Altankov, G.

Georgieva, M.

English

Varna Medical University Press: Journals

. 1 A D H E S I V E PROPERTIES OF LYMPHOCYTES — A POSSIBLE MECHANISM FOR T H E I R S E L E C T I V E DISTRIBUTION IN T H E ORGANISM G . A l t a n k o v , M . Georgieva K e y - w o r d s : ce l l adhesion — l y m p h o c y t e s — i m m o b i l i z e d substrate — p e r i p h e r a l l y m p h o i d organs . • , • * . ,. ' Although lymphocytes are usually referred to the category of so-called " m i -grat ing" or freely mobile cells, they remain for a certain time without fa i l at central or peripheral lymphoid organs (2). They can be seen here closely attached, i . e. adhered to underlying substrate. The reasons and concrete mechanisms of this event, recently called "lymphocyte homing" (4), are s t i l l not precisely enough determined. O n the other hand, it is an interesting fact that peripheral lympho-cytes (along w i t h other leukocytes) also may change their adhesive properties (AP) towards underlying substrate ( i . g. a glass plate) when they meet the anti-gen " i n v i t r o " (5). The intimate mechanisms of this phenomenon are s t i l l not c lar i f ied , too (6). That ' s why the investigation of lymphocyte A P " i n v i t r o " pre-sents a perspective direction of studying the physiology of these cells. I n the literature available there are not any exact statist ical data concerning the part of whole lymphoid population which possess A P , as wel l how A P change during cellular distribution in the organism. The aims of the present work were: 1 . To study at which degree peripheral lymphocytes show A P to glass surface in condition of short-term cult ivat ion " i n v i t r o " . 2. To perform a comparative stu-dy of A P lymphocytes obtained from various peripheral organs namely: blood, spleen, lymph nodes and Peyer 's plaques. Material and methods We studied lymphocyte A P from human blood taken from 19 healthy do-nors aged 28—42 years. As regards the second aim of our work for obvious rea-sons it was not possibly to perform comparative investigations of lymphocyte adhesiveness from different human lymphoid organs. That ' s why we accomplish-ed that only laboratory rats breed " W i s t a r " w i t h average weight 125 --15 g. Expe-riments were divided into 12 series in conformity to number of investigated ani-mals. Production of lymphocyte suspension H u m a n blood lymphocytes were separated after modified method of B o y u m (3) after, a borderline centrifugation w i t h Ficolurographin (m. w . 1,076) of d i l u -ted 1:1 by saline solution heparinized blood. A terminal concentration of 2.10 6 cells/ml was reached i n Difco-199 medium enriched wi th 10 % veal embryonal serum. The whole plate used i n experiments was sterilized and chemically clean. Lymphocyte v i t a l i t y was determined after the method wi th exclusion of the dye (0,23 % eosin-Y solution). Adhes ive properties of lymphocytes . 133 R a t lymphocytes were prepared from non-narcotized animals because it has been shown that the majority of narcoses cause cellular membrane changes. The lymphocytes were separated by the way described above. Spleen, Peyer's plaques, and mesenteric lymph nodes Were obtained postoperatively after k i l ­l ing the animals by gas emboly (20 m l air intracardiacly) . The organs were washed several times wi th a sterile saline solution. After that they were cut into small pieces (about 1:1 mm) and softly homogenized in Potter's homogenizer. T h i s suspension was filtrated through fine nylon net wi th pore size of 15 mm in order to remove the non-dispersed cells. W i t h a view to diminishing the admixture of other cells (erythrocytes, fibroblasts, mastocytes) we diluted the prepared cellu­lar suspension wi th nutr i t ive medium and centrifugalized it w i t h F i c o l urogra-phin (see above). A terminal concentration of 2,10 cells/ml wras reached. The v i t a l i t y of produced cellular suspensions ranged between 86 and 98 % . The pu­r i t y of obtained lymphocytes was over 90 %. The ratio of adhesive cells was de­termined after modified method of H a l l i d a y (1976) wi th Burker ' s chambers. There we put the lymphocyte suspensions. The incubation was 30 min at 37 °C in a humid chamber. We read the mean number of cells from a total of 20 middle-size squares before and after washing the chamber three times w i t h a saline solu­t ion. We determined the percentage of adhered cells according to the formula: о/ • M E A N N U M B E R O F C E L L S O N F I E L D A F T E R W A S H I N G /о a d h e s i o n - M E A N N U M B E R O F C E L L S O N F I E L D B E F O R E W A S H I N G X 1 0 0 The end results were processed stat ist ical ly after the method of var iat ional analysis . We found the levels of mean square deviation (S) and the confidential interval (p—0,05) according to the tabular method of Typet-Strelkov (1966). A n exemplary protocol resulted from one investigation only is shown on table 1 . T a b l e 1 % A D H E S I O N Rat number blood spleen J. nodes J Peyer's plaques 1 2 3 4 5 6 7 8 9 10 11 12 7,4 12,3 3 1 , 3 34 ,5 2 ,1 11,0 24 ,5 9 ,0 12,6 13,3 38,2 14,1 33,8 53,4 64,5 50,4 43 ,9 34,4 46 ,1 26,0 57,1 29,5 42 ,2 43 ,0 28,9 43,6 26,0 31 ,5 56,5 37,8 14.6 58,3 . 8 ,1 14,5 50,0 31 ,3 19,8 20 ,6 33 ,3 15,0 20 .7 Results and discussion The results received concerning human blood lymphocytes (see table 2) show that at an average 18,3=3,4 % of the whole blood lymphocyte population tend to adhere to glass. I n single individuals this percentage demonstrates a signifi­cant reproductiveness which is obvious also from the low rate of confidential 134 G . A l t a n k o v , M . Georgieva T a b l e 2 : Donor number Before washing . Af er washing % adhesion 1 1 1 r 2 pUVrf 3 I 6 7 10 11 12 13 14 15 16 17 5,70 10,95 2,13 10,01 7,23 6,31 9,12 4,63 8,35 в 3,85 4,64 9,30 25,80 20,30 9,10 0,54 2,33 0 ,56 0,70 0 ,82 0 ,67 0,78 1,50 1,80 2,11 2,32 0,73 1,31 2,40 3,10 1,50 3,25 A v e r a g e l e v e l 9,5 21 ,3 2l:l 11,3 10,6 8,6 32 ,4 21,0 16,6 22,0 19,0 28,2 25,8 12,0 7,4 35 ,6 18,3=—3,4 interval (^=3,4). The results concerning rat blood lymphocytes (—17,5 =±=3,3) are re lat ively near to the aforementioned ones. W i t h a v iew to solving the second aim we carried out a comparative investi­gation of lymphocyte A P from different peripheral lymphoid organs in rats . The end results show that these lymphocytes differ sharply in their adhesiveness as compared wi th blood lymphocytes. The percentage of adhered cells from spleen is at an average 44,4 3,8 %; of those from mesenteric lymph nodes 3 8 , 4 ± 5 , 2 %, and from Peyer's plaques 23,7 - 5 , 0 %, respectively. I t is obvious, that blood lymphocytes contain least adhesive cells (17,5 =±=3,8 % ) which depends most l ike ly on the fact that they are in the blood flow itself. I t is noteworthy, however, that approximately 1/5 of circulating lymphocytes display A P . I n our opinion the physiological explanation of this phenomenon is related to the level of microcirculatory unit and, especially, of postcapillary venules. I t is known that some blood lymphocytes abandon actively the blood flow (2) stick­ing to the vascular wal l and then move towards the interst i t ium. The impor­tant role of surface A P in the processes of lymphocyte movement is also proved i n other works (7). That ' s an entirely different state in peripheral lymphoid or­gans. I t is obvious that the percentage of adhered cells is significantly higher. I t ranges between 30 and 60 % wi th speen and lymph nodes and even exceedes these rates in some cases. T h i s increase of adhesiveness in lymphoid organs is most probably due to the "Lymphocyte homing" , i . e. the solid lymphocyte adhesion in follicles and their distribution in so-called "thymus dependent" and "thymus independent" zones (9). Thanks to Curt is ' et a l . (1973) works it could be clarif ied that this event is based on selective cell adhesion. On the other hand, the authors demonstrated that mature T - and B-lymphocytes could mutually influence their A P by secreting thermolabile factors, most probably, of protein nature. On the ground of the presented results and cited bibliographic data we could presume that the mechanism of lymphocyte distribution in the organism is humoral ly directly controlled by the own lymphoid system as it is realized Adhes ive properties of lymphocytes . 135 by the change of A P of mature immunocompetent cells. The fact that Peyer's pla­ques lymphocytes display re lat ively low A P is surprizing to a certain degree, indeed. I n them there is only a tendency but lack of stat ist ical ly reliable difference towards blood lymphocyte adhesion. I t takes unawareness because the immuno­competent cells are most compact and closely attached to underlying substrate, and, therefore, they must possess a high adhesiveness. We tend to explain this contradiction wi th the peculiarities of the method for lymphocyte preparation namely by "sof t " homogenization in case of which it is completely possible that most f i r m l y adhered cells cannot disperse at a l l . We can conclude that " i n v i t r o " received data concerning adhesive cell quan­t i ty from lymphoid organs are probably lowered towards their true levels in the organism. However, this doesn't impair our basic conclusion that functional lymphocyte redistribution in the organism is directly related w i t h the dynamic changes of their A P . R E F E R E N C E S 1 С T p е л к о в , Р . Б . Метод вычисления стандартной ошибки и доверительных интервалов при помощью таблицы. С у х у м и , Алашара , 1966. — 2. L i n g , N . P . S t i m u ­l a t i o n of l y m p h o c y t e s . М. , M e d i c i n a ( R u s s . ) , 1968. — З. В о у u m , A . J . Clin. Labor. Invest., 2 1 , 1968, S u p p l . 97 , 2 8 . - 4. H u d s o n , L . , F . С. H a y . P r a c t i c a l I m m u n o l o g y . B l a c k -w e l l S c i . P u b l i c a t i o n , 1980. — 5. H a 1 1 i d a y , W . G . Cancer Res . , 39 , 1979, 558. — 6. M a l u i s h , A . , W. Halliday. Cancer Re s . , 39, 1979, 625 . — 7. M с F а г 1 a n d , W . , H e i l m a n , D . , J . Moorhead. J . Ex peri m. Med., 124, 1966, 815. — 8. C u r ­t i s , A . , M . d е S о u s a. Nature New Biol., 224, 1973, 45 . — 9. P a r r o t , D . M . V . , M . A . d e S o u s a. Clin. Experim. Immunol., 8, 1971, 663—684 . А Д Г Е З И В Н Ы Е СВОЙСТВА ЛИМФОЦИТОВ К А К В О З М О Ж Н Ы Й М Е Х А Н И З М ИХ И З Б И Р А Т Е Л Ь Н О Г О Р А С П Р Е Д Е Л Е Н И Я В О Р Г А Н И З М Е Г . Алтынков, М . Георгиева Р Е З Ю М Е У 19 здоровых доноров исследованы периферические лимфоциты и вопрос о том, к а к а я част этих лимфоцитов проявляет адгезивные свойства к иммобилизированному субстрату — стеклу . У к а з ы в а е т с я на то , что в среднем 18,3 — , т. е. 3,4 % периферических лимфоцитов человека проявляет адгезивные с в о й с т в а . Проведено сопоставительное исследование количества адгезирующих клеток р а з л и ч ­ных периферических димфоидных органов крыс — крови, селезенки, мезентериальных лимфа­тических узлов и пейеровых бляшек . Процент адгезированных клеток с о с т а в л я е т соот­ветственно 17,5—3,3 ; 4 4 - 3 , 8 ; 3 8 , 4 - 5 , 2 и 23,7—=5,0. Обсуждается вопрос о лимфацитарной адгезии в физиологическом аспекте. Д е л а е т с я вывод , что функциональное разпределение иммунокомпетентных клеток организма н а х о ­дится в прямой зависимости от динамических изменений их адгезивных свойств . 

ADHESIVE PROPERTIES OF LYMPHOCYTES - A POSSIBLE MECHANISM FOR THEIR SELECTIVE DISTRIBUTION IN THE ORGANISM

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ADHESIVE PROPERTIES OF LYMPHOCYTES - A POSSIBLE MECHANISM FOR THEIR SELECTIVE DISTRIBUTION IN THE ORGANISM

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Varna Medical University Press: Journals