PROSTAGLANDINS

EFFECTS OF PROSTAGLANDINS ON LEUKOCYTE MIGRATION

Emi Shibuya, Kanjiro Masuda and Yasuho Izawa

Department of Ophthalmology, School of Medicine, University of Tokyo, Bunkyo-ku, Tokyo, Japan, ll3

Abstract Artificially synthetized prostaglandins ( PGE I ,PGE 2 PGF la, and PGF 2« ) were found, using Boyden's cham~er, to induce significant migration of polymorphonuclear leukocytes ( PMNs ) of the rabbit; PGF2«had greater effects than PGE I or E 2 . A typical dose dependent relationship was found between the PMNs migration and PGF 2~ concentrations. Indomethacin pretreatments of rabbits did not significantly alter the PMNs migration indicating that PGs synthetized in vivo was not involved in the migration. PGF2« was placed in the lower compartment opposite to PMNs and also in the upper compartment together with PMNs. No significant difference was found in the number of migrated PMNs between the two experimental conditions. PGs diffusion occurred across the millipore filter separating the two compartments where the concentrations were almost equal at the end of 3 hours incubation. It was thus concluded that PGs effects are to induce random PMNs movements rather than to initiate chemotactic directional migration.

Acknowledgements This investigation was supported by a Grant from the Ministry of Education and also by a Project Grant "Behcet's disease " from the Ministry of Health. Reprint requests to : Kanjiro Masuda M.D.

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Introduction Evidences have been accumulated to show a role of prostaglandins ( PGs ) in inflammatory reactions. PGs have been found in exudative tissue fluids o b t a i n e d in a variety of experimental inflammatory reactions, e.g. delayed inflammation due to carageenin in the rat ( 1 ), burned tissues in the dog ( 2 ), rabbit's aqueous humor in traumatic iritis ( 3 )-( 5 ). PGs like activity was also found in pleural inflammatory exudate in the rat induced by turpentine ( 6 ). Injection of PGs at a low concentration, produces v a s o d i l a t a t i o n ( 7 ) - ( 9 ) and injection at a high concentration induces a tissue reactions mimicking inflammatory responses ( 10 )-( 12 ). In human subjects, PGs were found at a high c o n c e n t r a t i o n in the inflammed skin ( 13 ), aqueous humor in uveitis ( 14 ), in thyroid tumor and intestine ( 15 5, ( 16 ). It is of interest to study PGs effects on leukocyte migration w h i c h is one of the cardinal symptoms of inflammation. Kally et al ( 17 ) ( 18 ) and Higgs et al ( 19 ), reported, through studies using Boyden's chamber, that PGE 1 induced significant polymorpho - leukocytes (PMNs) migration, hut PGA 1 and PGF2« did not show such effect. They concluded that PGE may be included in one of the chemotactic factors. Chemotaxis is defined as directional PMNs m o v e m e n t taking place from low towards high concentration of chemotactic substances ( 20 )-( 22 ). Since Kaley et al placed PGE 1 only in the chamber opposite to that containing PMNs, the experimental conditions were deemed insufficient to decide w h e t h e r PG induced PMNs m o v e m e n t was the chemotactic phenomenon as defined above or PG activated random cell movement. The present experiments were therefore undertaken to investigate this aspect using synthetized PGs. Materials

and Methods

i. P r o s t a g l a n d i n s (PGs) Syntetized PGs, i.e. PGEI, E2 , FI« and F2« , were obtained from Ono Pharmaceutical Company. They were dissolved in Hank's solution immediately before use to a final c o n c e n t r a t i o n of 100/3 ~g per ml. 2. Indomethacin Solution Chondroic sulphate was dissolved in citric acid Na2HPO4 -buffer solution(pH 4.6) and indomethacin (Nippon Merk Banyu Company) was dissolved in the above solution at a concentration of 12.5 mg per ml. 3. P o l y m o r p h o n u c l e a r Leukocytes (PMNs) PMNs were o b t a i n e d from albino r a b b i t s weighting 3-5 kg by a technique described by Hirsch ( 23 ). It entails intraperitoneal injection of 250mi of 0.06 per cent oyster

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glycogen saline and c o l l e c t i o n of the ascitic fluid three hours later. The ascitic fluid was i m m e d i a t e l y c e n t r i f u g e d and PMNs were suspended in 0.5 per cent ovalbumin-Hank's solution at a p o p u l a t i o n of 2 x 106 per ml. 4. A s s a y of PMNs m i g r a t i o n Effects of various solutions on leukocytes m i g r a t i o n w e r e assayed using a m o d i f i e d Boyden's chamber ( 24 ), consisting of two, 1 ml c o m p a r t m e n t s separated b y a millipore filter 0.65 um in pore size and 13 m m in diameter. The lower compartment was filled w i t h 0.i ml of sample to be assayed, 0.65 ml of Hank's solution and 0.25 ml of 2 per cent o v a l b u m i n - H a n k ' s solution ; the upper e o m p a r t m e n t was filled w i t h 1.0 ml of the PMNs suspension. After 3 hours incubation at 37 ° C , the m i l l i p o r e filter was fixed with alcohol and stained w i t h Mayer's Hematoxylin and o b s e r v e d under a light m i c r o s c o p e at a m a g n i f i c a t i o n of 400 times. The number of PMNs which m i g r a t e d into the filter to a depth of more than 30 um was counted in 5 d i f f e r e n t fields and average n u m b e r per field was calculated. A. Controls Effects of casein solutions, 0.002, 0.02, 0.2, 0.4 and 2 per cent were tested and the r e l a t i o n s h i p b e t w e e n casein concentrations and the effects on PMNs m i g r a t i o n w a s studied. On the basis of these studies, 0.2 per cent casein was chosen as a standard p o s i t i v e control for subsequent determinations. For a n e g a t i v e control, Hank's solution was used and for a blank control, a m i x t u r e of 2 per cent o v a l b u m i n 0.25 ml and Hank's solution 0.75 ml was used in order to m a t c h with p r o t e i n c o n c e n t r a t i o n of the PMNs suspension. B. Relative a c t i v i t y of samples The sample activity to induce PMNs m i g r a t i o n was exp r e s s e d as the relative activity w i t h regard to that of the standard p o s i t i v e control. The relative activity was calculated by the following formula : Activity

of sample

Positive

control

-

Activity

of blank

control X i00

activity

- Blank

control

activity

C. Experiments w i t h p r o s t a g l a n d i n s The following 4 types of e x p e r i m e n t s were carried out. a) C o m p a r i s o n of various PGs Effects on PMNs m i g r a t i o n of various PGs were compared using P G E I , E 2 , F I ~ and F2~. Final c o n c e n t r a t i o n s of these PGs were 10/3 ~g per ml. b) Dose d e p e n d e n c e of the effects Various c o n c e n t r a t i o n s of PGF2a were tested for their effects on PMNs m i g r a t i o n and the effects were correlated w i t h PGF2~ concentrations.

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c) P G F 2 a in the upper c o m p a r t m e n t In order to examine w h e t h e r PGs induce directional PMNs m i g r a t i o n due to c o n c e n t r a t i o n gradient or induce random migration, P G F 2 a was placed in the upper compartment together w i t h PMNs suspension and the results c o m p a r e d w i t h the effects of PGF 2 c o n t a i n e d in the lower compartment. In this series of experlments, 0.i ml of P G F 2 a solutions of various c o n c e n t r a t i o n s was added to 0.9 ml of PMNs suspension w h i c h was p r e p a r e d to give a final p o p u l a t i o n of 2 x i0 6 per ml in the m i x t u r e : the m i x t u r e was placed in the upper c o m p a r t m e n t of B o y d e n ' s chamber , and the lower compartment contained 0.75 ml Hank's solution and 0.25 ml, 2 per cent o v a l b u m i n solution. PGF 2a effects on PMNs migration was studied as above. d) D i f f u s i o n of P G F 2 a It was i n v e s t i g a t e d w h e t h e r initial c o n c e n t r a t i o n of PGF 2a was m a i n t a i n e d during three hours incubation period. PGF 2a 100/3 ug, was put into the lower c o m p a r t m e n t and PMNs suspension in the upper c o m p a r t m e n t as in the c o n v e n t i o n a l m e t h o d d e s c r i b e d above. Samples were taken from both compartments 30, 60, 120 and 180 minutes after the incubation. Samples were c e n t r i f u g e d at 3000 r.p.m, for 10 minutes at 4 o C and the supernatants were subjected to a r a d i o - i m m u n o assay of P G F 2 a , a c c o r d i n g to the m e t h o d of Hirata et al. .

D. Inhibition w i t h indomethacin It was thought possible that PMNs o b t a i n e d from rabbit were already under influence of endogenous PGs. In order to e l i m i n a t e this possibility, rabbits were injected w i t h indomethacin ( 2 ml, i.e.,25 m g ) i n t r a p e r i t o n e a l l y 3 times one day and one hour before g l y c o g e n injection and one hour before collection of the ascitic fluid. The PMNs thus obtained were subjected to the d e t e r m i n a t i o n s of migration.

Results A. Control e x p e r i m e n t s PMNs m i g r a t i o n was assayed using various c o n c e n t r a t i o n s of casein solution ( Figure 1 ). A linear relationship was found b e t w e e n the n u m b e r of m i g r a t e d PMNs cells and logarithm of casein c o n c e n t r a t i o n s in the range between 2 x 10 -3 and 4 x 1 0 - I per cent. On the basis of these results, 0.2 per cent casein was chosen as a p o s i t i v e control for s u b s e q u e n t experiments. PMNs m i g r a t i o n by this positive control was assayed on twelve o c c a s i o n s and PMNs m i g r a t i o n by Hank's solution was also tested ( Table 1 ). The results indicate that 0.2 per cent casein solution is an adequate positive control. B. Experiments w i t h PGs Effects of PGS on PMNs m i g r a t i o n was studied using 10 / 3 ug of PGE i , E 2 , F la and F 2a , and the results are

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120

~100

~'2",eo Œ60

~° ~ o 2~io-~

I

I

2~10 -2

Concentration

Figure i.

I

2 ,~10-1 4,, 10-~

2,,10 o

of Casein(% )

Relationship between the number of migrated PMNs per visual field and casein concentrations. Triplicated experiments were carried out for each concentration.

Table 1 Number of migrated PMNs per visual field for Hank's solution and 0.2 per cent casein solution. Negative Control (Hank's solution)

Mig r at ion (CelIs/V.F.) Mean + S.D.

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87 + 8 (n=12)

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given by open bars in Figure 2. All PGs used in these experiments induced significant PMNs migration ( P