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Blood was taken f r o m the f e m o r a l a r t e r y , f e m o r a l vein, veins of the spleen, l i v e r , kidney, s t o m a c h , and intestine and f r o m the r i g h t h e a r t of dogs on the 3rd and 7th days of aseptic inflammation (at the time of m a x i m a l accumulation of leukopotetins inthe blood) and the leukopoietteactivity of the s e r u m was studied. Inflammation was p r o duced by subcutaneous injection of turpentine. The leukopoietie activity of the venous blood flowing f r o m the v a r i o u s o r g a n s was c o m p a r e d with m a t of a r t e r i a l blood. Blood flowing f r o m the kidneys had the highest leukopoietic activity. Blood f r o m the veins of the spleen, l i v e r , s t o m a c h , and intestines had a lower content of leukopoietins than a r t e r i a l blood.

Endogenous s u b s t a n c e s capable of inducing leukocytosis or of stimulating leukopoiesis have been given many n a m e s depending on the conditions and methods used for their detection [3]. Opinions differ r e g a r d i n g the site of f o r m a t i o n of these s u b s t a n c e s . According to J a p a n e s e w o r k e r s , the f a c t o r inducing neutrophilic leukocytosis, the p r e s e n c e of which they d e m o n s t r a t e d in the blood of a n i m a l s a f t e r injection of typhoid vaccine, nucleic acids, and various other s u b s t a n c e s , is f o r m e d in the l i v e r [7, 13, 18]. The s a m e view is held, on the b a s i s of their own investigations, by B e e r [8] and Linke and S c h r i c k e r [14]. Ludwig et al. [15] c o n s i d e r that a f a c t o r inducing leukocytosis and found in the blood a f t e r r e p e a t e d l e u k o p h e r e s i s , is f o r m e d in the spleen. It has also been suggested that a factor stimulating the r e l e a s e of g r a n u l o c y t e s f r o m the bone m a r r o w into the circulating blood [11], and also a f a c t o r inactivating leukopoietins [6, 19, 21] or delaying their f o r m a t i o n [14], are f o r m e d in the spleen. Studies of the granuloeytopoietic activity of e x t r a c t s of various o r g a n s of intact a n i m a l s have shown that kidney tissue e x t r a c t has the highest activity [9, 10]. These w o r k e r s concluded that the chief s o u r c e of granulocyt0poietin found in the blood is the kidneys. Delmonte et al. [10] do not rule out the p o s s i b i l i t y that leukopietins may be s t o r e d in the kidneys. Since the content of leukopoietins in the blood was less than in the kidney t i s s u e s , they also accept that leukopoietins m a y be inactivated in the blood by an inhibitor. Menkin [16] c o n s i d e r s that f a c t o r s inducing leukocytosis and found in the blood and exudate during i n f l a m m a t o r y conditions are f o r m e d in the i n f l a m m a t o r y focus f r o m d a m a g e d leukocytes. The r e s u l t s of the w r i t e r s ' e a r l i e r investigations [4] conducted on dogs and h o r s e s with aseptic inflammation induced by turpentine d e m o n s t r a t e d an i n c r e a s e d blood level of leukopoietins capable of stimulating g r a n u l o c y t o p o i e s i s in intact a n i m a l s , The g r e a t e s t accumulation of leukopoietins in the blood of dogs was o b s e r v e d 3 and 7 days a f t e r injection of turpentine.

Pathophysiological L a b o r a t o r y , Central Institute of Hematology and Blood T r a n s f u s i o n , Ministry of Health of the USSR, Moscow. ( P r e s e n t e d b y A e a d e m i c i a n of the Academy of Medical Sciences of the USSR N. A. Fedorov.) T r a n s l a t e d f r o m Byulleten' ]~ksperimental'noi Biologii i Meditsiny~ Vol. 73, No. 3, pp. 2 3 - 2 6 , M a r c h 1972. Original article submitted May 24, 1971.

9 Consultants Bureau, a division of Plenum Publishing Corporation, 227 West ]7th Street, New York, N. Y. 100]]. All rights reserved. This article cannot be reproduced for any purpose whatsoever without permission of the publisher. A copy of this article is available from the publisher for $15.00.

252

a

To d i s c o v e r f r o m which organs the leukopoietins e n t e r the blood s t r e a m , in the investigation described below the leukopoietic activity was studied in s e r u m obtained f r o m the blood flowing f r o m various organs of dogs with aseptic inflammation.

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E x p e r i m e n t s were c a r r i e d out on 12 dogs (males and females) weighing 12-22 kg. Aseptic inflammation was induced Fig~ 1. Serum leukopoietic activity of by a single subcutaneous injection of 50% turpentine (diluted blood taken f r o m various v e s s e l s of with mineral oil) in a dose of 2.5 ml/10 kg body weight. Six dogs on 3rd (a) and 7th (b) days of dogs were investigated 3 days, and another six dogs 7 days aseptic inflammation. A b s c i s s a : 1) after the injection of turpentine. Under morphine and thiof e m o r a l a r t e r y ; 2) femoral vein; 3) pental a n e s t h e s i a the f e m o r a l a r t e r y and vein of the animals splenic vein; 4) hepatic vein; 5) renal were dissected, the abdomen was then opened, and blood vein; 6) gastric vein; 7) intestinal vein; samples were taken f r o m the veins of the kidney, liver, spleen, 8) right heart; ordinate, leukopoietic stomach, and small intestine, and f r o m the right h e a r t (through activity (in units). a catheter i n s e r t e d into the jugular vein), the f e m o r a l a r t e r y , and the f e m o r a l vein. The s e r u m leukopoietic activity was tested on Wistar r a t s . The leukopoietic activity was judged f r o m changes in granulocytopoiesis 3 days after a single intravenous injection of 1 ml of the test s e r u m [5]. An i n c r e a s e of 1% in the number of i m m a t u r e granulocytes in the bone m a r r o w c o m p a r e d with the initial value was taken as the unit of activity. Each sample of s e r u m was injected into 3 or 4 animals and the mean r e s u l t s calculated and subjected to statistical analysis. The leukopoietic activity of blood s e r u m f r o m the vessels specified above was c o m p a r e d with the activity of a r t e r i a l blood. The activity of 95 samples of s e r u m f r o m 319 r a t s was studied. EXPERIMENTAL

RESULTS

The mean values for the serum leukopoietin content in blood taken from the vessels specified are given in Fig. i. On the 3rd day after injection of turpentine the mean content of leukopoietins in a sample of serum taken from an artery was 38 units. In blood from the femoral vein and kidney the content of leukopoietins was higher than in arterial blood, but a significant increase was found only in blood from the renal vein (65 units, P < 0.01)o The content of [eukopoietins in blood from the splenic and hepatic veins was significantly reduced (25 and 3 units; P < 0.05 and 0.01 respectively). The leukopoietic activity in blood from the intestinal veins was lower than that of arterial blood, but the difference was not sLatistically significant. Similar changes were observed after 7 days but the difference from the arterial blood was significant only in the case of blood obtained from the renal vein, in which the leukopoietin content was 4 times higher (78 units, P < 0.01) than in arterial blood (21 units). The results show that leukopoietins enter the blood stream from the kidneys. These findings, indicating a role of the kidneys in leukopoietin formation, are in agreement with others published in the literature [9, I0]. The workers cited found a high content of leukopoietic activity in the kidney tissuew of intact animals. The active substance was thermostable and nonprotein in nature [i0]. In inflammation 2 leukopoietins were found to be present: one thermostable, the other thermolabile [16]. The possibility cannot be ruled out that thermostable leukopoietinis formed frorn disintegrated leukocytes in the focus of inflammation. The leukopoietic activity of breakdown products of leukocytes has been demonstrated repeatedly [i, 2, 12, 17, 20]. After passage through the liver and spleen, the leukopoietie activity of the serum is reduced, possibly because of storage of theleukopoietins in these organs. The lowered activity of the blood flowing from the stomach and intestine is probably due to excretion of leukopoietins via the gastro-intestinal tract. LITERATURE 1. 2.

CITED

V . A . Almazov and I. S. Freidlin, Pat. Fiziol., No. 6, 48 (1965). Yu. V. Belyanchikova, in: C u r r e n t P r o b l e m s in Hematology and Blood Transfusion [in Russian[, No. 41, Moscow (1970), p. 309.

253

3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.

254

M . G . Kakhetelidze, Pat. Fiziol., No. 5, 83 (1970). M . G . Kakheteiidze, A. N. Shlygin, et al., Pat. Fiziol., No. 2, 79 (1970). M . G . Kakhetelidze, and Z. M.Dolgina, Probl. GematoI., No. 2, 53 (1971). A. Semenov and N. Uskov, Arkh. Biol. Nauk, 5, No. 1, 1 (1897). M. Anan, T r a n s . Soc. Path. Japan, 26, 252 (1936). A . G . Beer, Folia Haemat. (Leipzig), 66, 222 (1942). H . R . Bierman, Ann. New York Acad. Sci., 1 ! 3, 753 (1964). L. Delmonte, W. C. Starbuck, and R. A. Liebelt, Am. J. Physiol., 215, 768 (1968). J . G . Gostomzyk, C. F e e s e r , and G. R u h e n s t r o t h - B a u e r , Kiln. Wsehr., 4..~2, 231 (1964). N. Hashem and F. S. Rosen, Lancet, 1, 201 (1964). Eo Komiya, Die Z e n t r a l n e r v o s e Regulation des Blutes, Stuttgart (1956). A. Linke and K. T. Schricker, in: 5 Kongress der Europaischen Geselischaft fiir H~imatoligie Colloquium, Berlin (1956), p. 228. F . C . Ludwig, M. E. Smoke, and J. S. Wellington, Ann. New York Aead. Sci., 136, 784 (1967). V. Menkin, Biochemical Mechanisms in Inflammation, Springfield (1956). H . L . Meyerhof, J. Physiol. (London), 98, 21 (1940). C. Muto, T r a n s . Soc. Path. Jap., 2-6, 246 (1936). J. Muto, Acta Haemat. Jap., 20, 348 (1957). A. Nettleship, Am. J. Clin. Path., 10, 265 (1940). B. Steinberg, Arch. Path., 65, 237 (1958).