table i), presence of antibodies to histone 49 days after the initial injection of antigen and about
Staining
7.
Bardawil, W. A., Toy, B. L., Hertig, A. 1958, p. 708.
T.
Amer J. Obstet. Gynec. April,
Methods
apportioned at random each lot containing both groups (table i), males and females. The antigen was commercial calf-thymus histone prepared by the method of Mirsky and Pollister,5 dissolved in 0-2 M hydrochloric acid, and adjusted to neutrality with 10 M sodium hydroxide. Rabbits in groupI received, in addition to the nucleoprotein antigen, an adjuvant based on that of Fischel et al.(table 11). Each dose contained the same constituents, but they were mixed in different proportions, and with the Fischel adjuvant a different antigen was used. Initial immunisation of the rabbits in group I was attempted by the intramuscular injection of two doses of the antigen-adjuvant combination described. Each rabbit in group II was given intravenous histone 50 mg. in aqueous solution, and those in group III were injected intramuscularly as their fellows in group I but received adjuvant alone. A month later the rabbits were given subcutaneous booster doses of histone 50 mg. Albino rabbits
were
to
*
Supported by Grant C-2451 from the U.S. Public Health Service, Bethesda, Maryland, U.S.A.
Toy, B. L., Galins, N., Bayles, Path. 1958, in the press. 2. Coons, A. H. Int. Rev. Cytol. 1956, 5, 1. 3. Friou, G. J., Finch, S. C., Detre, K. D. Fed. Proc. 1. Bardawil, W. A., T. B.
Amer. J.
1957, 16, 413. 4. Friou, G. J. Society of Clinical Investigation meetings, Atlantic City, N.J., 1957. 5. Mirsky, A. E., Pollister, A. W. J. gen. Physiol. 1946, 30, 117. 6. Fischel, E. E., Kabat, E. A., Stoerk, H. C., Bezer, A. E. J. Immunol. 1952, 69, 611.
Fig. 1-Autologous localisation of serum-gamma-globulin from rabbit Adj H-l. Note nuclear staining within superficial layers of epithelium. Fig. 2-Heterologous localisation of serum-gamma-globulin from rabbit Adj H-I. Human skin
as
tissue substrate. Note brilliant fluorescence of stained cellular nuclei.
889 TABLE I-EXPERIMENTAL GROUPS
15 days after the secondary dose. The antibody level was estimated crudely by the intensity of nuclear staining, and was observed to increase gradually over about 55 days, thereafter declining until staining became weak, irregular, or (as it often did) disappeared. A second booster injection, given to this rabbit about 10 weeks after the first, restored the capacity for intense intranuclear staining. Serum-antibodies in the hypersensitive rabbit are bound histochemically with intranuclear material in autologous, homologous, and heterologous tissues (figs. 1 and 2). In addition to staining bovine nuclei, from which the antigen had been derived, the serum-gamma-globulin of rabbit Adj H-1 reacted also with rabbit and human nucleoprotein. In group II rabbit H-3 died during the first week after injection. Rabbits H-1 and H-2 and the two controls in group III never produced antibodies measurable by the present technique. The participation of the rabbits’ own gammaglobulin was excluded by direct staining for this protein by techniques described elsewhere.7 Because a direct conjugate of serum-globulin from rabbit Adj H-1 was not available, auto-inhibition was not attempted. -
TABLE II-SENSITISING SOLUTION
.
Emulsifying
Modified from Fischel et al.6 by Duke Laboratories Inc.
agent made
identical with that of the so-called abnormal globulin or "L.E. factor " reported in human collagen disease. The heterophil reactivity observed is to be expected in view of the demonstration by Allfrey et al.that desoxyribose nucleic acid from various sources-including calf thymus and kidney, chick erythrocytes, paracentrotus and trout sperm, and wheat germ-can ,restore to nuclei their capacity for synthesis after it has been destroyed by desoxyribonuclease. Though nucleoproteins can mediate the most complex biological functions, they seem to remain sufficiently primitive to be immunologically non-
species-specific. If one of the so-called collagen diseases is shown to have definite basis in hypersensitivity against nucleoprotein, the experimentally noted lack of species specificity may be taken to imply that such allergy in the human patient may follow antigenic stimulation by viral, bacterial, or animal It has been found in this desoxyribonucleoprotein. that sera stain ordinary smears of laboratory positive the viridans streptococci, including group. Indeed, if a sensitisation non-specific against nucleoprotein is assumed, it is possible to construct a simple and orderly pathogenic theory to account for the classical morphological lesions found in the collagen diseases, embracing not only their character but also their topographical distribution in the a
’
body.
Demonstration of the intranuclear binding of nucleoprotein is but the first step in the establishment of nucleoprotein sensitivity as a factor in the pathogenesis of some of the collagen diseases. The cross-inhibition of serum from a patient with scleroderma provides additional suggestive evidence of a common process. The most arduous task of all, that of inducing clearcut clinical disease in animals artificially, has not yet been
accomplished. Summary
Serum from this rabbit has
successfully cross-inhibited staining from the gamma-globulin of a patient
a conjugate made with active scleroderma.
by
Collateral Tests At the apparent peak of nuclear staining intensity in Adj H-l, 35 days after the booster injection, a positive Arthus reaction (local oedema and necrosis) was obtained by the intradermal injection of histone 1-5 mg. An attempt to induce an Arthus reaction after the capacity for intranuclear staining had waned was fruitless, but an additional intradermal injection applied about a month after the ’second booster dose caused severe local haemorrhage and necrosis. Attempts to elicit similar responses from the rabbits in groups II and III failed. In attempted ring precipitations, rabbit and human sera were tested against neutral solutions of histone in multiple dilutions. The results were inconclusive because both test and control sera precipitated the antigen at practically all concentration levels, perhaps because of the presence of calcium and magnesium cations in the sera. L.E.-cell preparations made with serum from rabbit Adj H-1 did not show typical inclusion bodies. Blood-counts on the sensitised rabbit showed marked anxmia and leucopenia (lymphocytes 70%, segmented forms 29%, basophils 1%). Discussion
In the
rabbit (Adj H-l) hypersensitivity surviving induced histone, experimentally, led to the appearance of a circulating serum-antibody with a demonstrable reactive affinity for autologous, homologous, and heterologous intranuclear nucleoproteins. The histochemical behaviour of this antibody seems to be morphologically
to
test
The histochemical behaviour of sera from human patients with certain so-called collagen diseases has been simulated by samples from an animal sensitised against bovine histone. Evidence is presented that the observed effect depends on immunological reaction, and the relation to human disease is discussed. WADI A. BARDAWIL M.D.
Mexico
BENJAMIN Departments of Pathology, Harvard Medical School and St. Margaret’s Hospital, Boston, U.S.A. 8.
D.M.D.
L. TOY
Harvard
NORA GALINS Susquehanna.
B.A.
Allfrey, V., Mirsky, A. E., Osawa, S. The Nucleus and Protein Synthesis, in The Chemical Basis of Heredity (edited by W. D. McElroy and B. Glass); pp. 200 231, especially table 4, p. 221 (listing of species) and p. 218 ff. Baltimore, 1957.
" The fact that most students attend lectures has repercussions on their lecturers. Having a good attendance, teachers may assume that their students must learn a good deal from them, or else they would not attend. Accordingly, any lecturer who does not come into close contact with students in laboratory classes, or in other ways, can very well live in a pleasant dream-world about the effectiveness of his teaching. Perhaps the easiest and cheapest way to improve university teaching about one hundred per cent would be to make it compulsory for every lecturer to spend a good deal of time with his students immediately after each lecture to find out what they did, and did not, learn from it."-ARTHUR W. HAM, Canad. med. Ass. 1958, 78, 482.