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Age-related occurence of antibodies to cardiac tissue and vessels in the sera of healthy persons and patients with atherosclerosis
Mechanisms of Ageing and Development, 2 (1973) 333-343 © Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands
333
A G E - R E L A T E D O C C U R R E N C E OF ANTIBODIES TO CARDIAC TISSUE A N D VESSELS IN T H E SERA OF H E A L T H Y PERSONS A N D PATIENTS WITH ATHEROSCLEROSIS
I R I N A S. G O L O D
bnmunological Laboratory, Institute of Balneology and Physiotherapy, Ministry of Health, RSFSR, Sverdlovsk (U.S.S.R.) (Received February 8, 1973; in final form August 10, 1973)
SUMMARY
The paper concerns the frequency and intensity of reactions between watersoluble protein of aorta and myocardium and sera of persons of different age patterns. Specificity of the seropositive reactions observed has been demonstrated with the help of inhibition reactions and selective adsorption methods. An increased occurrence of reactions to cardiovascular tissue with age has been noted. Statistically significant values of the frequency of these reactions was found using sera of atherosclerotic patients. In the case of atherosclerosis of aorta and coronary vessels the reactions to protein of myocardium were predominant. In atherosclerosis of cerebral vessels and hypertension the reactions to aortic protein were paramount. Possible mechanisms of these phenomena are discussed.
INTRODUCTION
During ageing in humans there occurs a considerable deposition of protein in the vascular tissue 1. In the present study we investigated possible reactions of the protein components of the aorta and myocardium with the sera of healthy persons and patients suffering from atherosclerosis. M A T E R I A L A N D METHODS
Antigens Water-salt extracts of cardio-vascular tissue were used as antigens. Samples of thoracic aorta and myocardium showing atherosclerotic changes were taken to obtain the atherosclerotic antigens from persons dying from coronary thrombosis in the age-range 65-85. The "normal" antigens represented the water-salt extracts of cardiovascular tissue. The "normal" persons dying at the age of 20-23 years from accidental causes. The tissue was washed thoroughly and homogenized in 0.85 ~o solution of sodium chloride, and extracted at ÷ 4 ° C for 18-20 h. The supernatant
334 was obtained by centrifuging at 2000 >: g for 20 rain, and used as the antigen. The protein content was adjusted to 0.5-0.6 mg/ml. The sera to be investigated for possible antibody activity was inactivated in a water bath at +56°C for 30 rain. Antibody activity was demonstrated by means of Boyden's passive hemagglutination reaction (RPHA), using tannic acid treated rabbit erythrocytes as carrier cells. The sera under test were freed from heterophile antibodies to rabbit erythrocytes by adsorption techniques. Specificity of the passive hemagglutination reaction with the patients' sera was verified by an inhibition method. Serial two-fold dilutions of the serum under investigation were mixed with an equal volume of the tissue extract containing 0.6 mg of protein per ml. The mixture was incubated for 30 rain at room temperature, then 0.1 ml of antigen-coated, tanned erythrocytes was added, and the mixture was placed at '-4°C for 18 20 h. Specificity of the seropositive reactions to the antigens of the cardiovascular system was also studied by a selective adsorption method. The tissue used for adsorption was washed thoroughly in running water, homogenized and mixed with the serum to be investigated in the ratio I : 4. The mixture was kept at -+ 4'~C for 18-20 h, then the serum was separated from the tissue homogenate and checked for complete adsorption in the passive hemagglutination reaction. The sera under investigation have been tested in this reaction before and after activation at different temperatures. Data on destruction of organo-specific antibodies at +65°C 3,~ served as the basis for carrying out the experiments. RESULTS 700 persons aged 20-80 years were studied. Of these, 300 were healthy donors and 400 were patients with atherosclerosis. For healthy persons aged 20 49 the incidences of seropositive reactions are shown in Table I. Normal sera tended to react more with extracts of normal tissues
TABLE 1 PASSIVE HEMAGGLUTINATION REACTIONS WITH WATER SALT EXTRACTS OF CARDIOVASCULAR TISSUE AND BLOOD SERA OF HEALTHY DONORS OF DIFFERENT AGE GROUPS R P H A with water-salt extracts
Normal thoracic aorta Normal myocardium Atherosclerotic thoracic aorta Atherosclerotic myocardium
Frequency o f seropositive reactions ( ° o ) , in three age groups
Sero-positive reactions
20-29
30-39
40-49
~Z 2
P
28.95 18.62 11.57 1.66
15.05 20.2 6.44 2.15
25 25 15 10
3.0 2.14 0.7 7.8
0.05 0.05 0.05 0.05
335 TABLE II PASSIVE HEMAGGLUTINATION REACTIONS WITH WATER-SALT EXTRACTS OF CARDIOVASCULAR TISSUE AND BLOOD SERA OF ATHEROSCLEROTIC PATIENTS Diagnosis
Seropositive reactions ,4therosclerotic thoracic aorta
Atherosclerotic myocardium
Normal thoracic aorta
Normal myocardium
Z2
P
Z2
P
Z2
Z2
>0.05
21.1
<0.001 1 9 . 5
<0.001 2 6 . 3
<0.001
>0.05 >0.05
>0.05 >0.05
>0.05 >0.05
Atherosclerosis of aorta and coronary vessels 7.91 Atherosclerosis of brain vessels 17.3 Hypertension 17.3
<0.001 <0.001
2.74 4.6
P
1.24 7.8
2.1 0.77
P
t h a n with atherosclerotic tissues. The differences were most evident in the d o n o r s aged 20-29 a n d 30-39. W i t h the exceptions of reactions with the extract of n o r m a l thoracic aorta, the frequency of seropositive reactions was considerably higher in persons aged 20-29 years. A particularly high rate of increase of positive reactions was observed with the extract of atherosclerotic m y o c a r d i u m , increasing from 1.66 ~o at the age of 20-29 to 10~o at the age of 40-49. However, n o n e of the changes illustrated in Table I with sera from healthy d o n o r s reached statistical significance (P > 0.05). Also, the positive reactions occurred mainly at low serum dilutions. The m e a n titres of seropositive reactions with the extracts of a o r t a a n d m y o c a r d i u m did n o t exceed 1 : 8. As shown in Table II, the frequency of seropositive reactions with extracts of aorta a n d m y o c a r d i u m rose considerably when sera of atherosclerotic patients were employed. Sera from patients with severe aortic a n d c o r o n a r y atherosclerosis tended to react more frequently with the protein of atherosclerotic m y o c a r d i u m , while
TABLE Ill PASSIVE HEMAGGLUTINATION REACTIONS WITH WATER-SALT EXTRACTS OF AORTA AND MYOCARDIUM IN PERSONS SUFFERING FROM ATHEROSCLEROSIS OF CORONARY VESSELS OF DIFFERENT AGE GROUPS R P H A with water-salt extracts
Seropositive reaction frequency in two age groups 40~19
Atherosclerotic myocardium Normal myocardium Atherosclerotic thoracic aorta Normal thoracic aorta
50-59
Z2
P
Z2
P
22.1 29.8 13.6 8.3
< 0.001 ~<0.001 <0.01 <0.05
27,9 19.5 0.61 11.6
< 0.001 <0.001 >/0.05 <0.01
336 TABLE IV INFLUENCE OF SPECIFIC AND NON-SPECIFIC INHIBITION ON THE ACTIVITY OF BLOOD SERUM WITH THE EXTRACT OF NORMAL THORACIC AORTA hlve.s'tigatio, method
RPHA titre with extract of normal thoracic aorta RIHA with extract of normal thoracic aorta RIHA with liver extract RIHA with brain extract
Blood ser,m o f person.s' under observation I
2
3
4
5
6
7
,v
1:80 negative 1:80 1:80
1:80 negative 1:80 1:80
1:40 negative 1:40 1:40
1:40 negative 1:40 1:40
1:40 negarive 1:40 1:40
1:80 negarive 1:40 1:40
1:40 negative 1:40 1:40
1:160 negative I :l 60 1:160
sera from cases of cerebral atherosclerosis and hypertension tended to react with extracts from atherosclerotic aortas. Some clearly defined age differences in the nature and frequency of seropositive reactions are shown in Table Ill. At the age of 40-49 the reactions to the protein of unchanged myocardium ( Z z = 29.8)predominated, whereas at age 50-59 the reactions with atherosclerotic myocardium was most frequent (Z2 27.9), and occurred in 67.68 o/,, of cases. Removal of blood-groups specific antibodies by specific absorption did not affect the activity of the sera in the passive hemagglutination reaction with the extracts of aorta and myocardium. Specificity of seropositive reactions to the antigens of aorta and myocardium was demonstrated in the passive hemagglutination reaction by using the method of selective adsorption. The positive reactions with the extract of normal aorta were inhibited by prior absorption with this tissue but not by absorbing with extracts of liver or brain (Table IV). The positive reactions with the extract of atherosclerotic myocardium were inhibited by prior absorption with this antigen, but not by absorption with liver and brain (Table V). The positive reactions with the extract of the normal myocardium followed a similar pattern of specific absorption (Table VI). After the sera under test had been treated with homogenate of atherosclerotic thoracic aorta until no longer reactive with this tissue, they nevertheless remained active in relation to extracts of the normal thoracic aorta and atherosclerotic myocardium (Table VI). The lowering of activity of the sera in reaction with these antigens did not exceed 1-2 dilutions, which might be due to the presence of common antigenic components which, according to some investigators 5, are present in aorta and myocardium. The treatment of the sera with homogenate from normal myocardium did not alter the activity of the sera in the passive hemagglutination reaction with the extract of atherosclerotic aorta. The passive hemagglutination reaction with the control test-antigen (liver extract) remained negative meanwhile (Table VII).
R P H A with extract of atherosclerotic myocardium R I H A with liver extract R I H A with extract of atherosclerotic brain R I H A with extract of atherosclerotic myocardium R P H A titre with extract of normal myocardium R1HA with extract of normal myocarduim R I H A with extract of normal liver R I H A with extract of normal brain
Investigation method
1:32 1:32 1:32 neg. 1:128 neg. 1:32 1:32
1:16 1:16
neg.
1:32
neg.
1:32
1:32
2
1:16
1
1:64
1:64
neg.
1:64
neg.
1:64 1:64
1:64
3
1:128
1:128
neg.
1:128
neg.
1:64 1:64
1:64
4
Blood serum o f persons under observation
1:128
1:128
neg.
1:128
neg.
1:64 1:64
1:64
5
1:128
1:128
neg.
1:64
neg.
1:64 1:64
1:64
6
1:128
1:128
neg.
1:128
neg.
1:128 1:128
1:128
7
1:128
1:128
neg.
1:128
neg.
1:256 1:256
1:256
8
1:128
1:128
neg.
1:128
neg.
1:64 1:64
1:64
9
1:128
1:128
neg.
1:128
neg.
1:64 1:64
1:64
10
1:128
1:128
neg.
1:128
neg.
1:32 1:32
1:32
11
I N F L U E N C E O F SPECIFIC A N D N O N - S P E C I F I C I N H I B I T I O N O N T H E ACTIVITY OF B L O O D S E R U M IN R E A C T I O N W I T H T H E EXTRACTS OF M Y O C A R D I U M
TABLE V
-.-O
338 TABLE VI ACTIVITY OF H U M A N BLOOD SERUM IN REACTION WITH WATER-SALT EXTRACTS OF TISSUES AFTER TREATING THE SERA WITH HOMOGENATE OF ATHEROSCLEROTIC THORACIC AORTA Blood serum
1 2 3 4 5 6 7 8 9 10 11 12 13
R P H A titre with w a t e r salt extracts Tiss:le .................... usedJbr AtheroNormal Atheroadsorbtion sclerotie thoracic sclerotic thoracic aoFta myocardium
R P H A 1itre -Atherosclerotic thoracic
aorla
aorta
1:32 1:64 1:128 1:64 1:128 1:128 I :128 1:64 1:64 1:32 1:64 1:64 I :64
1:1o 1:16 1:64 1:64 1:32 1:16 1:64 neg. neg. 1:16 I :64 1:16 1:32
1:32 1:64 1:64 1:64 1:128 1:128 1:128 1:64 I :64 1:32 1:64 1:64 1:32
neg. neg. neg. neg. ncg. neg. neg. neg. neg. neg. neg. neg. neg.
.2 E ~ ~ 2 ~ .~ ~ o ~ .2 o ~ u ~ ~ ~_j ~ o -r
Absorption with normal thoracic aorta
with water salt exH'aets -Normal Atherothoracic sclerotic HOF[a IH|Y)cardillnl
1:16 1:16 1:32 1:32 1:32 1:16 I :64 neg. neg. 1:8 I :64 1:16 I :32
:16 :16 :32 :16 :128 :128 :64 :32 :32 :32 :32 :32 :32
r e m o v e d activity against the same
tissue. A d s o r p t i o n w i t h t i s s u e o f t h e a t h e r o s c l e r o t i c a o r t a a n d m y o c a r d i u m did n o t r e m o v e t h e a c t i v i t y o f s e r a in t h e p a s s i v e h e m a g g l u t i n a t i o n r e a c t i o n w i t h t h e e x t r a c t o f t h e n o r m a l a o r t a (see T a b l e VIII). TABLE VII I N F L U E N C E OF ADSORPTION WITH H O M O G E N A T E OF N O R M A L MYOCARDIUM ON THE ACTIVITY OF BLOOD SERUM 1N RPHA WITH THE EXTRACTS OF AORTA Blood
R P H A with water-salt extracts o f tissues
serllm
1 2 3 4 5 6 7 8 9 10 11 12
Atheroselerotie thoracic aorta (before ads'orption)
Liver
Atheroselerotie thoracic aorta (after adsorption)
Liver
1:64 1:64 1:32 1:32 1:128 1:128 1:64 1:128 1:16 1:64 1:32 1:32
neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg.
1:32 1:32 1:16 1:16 1:64 1:32 1:32 1:32 1:16 1:32 1:32 1:32
neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg.
1:32
neg.
1:16
1:32
1:16
neg.
1:32
1:64
1:32
1:16
1:16
1:8
neg.
1:16
1:8
1:32
1:16
neg.
1:32
1:32
1:128
1:64
neg.
1:128
5
1:64
4
Before adsorption After adsorption with homogenate of normal aorta After adsorption with homogenate of atherosclerotic thoracic aorta After adsorption with homogenate of normal myocarduim After adsorption with homogenate of atherosclerotic myocardium
3
1
o f thoracic aorta
2
Blood serum o f persons under investigation
R P H A with extract
1:16
1:64
1:64
neg.
1:64
6
1:32
1:64
1:32
neg.
1:64
7
1:32
1:64
1:64
neg.
1:128
8
1:16
1:16
1:8
neg.
1:16
9
1:16
1:32
1:32
neg.
1:64
10
1:8
1:32
1:32
neg.
1:32
11
1:64
1:64
1:32
neg.
1:128
12
INFLUENCE OF SELECTIVE ADSORPTION ON THE ACTIVITY OF S E R U M RPHA WITH THE EXTRACT OF N O R M A L THORACIC AORTA
TABLE VIII
1:16
1:64
1:16
neg.
1:64
13
R P H A with extract of normal myocardium R P H A with extracts of normal myocardium after treatment of sera under test with homogenate of normal myocardium R P H A with extract of atherosclerotic myocardium R P H A with extract of atherosclerotic myocardium after treatment of sera with normal myocardium
Investigation method
1:128
neg. 1:32
1:32
neg. 1:16
1:32
2
1:32
1
1:32
1:64
neg.
1:64
3
1:32
1:64
neg.
1:128
4
1:64
1:64
neg.
1:128
5
1:32
1:64
neg.
1:64
6
1:32
1:32
neg.
1:128
7
1:64
1:128
neg.
1:128
8
1:64
1:256
neg.
1:128
9
Blood serum o f persons sufferhlg f r o m atherosclerosi~' o f aorta and coronary vessels
1:64
1:64
neg.
1:128
10
1:64
1:64
neg.
1:128
11
1:64
1:64
neg.
1:128
12
BLOOD S E R U M OF A T H E R O S C L E R O T I C PATIENTS IN R P H A W I T H E X T R A C T S OF M Y O C A R D I U M A F T E R T R E A T M E N T W I T H H O M O G E N A T E OF N O R M A L M Y O C A R D 1 U M
TABLE IX
f,.~o
R P H A with extract o f atherosclerotic m y o c a r d i u m before w a r m i n g up the s e r u m after w a r m i n g up at ÷ 5 6 ° C " after w a r m i n g up at ÷ 6 5 ° C R P H A with extract of n o r m a l m y o c a r d i u m before w a r m i n g up after w a r m i n g up at + 5 6 ° C d u r i n g l h after w a r m i n g up at ÷ 6 5 c C R P H A with extract o f atherosclerotic thoracic a o r t a before w a r m i n g up to the serum after warming up at ÷ 5 6 " C after w a r m i n g up at + 6 5 ° C R P H A with extract of n o r m a l thoracic aorta before w a r m i n g up after w a r m i n g up at ÷ 5 6 ° C during 1 h after w a r m i n g up at ÷ 6 5 ° C
M e t h o d o f investigation
1:16 1:16 neg. 1:32 1:32 neg.
1:32 1:32 neg. 1:64 neg. 1:64
1:128 1:128 neg.
1:32 1:32 neg. 1:80 neg. 1:80
2
1:32 1:32 neg.
1
1:16 neg. 1:16
1:64 1:64 neg.
1:128 1:128 neg.
1:32 1:32 neg.
3
1:64 neg. 1:64
1:64 1:64 neg.
1:64 1:64 neg.
1:64 1:64 neg.
4
1:16 neg. 1:16
1:64 1:64 neg.
1:128 1:128 neg.
1:64 1:64 neg.
5
Blood serum o f persons under observation
1:160 neg. 1:160
1:64 1:64 neg.
1:128 1:128 neg.
1:64 1:64 neg.
6
1:80 neg. 1:80
1:64 1:64 neg.
1:64 1:64 neg.
1:64 1:64 neg.
7
1:80 neg. 1:80
1:64 1:64 neg.
1:64 1:64 neg.
1:128 1:128 neg.
8
1:40 neg. 1:40
1:64 1:64 neg.
1:64 1:64 neg.
1:128 1:128 neg.
9
1:40 neg. 1:40
1:128 1:128 neg.
1:128 1:128 neg.
1:64 1:64 neg.
10
1:40 neg. 1:40
1:128 1:128 neg.
1:16 1:16 neg.
1:32 1:32 neg.
11
1:32 1:32 neg. 1:16 1:16 neg.
1:32 1:32 neg.
13
1:32 1:32 neg.
12
INFLUENCE OF DIVERSE TEMPERATURES ON THE ACTIVITY OF BLOOD SERUM OF PERSONS UNDER OBSERVATION IN RPHA WITH EXTRACTS OF AORTA AND MYOCARDIUM
TABLE X
L~3
342 Table IX shows that after adsorption with normal myocardial homogenate, the sera did not interact with the extract of this same tissue. At the same time adsorption with normal myocardial homogenate did not affect the activity of sera in the passive hemagglutination reaction with the extracts of atherosclerotic myocardium. Heating the sera at t 56"C for one hour did not affect their activity in the reaction with the antigens of aorta and myocardium. Heating to -t 65'C for one hour abolished the activity (Table X). DISCUSSION The rise with age in frequency of detection of organo-specific antibodies has been observed by various authors 6-s. The present investigations document a rise with age in the frequency of seropositive reactions to the antigens of the heart and vessels. The frequency of positive reactions to the antigens in the sera o f patients with atherosclerosis is statistically significant. Specificity o f the serum substances reacting with the tissue extracts as well as their ability to preserve activity at +56°C but to be destroyed at +65°C indicates that they are organo-specific antibodies. It is a known fact that the occurrence of the destructive and dystrophic changes in the aorta and other vessels increases with age. The change of protein components of the vessel walls and particularly the precipitation of protein in the inner envelope of the aorta creates conditions leading to auto-immunologic reactions to protein components of the vessels. In our investigations an increased incidence of antivascular antibodies has been found in atherosclerosis of cerebral vessels and in hypertension. The antivascular antibodies produce a toxic action and increase the cardiac permeability 9. Fixation of these antibodies in the walls of cerebral vessels may increase vascular permeability and lead to thrombogenesis 1°. The influence of hypertension is realized through the increase of vascular permeability 11. One may presume that the increased production of vascular antibodies in hypertension may contribute to the increase in vascular permeability and to progressive pathological processes. Morphological changes in coronary arteries have been observed in persons aged 30-39 years 1. The change in the myocardial blood supply affects also the properties of morphogenesis and, consequently, the antigenic structure of this tissue, which may lead to more frequent reactions to the myocardial antigens with age. In the ischaemic stage of coronary atherosclerosis the protein component of myocardium is disturbed. This is manifested by the change of nuclei and mitochondria of the muscle fibers 1. The appearance of immunological activity in the serum of patients suffering from atherosclerosis in relation to the nucleus function of myocardium has been established in the course of prior investigations 12. In the present work a certain age pattern dynamics to the antigens of myocardium and atherosclerosis has been established. In the age-group 40-49 reactions to the normal myocardial antigens were predominate, while in the group 50-59 the most frequent reactions were those to protein of atherosclerotic myocardium.
343 I n the course o f illnesses f e a t u r i n g i m m u n e d i s t u r b a n c e s there has been n o t e d a n increased activity o f lysosomes in m y o c a r d i u m 13. It is quite possible t h a t increased excretion o f l y s o s o m a l h y d r o l a s e s leads to a higher p r o t e o l y s i s o f the tissues a n d change in their structure, which c o u l d facilitate the f o r m a t i o n o f a u t o - a n t i g e n s a n d a u t o - a n t i b o d i e s . A n t i g e n - a n t i b o d y complexes so f o r m e d m a y d a m a g e the tissues 14. T a k i n g into c o n s i d e r a t i o n the m o r e frequent occurrence with age a n d atherosclerosis o f the reactions to the antigens o f a o r t a a n d m y o c a r d i u m , one m a y p r e s u m e t h a t with ageing the possibility o f f o r m a t i o n o f the a n t i g e n - a n t i b o d y complexes a n d their h a r m f u l effect o n the h e a r t a n d vessels rises considerably.
REFERENCES 1 G. G. Avtandilov, Dynamics of Atherosclerosic Process in Man, Medicina, Moscow, 1970, p. 173. 2 S. V. Boyden, The adsorption of proteins on erythrocytes treated with tannic acid and subsequent hemagglutination by antiprotein sera, J. exp. Med., 93 (2) (1951) 107-120. 3 S. V. Boyden, Natural antibodies and the immune response, Adv. Immunol., 5 (1966) 1-24. 4 D. C. Dumonde, Tissue-specific antigens, Adv. lmmunol., 5 (1966) 245-384. 5 D. G, Grigoryan et al., Immunologic and biochemical investigation of the dog's heart and aorta tissues with the experimental atherosclerosis, Bull. Exp. Biol. Med., 66 (8) (1968) 29-33. 6 S.A. Korol and M.P. Pintchuk, On auto-immune reactions in persons of different age patterns, I X Scientific Con]erence on Age Morphology, Physiology and Biochemistry, Moscow, 1969, pp. 350-351. 7 U. Serrafini, G. Torrigiani and C. Mascela, L'incidenza degli auto-anticorpi nella popolazione normale, Folia Allergol., 12 (2) (1965) 79-93. 8 G. Thomas, I. Lawrence, C. Ralph and J. Williams, Human antiglobulin and pepsin-digested 7-globulins, J. Exp. Med., 125 (2) (1967) 233-248. 9 M. G. Kishov, Haemorrhagic effect of angiocytotoxins, Cor Vasa, 11 (3) (1969) 204-219. 10 E. G. Larsky, The role of biochemical investigations in the study of the problem of transient disturbances of the brain blood circulation, Bull. Akad. Med. Sci. USSR, 6 (1967) 40-44. 11 A. L. Myasnikov, Hypertonic Illness and Atherosclerosis, Moscow, 1962. 12 1. S. Golod, The study of immunological activity of blood serum in relation to the nuclear fraction of the cardial muscle at atherosclerosis, Cardiologia, 11 (3) (1971) 65-69. 13 G. Weissmann, Lysosomes auto-immune phenomena and disease of connective tissue, Lancet, 2 (7374) (1964) 1373-1375. 14 F. J. Dixon, Antigen-antibody complexes and auto-immunity, Ann. N.Y. Acad. Sci., 124 (1965) 162 166.
333
A G E - R E L A T E D O C C U R R E N C E OF ANTIBODIES TO CARDIAC TISSUE A N D VESSELS IN T H E SERA OF H E A L T H Y PERSONS A N D PATIENTS WITH ATHEROSCLEROSIS
I R I N A S. G O L O D
bnmunological Laboratory, Institute of Balneology and Physiotherapy, Ministry of Health, RSFSR, Sverdlovsk (U.S.S.R.) (Received February 8, 1973; in final form August 10, 1973)
SUMMARY
The paper concerns the frequency and intensity of reactions between watersoluble protein of aorta and myocardium and sera of persons of different age patterns. Specificity of the seropositive reactions observed has been demonstrated with the help of inhibition reactions and selective adsorption methods. An increased occurrence of reactions to cardiovascular tissue with age has been noted. Statistically significant values of the frequency of these reactions was found using sera of atherosclerotic patients. In the case of atherosclerosis of aorta and coronary vessels the reactions to protein of myocardium were predominant. In atherosclerosis of cerebral vessels and hypertension the reactions to aortic protein were paramount. Possible mechanisms of these phenomena are discussed.
INTRODUCTION
During ageing in humans there occurs a considerable deposition of protein in the vascular tissue 1. In the present study we investigated possible reactions of the protein components of the aorta and myocardium with the sera of healthy persons and patients suffering from atherosclerosis. M A T E R I A L A N D METHODS
Antigens Water-salt extracts of cardio-vascular tissue were used as antigens. Samples of thoracic aorta and myocardium showing atherosclerotic changes were taken to obtain the atherosclerotic antigens from persons dying from coronary thrombosis in the age-range 65-85. The "normal" antigens represented the water-salt extracts of cardiovascular tissue. The "normal" persons dying at the age of 20-23 years from accidental causes. The tissue was washed thoroughly and homogenized in 0.85 ~o solution of sodium chloride, and extracted at ÷ 4 ° C for 18-20 h. The supernatant
334 was obtained by centrifuging at 2000 >: g for 20 rain, and used as the antigen. The protein content was adjusted to 0.5-0.6 mg/ml. The sera to be investigated for possible antibody activity was inactivated in a water bath at +56°C for 30 rain. Antibody activity was demonstrated by means of Boyden's passive hemagglutination reaction (RPHA), using tannic acid treated rabbit erythrocytes as carrier cells. The sera under test were freed from heterophile antibodies to rabbit erythrocytes by adsorption techniques. Specificity of the passive hemagglutination reaction with the patients' sera was verified by an inhibition method. Serial two-fold dilutions of the serum under investigation were mixed with an equal volume of the tissue extract containing 0.6 mg of protein per ml. The mixture was incubated for 30 rain at room temperature, then 0.1 ml of antigen-coated, tanned erythrocytes was added, and the mixture was placed at '-4°C for 18 20 h. Specificity of the seropositive reactions to the antigens of the cardiovascular system was also studied by a selective adsorption method. The tissue used for adsorption was washed thoroughly in running water, homogenized and mixed with the serum to be investigated in the ratio I : 4. The mixture was kept at -+ 4'~C for 18-20 h, then the serum was separated from the tissue homogenate and checked for complete adsorption in the passive hemagglutination reaction. The sera under investigation have been tested in this reaction before and after activation at different temperatures. Data on destruction of organo-specific antibodies at +65°C 3,~ served as the basis for carrying out the experiments. RESULTS 700 persons aged 20-80 years were studied. Of these, 300 were healthy donors and 400 were patients with atherosclerosis. For healthy persons aged 20 49 the incidences of seropositive reactions are shown in Table I. Normal sera tended to react more with extracts of normal tissues
TABLE 1 PASSIVE HEMAGGLUTINATION REACTIONS WITH WATER SALT EXTRACTS OF CARDIOVASCULAR TISSUE AND BLOOD SERA OF HEALTHY DONORS OF DIFFERENT AGE GROUPS R P H A with water-salt extracts
Normal thoracic aorta Normal myocardium Atherosclerotic thoracic aorta Atherosclerotic myocardium
Frequency o f seropositive reactions ( ° o ) , in three age groups
Sero-positive reactions
20-29
30-39
40-49
~Z 2
P
28.95 18.62 11.57 1.66
15.05 20.2 6.44 2.15
25 25 15 10
3.0 2.14 0.7 7.8
0.05 0.05 0.05 0.05
335 TABLE II PASSIVE HEMAGGLUTINATION REACTIONS WITH WATER-SALT EXTRACTS OF CARDIOVASCULAR TISSUE AND BLOOD SERA OF ATHEROSCLEROTIC PATIENTS Diagnosis
Seropositive reactions ,4therosclerotic thoracic aorta
Atherosclerotic myocardium
Normal thoracic aorta
Normal myocardium
Z2
P
Z2
P
Z2
Z2
>0.05
21.1
<0.001 1 9 . 5
<0.001 2 6 . 3
<0.001
>0.05 >0.05
>0.05 >0.05
>0.05 >0.05
Atherosclerosis of aorta and coronary vessels 7.91 Atherosclerosis of brain vessels 17.3 Hypertension 17.3
<0.001 <0.001
2.74 4.6
P
1.24 7.8
2.1 0.77
P
t h a n with atherosclerotic tissues. The differences were most evident in the d o n o r s aged 20-29 a n d 30-39. W i t h the exceptions of reactions with the extract of n o r m a l thoracic aorta, the frequency of seropositive reactions was considerably higher in persons aged 20-29 years. A particularly high rate of increase of positive reactions was observed with the extract of atherosclerotic m y o c a r d i u m , increasing from 1.66 ~o at the age of 20-29 to 10~o at the age of 40-49. However, n o n e of the changes illustrated in Table I with sera from healthy d o n o r s reached statistical significance (P > 0.05). Also, the positive reactions occurred mainly at low serum dilutions. The m e a n titres of seropositive reactions with the extracts of a o r t a a n d m y o c a r d i u m did n o t exceed 1 : 8. As shown in Table II, the frequency of seropositive reactions with extracts of aorta a n d m y o c a r d i u m rose considerably when sera of atherosclerotic patients were employed. Sera from patients with severe aortic a n d c o r o n a r y atherosclerosis tended to react more frequently with the protein of atherosclerotic m y o c a r d i u m , while
TABLE Ill PASSIVE HEMAGGLUTINATION REACTIONS WITH WATER-SALT EXTRACTS OF AORTA AND MYOCARDIUM IN PERSONS SUFFERING FROM ATHEROSCLEROSIS OF CORONARY VESSELS OF DIFFERENT AGE GROUPS R P H A with water-salt extracts
Seropositive reaction frequency in two age groups 40~19
Atherosclerotic myocardium Normal myocardium Atherosclerotic thoracic aorta Normal thoracic aorta
50-59
Z2
P
Z2
P
22.1 29.8 13.6 8.3
< 0.001 ~<0.001 <0.01 <0.05
27,9 19.5 0.61 11.6
< 0.001 <0.001 >/0.05 <0.01
336 TABLE IV INFLUENCE OF SPECIFIC AND NON-SPECIFIC INHIBITION ON THE ACTIVITY OF BLOOD SERUM WITH THE EXTRACT OF NORMAL THORACIC AORTA hlve.s'tigatio, method
RPHA titre with extract of normal thoracic aorta RIHA with extract of normal thoracic aorta RIHA with liver extract RIHA with brain extract
Blood ser,m o f person.s' under observation I
2
3
4
5
6
7
,v
1:80 negative 1:80 1:80
1:80 negative 1:80 1:80
1:40 negative 1:40 1:40
1:40 negative 1:40 1:40
1:40 negarive 1:40 1:40
1:80 negarive 1:40 1:40
1:40 negative 1:40 1:40
1:160 negative I :l 60 1:160
sera from cases of cerebral atherosclerosis and hypertension tended to react with extracts from atherosclerotic aortas. Some clearly defined age differences in the nature and frequency of seropositive reactions are shown in Table Ill. At the age of 40-49 the reactions to the protein of unchanged myocardium ( Z z = 29.8)predominated, whereas at age 50-59 the reactions with atherosclerotic myocardium was most frequent (Z2 27.9), and occurred in 67.68 o/,, of cases. Removal of blood-groups specific antibodies by specific absorption did not affect the activity of the sera in the passive hemagglutination reaction with the extracts of aorta and myocardium. Specificity of seropositive reactions to the antigens of aorta and myocardium was demonstrated in the passive hemagglutination reaction by using the method of selective adsorption. The positive reactions with the extract of normal aorta were inhibited by prior absorption with this tissue but not by absorbing with extracts of liver or brain (Table IV). The positive reactions with the extract of atherosclerotic myocardium were inhibited by prior absorption with this antigen, but not by absorption with liver and brain (Table V). The positive reactions with the extract of the normal myocardium followed a similar pattern of specific absorption (Table VI). After the sera under test had been treated with homogenate of atherosclerotic thoracic aorta until no longer reactive with this tissue, they nevertheless remained active in relation to extracts of the normal thoracic aorta and atherosclerotic myocardium (Table VI). The lowering of activity of the sera in reaction with these antigens did not exceed 1-2 dilutions, which might be due to the presence of common antigenic components which, according to some investigators 5, are present in aorta and myocardium. The treatment of the sera with homogenate from normal myocardium did not alter the activity of the sera in the passive hemagglutination reaction with the extract of atherosclerotic aorta. The passive hemagglutination reaction with the control test-antigen (liver extract) remained negative meanwhile (Table VII).
R P H A with extract of atherosclerotic myocardium R I H A with liver extract R I H A with extract of atherosclerotic brain R I H A with extract of atherosclerotic myocardium R P H A titre with extract of normal myocardium R1HA with extract of normal myocarduim R I H A with extract of normal liver R I H A with extract of normal brain
Investigation method
1:32 1:32 1:32 neg. 1:128 neg. 1:32 1:32
1:16 1:16
neg.
1:32
neg.
1:32
1:32
2
1:16
1
1:64
1:64
neg.
1:64
neg.
1:64 1:64
1:64
3
1:128
1:128
neg.
1:128
neg.
1:64 1:64
1:64
4
Blood serum o f persons under observation
1:128
1:128
neg.
1:128
neg.
1:64 1:64
1:64
5
1:128
1:128
neg.
1:64
neg.
1:64 1:64
1:64
6
1:128
1:128
neg.
1:128
neg.
1:128 1:128
1:128
7
1:128
1:128
neg.
1:128
neg.
1:256 1:256
1:256
8
1:128
1:128
neg.
1:128
neg.
1:64 1:64
1:64
9
1:128
1:128
neg.
1:128
neg.
1:64 1:64
1:64
10
1:128
1:128
neg.
1:128
neg.
1:32 1:32
1:32
11
I N F L U E N C E O F SPECIFIC A N D N O N - S P E C I F I C I N H I B I T I O N O N T H E ACTIVITY OF B L O O D S E R U M IN R E A C T I O N W I T H T H E EXTRACTS OF M Y O C A R D I U M
TABLE V
-.-O
338 TABLE VI ACTIVITY OF H U M A N BLOOD SERUM IN REACTION WITH WATER-SALT EXTRACTS OF TISSUES AFTER TREATING THE SERA WITH HOMOGENATE OF ATHEROSCLEROTIC THORACIC AORTA Blood serum
1 2 3 4 5 6 7 8 9 10 11 12 13
R P H A titre with w a t e r salt extracts Tiss:le .................... usedJbr AtheroNormal Atheroadsorbtion sclerotie thoracic sclerotic thoracic aoFta myocardium
R P H A 1itre -Atherosclerotic thoracic
aorla
aorta
1:32 1:64 1:128 1:64 1:128 1:128 I :128 1:64 1:64 1:32 1:64 1:64 I :64
1:1o 1:16 1:64 1:64 1:32 1:16 1:64 neg. neg. 1:16 I :64 1:16 1:32
1:32 1:64 1:64 1:64 1:128 1:128 1:128 1:64 I :64 1:32 1:64 1:64 1:32
neg. neg. neg. neg. ncg. neg. neg. neg. neg. neg. neg. neg. neg.
.2 E ~ ~ 2 ~ .~ ~ o ~ .2 o ~ u ~ ~ ~_j ~ o -r
Absorption with normal thoracic aorta
with water salt exH'aets -Normal Atherothoracic sclerotic HOF[a IH|Y)cardillnl
1:16 1:16 1:32 1:32 1:32 1:16 I :64 neg. neg. 1:8 I :64 1:16 I :32
:16 :16 :32 :16 :128 :128 :64 :32 :32 :32 :32 :32 :32
r e m o v e d activity against the same
tissue. A d s o r p t i o n w i t h t i s s u e o f t h e a t h e r o s c l e r o t i c a o r t a a n d m y o c a r d i u m did n o t r e m o v e t h e a c t i v i t y o f s e r a in t h e p a s s i v e h e m a g g l u t i n a t i o n r e a c t i o n w i t h t h e e x t r a c t o f t h e n o r m a l a o r t a (see T a b l e VIII). TABLE VII I N F L U E N C E OF ADSORPTION WITH H O M O G E N A T E OF N O R M A L MYOCARDIUM ON THE ACTIVITY OF BLOOD SERUM 1N RPHA WITH THE EXTRACTS OF AORTA Blood
R P H A with water-salt extracts o f tissues
serllm
1 2 3 4 5 6 7 8 9 10 11 12
Atheroselerotie thoracic aorta (before ads'orption)
Liver
Atheroselerotie thoracic aorta (after adsorption)
Liver
1:64 1:64 1:32 1:32 1:128 1:128 1:64 1:128 1:16 1:64 1:32 1:32
neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg.
1:32 1:32 1:16 1:16 1:64 1:32 1:32 1:32 1:16 1:32 1:32 1:32
neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg.
1:32
neg.
1:16
1:32
1:16
neg.
1:32
1:64
1:32
1:16
1:16
1:8
neg.
1:16
1:8
1:32
1:16
neg.
1:32
1:32
1:128
1:64
neg.
1:128
5
1:64
4
Before adsorption After adsorption with homogenate of normal aorta After adsorption with homogenate of atherosclerotic thoracic aorta After adsorption with homogenate of normal myocarduim After adsorption with homogenate of atherosclerotic myocardium
3
1
o f thoracic aorta
2
Blood serum o f persons under investigation
R P H A with extract
1:16
1:64
1:64
neg.
1:64
6
1:32
1:64
1:32
neg.
1:64
7
1:32
1:64
1:64
neg.
1:128
8
1:16
1:16
1:8
neg.
1:16
9
1:16
1:32
1:32
neg.
1:64
10
1:8
1:32
1:32
neg.
1:32
11
1:64
1:64
1:32
neg.
1:128
12
INFLUENCE OF SELECTIVE ADSORPTION ON THE ACTIVITY OF S E R U M RPHA WITH THE EXTRACT OF N O R M A L THORACIC AORTA
TABLE VIII
1:16
1:64
1:16
neg.
1:64
13
R P H A with extract of normal myocardium R P H A with extracts of normal myocardium after treatment of sera under test with homogenate of normal myocardium R P H A with extract of atherosclerotic myocardium R P H A with extract of atherosclerotic myocardium after treatment of sera with normal myocardium
Investigation method
1:128
neg. 1:32
1:32
neg. 1:16
1:32
2
1:32
1
1:32
1:64
neg.
1:64
3
1:32
1:64
neg.
1:128
4
1:64
1:64
neg.
1:128
5
1:32
1:64
neg.
1:64
6
1:32
1:32
neg.
1:128
7
1:64
1:128
neg.
1:128
8
1:64
1:256
neg.
1:128
9
Blood serum o f persons sufferhlg f r o m atherosclerosi~' o f aorta and coronary vessels
1:64
1:64
neg.
1:128
10
1:64
1:64
neg.
1:128
11
1:64
1:64
neg.
1:128
12
BLOOD S E R U M OF A T H E R O S C L E R O T I C PATIENTS IN R P H A W I T H E X T R A C T S OF M Y O C A R D I U M A F T E R T R E A T M E N T W I T H H O M O G E N A T E OF N O R M A L M Y O C A R D 1 U M
TABLE IX
f,.~o
R P H A with extract o f atherosclerotic m y o c a r d i u m before w a r m i n g up the s e r u m after w a r m i n g up at ÷ 5 6 ° C " after w a r m i n g up at ÷ 6 5 ° C R P H A with extract of n o r m a l m y o c a r d i u m before w a r m i n g up after w a r m i n g up at + 5 6 ° C d u r i n g l h after w a r m i n g up at ÷ 6 5 c C R P H A with extract o f atherosclerotic thoracic a o r t a before w a r m i n g up to the serum after warming up at ÷ 5 6 " C after w a r m i n g up at + 6 5 ° C R P H A with extract of n o r m a l thoracic aorta before w a r m i n g up after w a r m i n g up at ÷ 5 6 ° C during 1 h after w a r m i n g up at ÷ 6 5 ° C
M e t h o d o f investigation
1:16 1:16 neg. 1:32 1:32 neg.
1:32 1:32 neg. 1:64 neg. 1:64
1:128 1:128 neg.
1:32 1:32 neg. 1:80 neg. 1:80
2
1:32 1:32 neg.
1
1:16 neg. 1:16
1:64 1:64 neg.
1:128 1:128 neg.
1:32 1:32 neg.
3
1:64 neg. 1:64
1:64 1:64 neg.
1:64 1:64 neg.
1:64 1:64 neg.
4
1:16 neg. 1:16
1:64 1:64 neg.
1:128 1:128 neg.
1:64 1:64 neg.
5
Blood serum o f persons under observation
1:160 neg. 1:160
1:64 1:64 neg.
1:128 1:128 neg.
1:64 1:64 neg.
6
1:80 neg. 1:80
1:64 1:64 neg.
1:64 1:64 neg.
1:64 1:64 neg.
7
1:80 neg. 1:80
1:64 1:64 neg.
1:64 1:64 neg.
1:128 1:128 neg.
8
1:40 neg. 1:40
1:64 1:64 neg.
1:64 1:64 neg.
1:128 1:128 neg.
9
1:40 neg. 1:40
1:128 1:128 neg.
1:128 1:128 neg.
1:64 1:64 neg.
10
1:40 neg. 1:40
1:128 1:128 neg.
1:16 1:16 neg.
1:32 1:32 neg.
11
1:32 1:32 neg. 1:16 1:16 neg.
1:32 1:32 neg.
13
1:32 1:32 neg.
12
INFLUENCE OF DIVERSE TEMPERATURES ON THE ACTIVITY OF BLOOD SERUM OF PERSONS UNDER OBSERVATION IN RPHA WITH EXTRACTS OF AORTA AND MYOCARDIUM
TABLE X
L~3
342 Table IX shows that after adsorption with normal myocardial homogenate, the sera did not interact with the extract of this same tissue. At the same time adsorption with normal myocardial homogenate did not affect the activity of sera in the passive hemagglutination reaction with the extracts of atherosclerotic myocardium. Heating the sera at t 56"C for one hour did not affect their activity in the reaction with the antigens of aorta and myocardium. Heating to -t 65'C for one hour abolished the activity (Table X). DISCUSSION The rise with age in frequency of detection of organo-specific antibodies has been observed by various authors 6-s. The present investigations document a rise with age in the frequency of seropositive reactions to the antigens of the heart and vessels. The frequency of positive reactions to the antigens in the sera o f patients with atherosclerosis is statistically significant. Specificity o f the serum substances reacting with the tissue extracts as well as their ability to preserve activity at +56°C but to be destroyed at +65°C indicates that they are organo-specific antibodies. It is a known fact that the occurrence of the destructive and dystrophic changes in the aorta and other vessels increases with age. The change of protein components of the vessel walls and particularly the precipitation of protein in the inner envelope of the aorta creates conditions leading to auto-immunologic reactions to protein components of the vessels. In our investigations an increased incidence of antivascular antibodies has been found in atherosclerosis of cerebral vessels and in hypertension. The antivascular antibodies produce a toxic action and increase the cardiac permeability 9. Fixation of these antibodies in the walls of cerebral vessels may increase vascular permeability and lead to thrombogenesis 1°. The influence of hypertension is realized through the increase of vascular permeability 11. One may presume that the increased production of vascular antibodies in hypertension may contribute to the increase in vascular permeability and to progressive pathological processes. Morphological changes in coronary arteries have been observed in persons aged 30-39 years 1. The change in the myocardial blood supply affects also the properties of morphogenesis and, consequently, the antigenic structure of this tissue, which may lead to more frequent reactions to the myocardial antigens with age. In the ischaemic stage of coronary atherosclerosis the protein component of myocardium is disturbed. This is manifested by the change of nuclei and mitochondria of the muscle fibers 1. The appearance of immunological activity in the serum of patients suffering from atherosclerosis in relation to the nucleus function of myocardium has been established in the course of prior investigations 12. In the present work a certain age pattern dynamics to the antigens of myocardium and atherosclerosis has been established. In the age-group 40-49 reactions to the normal myocardial antigens were predominate, while in the group 50-59 the most frequent reactions were those to protein of atherosclerotic myocardium.
343 I n the course o f illnesses f e a t u r i n g i m m u n e d i s t u r b a n c e s there has been n o t e d a n increased activity o f lysosomes in m y o c a r d i u m 13. It is quite possible t h a t increased excretion o f l y s o s o m a l h y d r o l a s e s leads to a higher p r o t e o l y s i s o f the tissues a n d change in their structure, which c o u l d facilitate the f o r m a t i o n o f a u t o - a n t i g e n s a n d a u t o - a n t i b o d i e s . A n t i g e n - a n t i b o d y complexes so f o r m e d m a y d a m a g e the tissues 14. T a k i n g into c o n s i d e r a t i o n the m o r e frequent occurrence with age a n d atherosclerosis o f the reactions to the antigens o f a o r t a a n d m y o c a r d i u m , one m a y p r e s u m e t h a t with ageing the possibility o f f o r m a t i o n o f the a n t i g e n - a n t i b o d y complexes a n d their h a r m f u l effect o n the h e a r t a n d vessels rises considerably.
REFERENCES 1 G. G. Avtandilov, Dynamics of Atherosclerosic Process in Man, Medicina, Moscow, 1970, p. 173. 2 S. V. Boyden, The adsorption of proteins on erythrocytes treated with tannic acid and subsequent hemagglutination by antiprotein sera, J. exp. Med., 93 (2) (1951) 107-120. 3 S. V. Boyden, Natural antibodies and the immune response, Adv. Immunol., 5 (1966) 1-24. 4 D. C. Dumonde, Tissue-specific antigens, Adv. lmmunol., 5 (1966) 245-384. 5 D. G, Grigoryan et al., Immunologic and biochemical investigation of the dog's heart and aorta tissues with the experimental atherosclerosis, Bull. Exp. Biol. Med., 66 (8) (1968) 29-33. 6 S.A. Korol and M.P. Pintchuk, On auto-immune reactions in persons of different age patterns, I X Scientific Con]erence on Age Morphology, Physiology and Biochemistry, Moscow, 1969, pp. 350-351. 7 U. Serrafini, G. Torrigiani and C. Mascela, L'incidenza degli auto-anticorpi nella popolazione normale, Folia Allergol., 12 (2) (1965) 79-93. 8 G. Thomas, I. Lawrence, C. Ralph and J. Williams, Human antiglobulin and pepsin-digested 7-globulins, J. Exp. Med., 125 (2) (1967) 233-248. 9 M. G. Kishov, Haemorrhagic effect of angiocytotoxins, Cor Vasa, 11 (3) (1969) 204-219. 10 E. G. Larsky, The role of biochemical investigations in the study of the problem of transient disturbances of the brain blood circulation, Bull. Akad. Med. Sci. USSR, 6 (1967) 40-44. 11 A. L. Myasnikov, Hypertonic Illness and Atherosclerosis, Moscow, 1962. 12 1. S. Golod, The study of immunological activity of blood serum in relation to the nuclear fraction of the cardial muscle at atherosclerosis, Cardiologia, 11 (3) (1971) 65-69. 13 G. Weissmann, Lysosomes auto-immune phenomena and disease of connective tissue, Lancet, 2 (7374) (1964) 1373-1375. 14 F. J. Dixon, Antigen-antibody complexes and auto-immunity, Ann. N.Y. Acad. Sci., 124 (1965) 162 166.