Role of nuclear antigens and antinuclear antibodies in inflammation

Biochemical Pharmacology, Supplement, pp. 77-86. Pergamon Press. 1968. Printed in Great Britain

ROLE OF N U C L E A R ANTIGENS AND A N T I N U C L E A R ANTIBODIES IN INFLAMMATION* EUGENE V. BARNETTt Department of Medicine, UCLA School of Medicine, Los Angeles, California Abstract--The sera of patients with lupus erythematosus and other "autosensitivity" diseases, such as rheumatoid arthritis, frequently contain not only the LE cell factor but antinuclear antibodies directed against denatured and native DNA, Sm antigen of the nucleus, protein associated with RNA, and probably many other nuclear antigens. The patients rarely have antibody only to a single nuclear antigen and, furthermore, these antinuclear antibodies are found to be heterogeneous immunoglobulins of the 7G, ~,A, and 7M immunoglobulin classes. Data are presented which suggest that at least in rheumatoid arthritis the antibodies may be the result rather than the cause of the disease. Similar antibodies have been elicited by immunization of animals with killed bacteria, human serum, and DNA components conjugated to carrier proteins. Animals with such experimentally induced antibodies do not acquire "human" aiseases and it is of further interest that such antinuclear antibodies are found in low concentrations in humans without disease. The above data, taken together with the observations that antinuclear antibodies are generally incapable of injuring intact cells, initially suggest that these antibodies have limited pathogenetic significance. This may not be the case, however, because there is increasing evidence that nuclear antigens and antinuclear antibodies may be one of a series of antigen-antibody combinations capable of eliciting inflammation of the synovium within the joint and along the basement membrane of the renal glomerulus. One animal disease which may bear on this question is Aleutian disease of mink. We have recently had the opportunity to examine 16 pairs of mink sera taken before and after the onset of the disease. Nuclear antigen (DNA) was detected in all these sera both before and after onset of the disease. Only in the diseased animals was antinuclear antibody detectable. It has been suggested by others that the vasculitis and other immunopathologic features of Aleutian disease are due to circulating antigen-antibody complexes even though the disease can be transmitted from mink to mink by filterable material. ANTINUCLEAR ANTIBODIES NUCLEAR antigens are those constituents of the cell nucleus capable of reacting in a variety of serologic tests with a n t i n u c l e a r antibodies. The lupus erythematosus (LE) cell factor is one o f m a n y such antinuclear antibodies and it appears to be specific for native nucleoprotein. 1 The clinical test to detect the LE cell p h e n o m e n o n depends on the availability of free leukocyte nuclei plus the LE cell factor in the patient's serum. This c o m b i n a t i o n results in a h o m o g e n e o u s l y staining hematoxylin body which is phagocytized by p o l y m o r p h o n u c l e a r leukocytes to yield the characteristic LE cell. The LE cell factor is only one of the a n t i n u c l e a r antibodies present in y-globulin of * This work is supported in part by research and training grants from the National Institutes of Health, Public Health Service. t Associate Professor of Medicine; recipient of a Senior Investigatorship from the Arthritis Foundation. 77

78

EUGENE V. BARNETT

certain patients that can be detected by immunofluorescence tests. The immunofluorescence tests depend on a source of nuclei, such as leukocytes in human blood smears, which are available in the laboratory. A drop of the patient's serum is placed on the slide and antinuclear factors then bind to the leukocyte. The slide is washed free of non-antibody proteins. A histochemical stain is then applied. This stain consists of rabbit antibody to human v-globulin which has been conjugated with fluorescein isothiocyanate. This preparation is then viewed through the fluorescence microscope. A variety of immunofluorescence patterns are found when the sera of many patients with collagen-vascular diseases are examined. Some sera yield shaggy nuclear fluorescence patterns and others, solid, speckled, or nucleolar patterns (Fig. 1). The factors influencing the patterns achieved by antinuclear factors in immunofluorescence tests include the specificity of the antibody for different nuclear constituents (such as DNA, nucleoprotein, etc.), the immunoglobulin class of the antinuclear factor, and diagnosis of the patient from which the antinuclear factors were taken (Y. B. Bickel, E. V. Barnett and C. M. Pearson, unpublished data). Quantitative factors, such as the fluorescein-protein ratio of the antibody to human 7-globulin used in the test and the concentration of the various antibodies, also play a role. Furthermore, there are qualitative factors to be considered, such as the observer error in interpreting the immunofluorescence pattern, whether leukocyte nuclei or liver nuclei are used, and the fixative used (alcohol or acetone). We reported 2--4 the immunoglobulin classes of antinuclear antibodies as found in different collagen-vascular diseases such as systemic lupus erythematosus, rheumatoid arthritis, and juvenile rheumatoid arthritis. From the data obtained we suggested that the lupus patients had been extensively immunized by nuclear antigens at the time of initial diagnosis while the rheumatoid arthritis patients appeared to be immunized during the course of their disease. Others have suggested that these antinuclear antibodies are not the result of immunization but rather are due to some genetic defect. However, since similar antibodies can be elicited in lower animals by immunization with nuclear antigens appropriately conjugated to carrier proteins, a, ~ we felt that the immunization hypothesis was more attractive. NUCLEAR

ANTIGENS

AS I M M U N O G E N S

To investigate how this immunization might come about, we studied which human serum protein fractions were capable of eliciting antinuclear antibodies in rabbits 7 (Table 1). Whole human serum in complete Freund's adjuvant consistently elicited TABLE 1. ANTINUCLEAR ANTIBODIES IN RABBITS IMMUNIZED WITH VARIOUS ANTIGENS Antigen Whole human serum Human Cohn fraction 1 Human Cohn fraction II Human Cohn fraction Ill Human Cohn fraction IV-I Human Cohn fraction IV-4 Human Cohn fraction V Human complement protein Single-strand rabbit D N A Single-strand human DNA Freund's-adjuvant controls

No. of sera tested 18 2 21

6 4 4 4 6 5 5 4

No. of sera positive 15 0 0

2 1 1 0 0 0 0 0

.ll

NOMOGENOUS ("Solid")

SPECKLED

~iA-

50t~ne soluble protein

n s~one

~r

M~ M ~ F ~ A N O ~ } S

~m ~ , ~*~bQggy")

NUCLEOLAR

? prO~eJ~ Os~ot:iated wifh RNA

Fl(;. 1. Patterns o[" nuclear fluorescence seen in indirect immunofluorescence tesls for antinuclear antibodies.

Bio-S.pp. facing page 7,~

FI(;. 6. Ivhiorescence ill renal glonlcruhls frolll p~llient wJl]l s~SlellliC hlpus erylllelllal,~u,~, ,,B.iJncd dil'ecil} with fltioresceJn-cttnjugalcd

antJnuclear antibocl}.

Fill. "7. ~:/me area Lis |-Jg. 6 slLlilletl w i l h acridine o r a n g e Io delecl Oucle~lr 111Ll[¢rJ~ll ,'lJOllg lhe IILISOIll~I1 l111¢lllhra he.

Immunology

79

antibody capable of reacting with human leukocyte nuclei in the immunofluorescence test. Cohn fractions III and IV may also have induced antinuclear antibodies. DNA in Freund's adjuvant did not induce antinuclear antibodies. A minority of rabbit anti-whole-human-serum antibodies were shown in addition to contain complementfixing antibody to DNA, with specificity similar to that seen in the sera of patients with systemic lupus erythematosus. This strongly suggested that even normal human serum might contain nuclear antigens complexed to some carrier protein and therefore be capable of eliciting the induction of antinuclear antibodies. SEROLOGIC DETECTION OF NUCLEAR ANTIGENS To test this more directly, we utilized a human antibody from patient V with systemic lupus erythematosus 8 (Fig. 2). At a dilution of 1:300 this antibody was shown to give reproducible complement-fixation curves with single-strand D N A of calf thymus origin. This human antibody also reacted with calf thymus nucleoprotein and native DNA but gave only equivocal complement fixation with RNA and histone (Fig. 3). The human antibody was shown to fix complement in the presence of antigens in human joint fluids (Fig. 4). This reaction was abolished if the joint fluids were incubated with deoxyribonuclease prior to testing. 100"

s0.

%

-,.:,..~:-~.,.,

60

c,

~?k'~\".,

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S.S. CALF THYMUS

0.01 DNA/ml

Multiple determinations of complement fixation by serum V (calf thymus).

(1:300)

and single-strand DNA

Certain normal human sera from patients with collagen-vascular disease and certain joint fluid specimens also were shown to contain antigens, in varying concentrations, capable of complement-fixing reaction with this human antibody (Fig. 5). The nuclear antigen content of the pathologic sera is shown in Table 2. This does not imply that these pathologic sera actually contain single-strand calf thymus DNA, but rather that they contain an antigen which fixes complement with human antibody to the same degree as would be achieved with single-strand calf thymus DNA in the concentrations shown. The antigen content measured did not correlate with the absence or presence of nephritis in systemic lupus erythematosus, at least at the time when the specimens were obtained from these patients. Tan e t aL 9 have data from two patients with systemic

80

EUOENE V. BARNETT

100"I I~. . . . . . . , f-'-":-" "'~ . . . . . . . . . . . . . . . ""',

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-o- . . . . . .

~ ' " ' % % 0 %% •

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Fla. 3. Complement fixation by serum V (1:100) and single-strand native calf thymus DNA, calf thymus nucleoprotein, calf thymus RNA, and calf thymus histone. 100

75 JF-3

% C'

50

FIX.

20 JF-3 Rx D N A s e JF-8 Px DNAse _- ' 1:100

_-

~ --

1:300

1:900

DILUTION

OF HUMAN

,

1.2700

SPECIMEN

FIG. 4. Complement fixation with serum E (1:300) and synovial fluid specimens from patients with gout, tested before and after incubation with deoxyribonuclease (DNAse).

lupus erythematosus and glomerulonephritis, which strongly suggest that the D N A content of the serum markedly increases with exacerbations of the nephritis. They examined many normal human sera, using precipitating antibodies obtained from patients with systemic lupus erythematosus, and were unable to detect nuclear antigens. In our study (Table 3), it appeared that normal human sera rarely contained more than 0.3 t~g/ml equivalent of single-strand calf thymus DNA, and none tested contained more than 3.5 tzg/ml. A pool of normal human serum which had been used to immunize rabbits to elicit antinuclear antibodies was shown to contain 1.5 t~g/ml

81

Immunology 100'

[] J F - 9

75

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FIG. 5. Complement fixation with serum E (1:300) and specimens of normal human sera (NS-I, NS-5, and NS-8), abnormal human serum (A-14, A-35, A-36, and A-21), and synovial fluid (JF-2 JF-4, and JF-9).

TABLE 2. NUCLEAR ANTIGEN CONTENT OF PATHOLOGIC HUMAN SERA

Disease RA SLE ÷ nephritis SLE without nephritis Systemic mastocytosis Uremia with chronic glomerulonephritis RA SLE ÷ nephritis SLE without nephritis Vasculitis, local Rheumatic heart Psoriasis with R A Gout Scleroderma

Number tested 2 1 2 1 1 4 5 5 1 I 2 1 1

Equivalence to single-strand calf thymus D N A 0,g/~) 15"0, 2"3 8"6 16"0, 13"5 4'9 9'0

1

<0"2

TABLE 3. NUCLEAR ANTIGEN CONTENT OF NORMAL HUMAN SERA Sera R.K. G.D. G.W. D.A. D.C. D.G. Others (8) Pool of "normal serum"

Equivalence to single-strand calf thymus D N A (~g/~) 3"50 2-60 1"94 0"80 0.28 0-26 <0.26 1-5

82

EUGENE V. BARNETT

equivalent of single-strand calf thymus DNA. We have documented in our laboratory that essentially no DNA is released into the plasma or serum by aging blood or by in vitro hemolysis. 9 Furthermore, plasma from outdated whole human blood was extracted with hot perchloric acid and the extract was tested in the diphenylamine test for deoxyribose after concentration by pressure dialysis; the original material contained 0.8 t~g of DNA/ml. These data taken with our experience with rabbits immunized with whole human serum strongly suggest that small amounts of nuclear antigens including D N A are present in normal serum and that the amounts detected are probably not the result of leukocyte breakdown in vitro. The large amounts of nuclear antigen detected in pathologic sera by the complement-fixation test were certainly in accord with similar data obtained by the less sensitive precipitin technique by Tan et al. 9 In addition, we immunized rabbits with single-strand calf thymus D N A in an electrostatic complex with methylated rabbit serum albumin, e, 10 The rabbits produced ~,M complement-fixing antibody specific for single-strand and native calf thymus DNA with no reactivity with RNA or histone. Such antibody gave reactivity with T4 phage DNA, suggesting some specificity for the cytosine determinants of single-strand calf thymus DNA. With the use of such rabbit antibody we detected nuclear antigen in eight of fourteen abnormal sera, but the concentrations detected were much lower than those achieved by using antibody from patients with lupus erythematosus. ROLES OF NUCLEAR ANTIGENS AND ANTINUCLEAR ANTIBODIES In inflammation

It would appear that normal individuals may have nuclear antigens and even antinuclear antibodies 11-18 in low concentrations, A variety of patients with collagenvascular disease have large amounts of nuclear antigens 9 and antinuclear antibodies at various stages of their disease. ~2 How then can we incriminate nuclear antigens and and antinuclear antibodies in the inflammation seen in rheumatologic disorders? It has been shown that antigen can combine with antibody, fix components of the complement system, and initiate a series of cytologic and cytotoxic reactions as well as influence vascular permeability, ~4 in other words, initiate the very complex series of events that we call "inflammation." We, and others, 15 have detected nuclear antigens in high concentration in synovial fluids (Table 4). The nuclear antigen concentrations reported here in terms of DNA did not correlate with the leukocyte counts of the synovial fluids. Furthermore, we have shown that rheumatoid synovial fluids (Table 5) contain antinuclear antibodies as well as rheumatoid factors, ~6 and that rheumatoid synovial fluids consistently have depressed complement levels as compared to serum levels. It has been suggested that the presence of both nuclear antigens and antibodies in sufficient quantity may be necessary to initiate or propagate the inflammation. 17, 18 Nuclear antigens and antinuclear antibodies may indeed be responsible, in part at least, for synovial inflammation in rheumatoid arthritis. In nephritis

Another site for inflammation in rheumatic disease is the glomerulus in systemic lupus erythematosus. It has been shown that the glomerular lesions contain immunoglobulins and complement components, and it is increasingly evident that

Immunology

83

TABLE 4. NUCLEAR ANTIGEN CONTENT OF SYNOVIAL FLUID SPECIMENS

Disease

Number tested

Equivalence to single-strand calf thymus D N A (/~g/ml)

3 1 2 l 1

74-3, 31-8, 7.6 37.8 42.6, 0'42 2.9 0'2

RA SLE Gout Reiter's Infection

TABLE 5. ANTINUCLEAR FACTORS ( A N F ) AND RHEUMATOID FACTORS ( R F ) IN SERUM AND SYNOVIAL FLUID OF ADULTS WITH RHEUMATOID ARTHRITIS ANF

RF

4 2 8 7

4 0 13 4

Serum only Synovial fluid only Both serum and synovial fluid Neither

a n t i n u c l e a r a n t i b o d i e s are a m o n g the i m m u n o g l o b u l i n s present. 19-22 It is i m p o r t a n t to d e m o n s t r a t e t h a t n u c l e a r a n t i g e n s are also p r e s e n t at this site i f we are to establish t h a t the m e c h a n i s m for i n f l a m m a t i o n is the s e q u e s t r a t i o n o f n u c l e a r a n t i g e n - a n t i n u c l e a r a n t i b o d y c o m p l e x e s . 22 T o a p p r o a c h this p r o b l e m we e x a m i n e d s u c h g l o m e r u l i in i m m u n o f l u o r e s c e n c e tests utilizing a fluorescein c o n j u g a t e o f the ~,-globulin c o m p o n e n t o f a p o o l o f l u p u s sera ( T a b l e 6). S u c h a r e a g e n t is c a p a b l e o f d e t e c t i n g a v a r i e t y o f n u c l e a r antigens. I n a d d i t i o n , we used t h e h i s t o c h e m i c a l stain, a c r i d i n e o r a n g e , f o r the d e t e c t i o n o f n u c l e a r a n t i g e n s in these sections. Fig. 6 s h o w s the b a s e m e n t m e m b r a n e in t h e l u p u s g l o m e r u l u s b r i g h t l y stained w i t h a n t i n u c l e a r a n t i b o d y , d e m o n s t r a t i n g the p r e s e n c e o f n u c l e a r antigens. Fig. 7 s h o w s the s a m e a r e a b r i g h t l y stained w i t h a c r i d i n e o r a n g e . O n l y in p a t i e n t s w i t h l u p u s n e p h r i t i s did we find e v i d e n c e to suggest t h a t n u c l e a r antigens, a n t i n u c l e a r a n t i b o d i e s , a n d c o m p l e m e n t c o m p o n e n t s , as well as f i b r i n o g e n , m i g h t be i n v o l v e d in the lesion. TABLE 6. GLOMERULAR LOCALIZATION OF IMMUNOLOGIC FACTORS IN RENAL BIOPSIES*

Pt. R.C. B.M.

P.M. S.G. C.B.

J.N. S.W. N.S. R.S.

V.B.

Disease Chronic inflammation and scar. Acute glomerulonephritis Subacute and chronic glomerulonephritis Subacute and chronic glomerulonephritis C h r o n i c glomerulonephritis Malign. nephrosclerosis Lupus nephritis Lupus nephritis Lupus nephritis Lupus nephritis (?)

Serum ANF

"Nuclear" AG A.O.

G

-?

. !

-

i i i

~ !-i~

tr. ~ b.v. r- + + + Jtr.

.

.

.

fl~C

.

Serum C'

Fibr.

T

-

~,

non-sp.

i ; ] ---inon-sp. ~z

----Jz -- ?

* Y. B. Bickel, E. V. Barnett and R. L. Vernier, unpublished data.

F i J -i ltr. + b.v. + + [ J tr.

~,

--

1' 1" $ $ $ 1"

+ + -non-sp. glom.

84

EUGENE V. BARNETT

INFLUENCE OF RHEUMATOID FACTOR IN NEPHRITIS Davis ~3 suggested that rheumatoid factor may protect patients with systemic lupus erythematosus from renal involvement. He suggested this on the basis of his obervation that rheumatoid factor in certain experimental situations inhibited complement fixation by antigen-antibody complexes. In our experience (Table 7), the detection of rheumatoid factor bore no relation to the presence or absence of renal disease in systemic lupus erythematous. TABLE 7.

INCIDENCE OF RENAL DISEASE AND RHEUMATOID FACTORS

(RF)

IN SYSTEMIC

LUPUS ERYTHEMATOSUS R e n a l disease RF

Present

Absent

Total

14 13 27

13 1l 24

27 24 51

Present Absent Total

SLE SLE with nephritis S L E w i t h o u t nephritis

Neg 13 11

80 3 2

ALEUTIAN

160 4 2

DISEASE

320 4 2

640 0 2

RF titer 1280 1 1

2560 0 2

5120 2 0

5120 0 2

OF MINK

Even if further investigations support these suggestions of the pathogenetic significance of nuclear antigen-antibody interactions, we still have not answered the question as to the origin of either the antigens or the antibodies. It is certainly possible that the immunogen which initially elicits the antinuclear antibody is not an autologous nuclear material but rather infectious nuclear material. One animal disease which may bear on this question is Aleutian disease of mink. This disease is characterized by vasculitis, nephritis, and hypergammaglobulinemia. 24, 25 The disease can be transferred from mink to mink by ultrafiltrates of tissues or serum. We recently had the opportunity of studying mink sera supplied by Dr. Ralph C. Williams, Jr., and his associates. Nuclear antigens in mink sera

Utilizing human lupus serum as the reagent capable of detecting nuclear antigen, we calculated that there was over 100/~g/ml equivalent of single-strand calf thymus D N A in one pool of serum taken before onset of and a comparable amount in a second pool taken after the onset of Aleutian disease. When similar pools were extracted with perchloric acid and the extracts tested with the diphenylamine reagent for deoxyribose, there was 92/xg/ml prior to disease and 40 tzg/ml after onset. Utilizing rabbit antibody capable of detecting single-strand and native calf thymus DNA, we tested 16 specimens of mink sera obtained prior to onset and another 16 obtained after onset of the disease; the mean level was 2-7 and 2.3 t~g/ml (equivalent to single-strand calf thymus DNA) before and after onset of disease, respectively, or 8"9 and 6.6/xg/ml (equivalent to native calf thymus DNA). In addition, these mink sera had DNA antigen detectable in precipitin tests with both human and rabbit antibodies.

Immunology

85

A n t i n u e l e a r antibodies in m i n k sera

It was p a r t i c u l a r l y startling to detect a n t i n u c l e a r a n t i b o d i e s in these sera by i m m u n o fluorescence tests. W h e r e a s only seven o f fifteen m i n k sera tested before onset o f the disease h a d w e a k l y positive a n t i n u c l e a r antibodies, all sixteen sera h a d strongly positive tests for a n t i n u c l e a r a n t i b o d i e s after onset. F u r t h e r m o r e , n o n e o f the sera tested before onset h a d p r e c i p i t a t i n g a n t i b o d y for single-strand calf t h y m u s D N A . while seven o f thirteen tested after onset h a d precipitating a n t i b o d y detectable by 5 0 / z g / m l single-strand calf t h y m u s D N A (shown to be protein-free b y the L o w r y technique). R O L E O F V I R E M I A IN A U T O I M M U N I T Y A t present, we have no evidence that the nuclear antigen detected in these sera is associated with the infectious m a t e r i a l c a p a b l e o f t r a n s m i t t i n g the disease rather t h a n with the p r o d u c t s o f cell b r e a k d o w n as the result o f disease, but the detection o f such antigen in the sera p r i o r to onset o f the disease s u p p o r t s the f o r m e r possibility. In any case, here is an infectious disease which includes viremia, nuclear antigenemia, vasculitis, nephritis, h y p e r g a m m a g l o b u l i n e m i a , and the a p p e a r a n c e o f a n t i n u c l e a r antibodies. The n u m e r o u s investigations o f nuclear antigens a n d a n t i n u c l e a r antibodies in h u m a n disease has tended to incriminate these factors in i n f l a m m a t o r y processes. T h e relationship o f possible viremia to these factors is unclear at present, b u t further investigation is certainly w a r r a n t e d . REFERENCES 1. E. V. BARNETT, Calif. Med. J. 104, 463 (1966). 2. E. V. BARNETT, J. J. CONDEMI, J. P. LEDDY and J. H. VAUGHAN, dr. elin. hwest. 43, 1104 (1964). 3. E. V. BARNETT, J. P. LEDDY, J. J. CONDEMI and J. H. VAUGHAN, Ann. IV, Y. Avad. Sci. 124, 896

(1965). 4. E. V. BARNETT,J. J. CONDEMI, R. F. JACOX, F. S. NORTH and J. H. VAUGHAN, Attn. int. Med. 63, 100 (1965). 5. V. P. BUTLER, JR., S. M. BEISER, B. F. ERLANGER, S. W. TANNENBAUM,S. COHEN and A. BENDICH, Proc. hath. Acad. Sci. U.S.A. 48, 1597 (1962). 6. O. J. PLESCIA,W. BRAUNand N. C. PALCZUK,Proc. natn. Acad. Sci. U.S.A. 52, 279 (1964). 7. E. V. BARNETTand J. H. VAU~HAN,J. exp. Med. 123, 733 (1966). 8. E. V. BARNETT,Arthritis Rheum. 9, 488 (1966). 9. E. M. TAN, P. H. SCHUR, R. I. CARRand H. G. KUNKEL,J. clin. Invest. 45, 1732 (1966). 10. B. D. STOLLARand A. L. SANOBERG,J. Immun. 96, 755 (1966). 11. R. J. LAFFIN, W. A. BARDAWIL,W. N. PACHASand J. S. MCCARTHY, Am. J. Path. 45, 465 (1964). 12. A. S. TOWNES,C. R. STEWART, JR. and A. G. OSLER, Bull. Johns Hopkins Hosp. 112, 183 (1963). 13. R. J. CAMMARATA,G. P. RODNANand R. H. FENNELL, J. Am. reed. Ass. 199, 455 0967). 14. C. G. COCHRANEand P. A. WARD, in International SympoMum Immunopathology, p. 433. Grune & Stratton, New York (1965). 15. T. J. PEKIN, JR., T. I. MALININand N. J. ZVAIFLER, Ann. int. Med. 65, 1229 (1966). 16. E. V. BARNETT, J. BIENENSTOCKand K. J. BLOCH, J. Am. reed. Ass. 198, 143 (1966). 17. E. V. BARNETT, J. BIENENSTOCKand K. J. BLOCH, Arthritis Rheum. 7, 726 (1964). 18. N. J. ZVAIFLER, Arthritis Rheum. 8, 289 (1965). 19. P. FREEDMANand A. S. MARKOWITZ, Br. reed. J. 5286, 1175 0962). 20. C. KRISHNAN and M. KAPLAN, J. clin. Invest. 46, 569 (1967). K. SVEC, J. BLAIRand M. KAPLAN, J. clin. Invest. 46, 558 (1967), D. KOFFLER, P. SCHURand H. G. KUNKEL, Trans. Ass. Am. Physicians, May 2, 1967. J. S. DAVIS,rV, Arthritis Rheum. 9, 631 0966). J. B. HENSON,R. W. LEADER, J. R. GORHAMand G. A. PADGETT, Path. Veterinaria 3, 289 (1966). 25. D. D, PORTER, W. J. DIXON and A. E. LARSEN, ./. exp. Med. 121,889 (1965).

21. 22. 23. 24.

86

EUGENE V. BARNETT

COMMENTS Dr. Barnett's thesis was that DNA-antibody complexes fix complement and attach to cells, thereby inducing cell damage. The interaction of antibody with intact cells may not produce cell damage because of the lack of availability of cellular D N A in the extracellular environment. Dr. Johnson raised the possibility that nuclear antigens can be released at sites of cell damage and act as adjuvants to unrelated antigens. Dr. Johnson wanted to know if the release of nuclear antigen could be related to the shower of different antibodies occurring in certain diseases like lupus erythematosus. Dr. Barnett emphasized the studies by other groups of workers showing both a decreased and an increased antibody response to exogenously administered antigens in patients with lupus erythematosus. The question is unanswered at present, but one discussant emphasized the studies by Dixon's group at La Jolla, California, in which NZB-F1 hybrid mice with glomerulonephrit~s had DNAanti-DNA complexes plus other nuclear material in renal glomeruli. Dr. Barnett emphasized his own preliminary studies in which Aleutian mink disease has many immunologic similarities to the disease occurring in NZB mice. The need for greater correlative studies in this area was emphasized and agreed upon by several participants. Although Aleutian mink fail to get arthritis, one participant suggested that such animals be traumatized at various joints to determine if they subsequently get chronic arthritis, especially since they may have the antigen-antibody complexes necessary to elicit such a response. Dr. Weissmann stated that D N A stimulates blast transformation in patients with systemic lupus erythematosus if their lymphocytes are cultured with appro!arnate antigens. One of the mechanisms whereby joints are injured in diseases in which delayed hypersensitivity plays a role is through the transformation of small lymphocytes into large lymphocytes (blastoid cells) that are rich in acid hydrolases. There is no direct effect, in intact cells, of antigen-antibody complexes on lysosomes unless these antigen-antibody complexes are phagocytosed in the presence of complement. Antigens and antibodies, either alone or as complexes, have no effect on isolated lysosomes and cells. Apparently, antigen-antibody complexes effect the transformation of lymphocytes very much the way that phytohemagglutinin does, by inducing the tlansformation of a naive lymphocyte, which cannot injure tissue, to a blastoid cell, which can injure tissue. By estimate, the acid phosphatase and arylsulfatase activities of a transformed lymphocyte are roughly a third to a quarter of those of a polymorphonuclear leucocyte. It is not known how an aggressive lymphocyte becomes cytotoxic, and it is unclear how lymphocytes injure cells. It is clear that the transformed lymphocyte probably is not phagocytic. It may be that hydrolases gain access to other cells by extrusion of the entire granule. Dr. Cline did not think one needed to invoke lysosomal changes in lymphocytes in order for them to be cytotoxic. Lymphocytes sensitized to L-cells group around the L-cell, and the L-ccll dies. These presumably do not have the hypertrophied lysosomal system that occurs in blast-transformed lymphocytes.