TRRDMBDSIS RESEARCH15; 297-307 Pergsmon Press

Ltd.1979. Printed in Grert.Mtb

TX-MIGRATION

fNHIBITDRYACTIVITYOF COHCAUAVALIW-AIN VIVO AND IN VITRO HDDIFICATIO#S SYDIPYRTDAMDLEANDACET%SALICYLICACfD

STIMULATED HUMAN LyHoflocyTEs.

E.

Coeugniet

Laboratory of clLnica1Immunology, MedicalDepartatant TAI Rigshospltalet, UniversftyHospital, Copenhagen, Denmark. x1 (Received 11.12.1978; in revised form 2.2.1979 Acceptedby EditorN. Goossens)

ItaTRoDDCTIoN Lysrghokines are msdiatorsof influrutionreleasedfrcm antigen or from sensitized lymphocytes stimulatedwith specific non-sensitized lymphocytes stimulated with mitcgens.The multiple biological activities of lymphokines couldpartically explainthe thraabotic processin the infmtory damageof cell-mediated iaaunereactionsand the beneficial effectof thrombocvte antiaggregant and anticoagulant therapy(1, 4, 12, 13, 14, 15). In vitroand in viva drugswith antiaggregant activitysuch as dipyridamole (DIPY),acetylsallcplic acid (ASA) or its soluble derivative Aysineacetylsalicylate (IASA)have a dwressive x1 New address:Set. Josef Hospital,509 Leverkusen, FederalRepublicof Germany. 297

effect on lymphokine production by lymphocytes stimulated with Concanavalin-A (Con-A) (4, 8). The present study examines the effect of DIPY, ASA and lymphokines on the ability of thrombocyte to migrate (18). MATERIALS AND METHODS Subjects and drug treatment: Venous blood lymphocyteswere isolated from healthy adult humans of both sexes and used for Con-A induced production of leucocyte migration inhibitory activity (LMIA)-rich and control supernatants as described elsewhere (4, 6, 7). The supernatants, pooled and lyophilized, were the "standard" LMIA preparations for all experiments. Fourteen subjects, male and female, healthy or with immunoinflammatory diseases were treated either with one drug or with the drug combinations (Table 1).

TABLE 1 Data of 14 Cases studied

Case no. 1 3 4 5 6 : 9 :: 12 13 14

Sex

Age

Diagnosis

9 d d 9 d 9 9 d 0 9 9 9 9 9

32 33 33 68 32 26 56 34 31 22 54 32 68 28

RA H

H

H H AA BA BA PA BA BA BA H LED

BA- rheumatoid arthritis Ii * healthy AA= allergic arthritis during Salmonella infection LED = disseminated lupus erythematosus The drugs were given orally in four daily doses at equal intervals. Blood was drawn before treatment (control)and at 1, 2, 4, 12, 24 and 48 h after the commencementof treatment. The same subjects were tested both during ASA, DIPY and combined ASA + DIPY administration.The interval between each treatment period was 7 days. The in vitro and in vivo production and effect of LMIA/T!4IAwere tested as well as the modification of these by ASA, DIPY and ASA + DIPY therapy. Preparation of drugs: For in vitro assays on thrombocytes, mpyridamole (DIPY) (Persantin@,Boehringer Ingelheim, Germany) and LASA (Aspegic9,Egic, Montargis, France) were .

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diluted/dissolved in drug-free thromhocyte poor plasma (TPP) from the study patients with penicillin 67 UI/ml and streptomycin 67 ug/ml (Difco Laboratories, Michigan, USA). The concentrations employed were comparable with clinically relevant plasma concentrations as shown in Table 2. TABLE 2 Final Concentrations (in vitro) of chemical Compound used in the Experiments. Clinically relevant Plasma Levels

Drugs

Concentrations used in the experiments

DIPY (pg/ml) LASA (pg/ml)

Therapeutic plasma concentrations l-lo

3

100-300

With respect of the salicylate levels obtained, 0.9 g of LASA was calculated in 0.5 g ASA equivalents.All drug preparations were immediately used for experiments. For in vitro assays on leucocytes the drugs were diluted/dissolved in tissue culture medium 199 (TC 199) (4, 5, 8). The LMIA of the lyophilized supernatants was tested after redissolution in TC 199 at a concentration corresponding to the initial supernatants (IC)I three times concentrated (3 x Ic), three times diluted (X/3), nine times diluted (X/9) and 27 times diluted (X/27). The technique has been previously described (5, 6). In assays with drugs we used only IC supernatants. For oral administration the subjects were given coated tablets each containing 100 mg DIPY and tablets each containing 590 mg ASA (MagnylB, Fh. Nord, Copenhagen, Denmark). The doses were 8-10 mg/kg/day at least 2 hours after the last meal and 1 hour before the next. Leucocyte migration assay: The method of Clausen was used with some modifications (4, 5, 6). In this system the leucocytes migrate under an agarose medium containing nutritive substances. Thrombocyte migration assay: Blood was obtained from patients who had not taken any drugs for at least one week and again after drug administration (as described in "Subjects and drug treatment") . The blood was mixed with 3 per cent trisodium citrate (1 part to 9 parts of blood) and thrombocyte rich plasma (TRP) was prepared by centrifugation of whole blood at 150 g for 10 min. The TRP was once more centrifuged at 150 g for 5 min in order to reduce the red and white cell contamination, The number of thrombocytes was adjusted to 300,000 per mm3 with autologous TPP, prepared by centrifuging a portion of the original blood at 2400 g for 30 min. TRP was collected in a suitable number of siliconized 20 pl glass capillary tubes (Drummond Memocaps@, Drummond Scientific Supplies, Broomal, Pa., USA). After sealing one end in a flame and centrifuging at 1200 g

300

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OF HUM?;

LYMPHOCYTES

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for 3.5 min the capillary tube was cut immediately above the surface of the thrombocyte button. The segment containing the button was placed in a 0.5 ml clear plastic circular migration chamber (disposable polystyrene chamber for cell migration, Sterilin Ltd., Middlesex, England) and anchored with a small amount of silicone grease (Dow Corning Corp., USA). The migration chamber was then filled with TPP and covered on the open side with a second siliconized cover glass. After 24 hours, incubation at room temperature the migration area of the thrombocytes around the opening of the capillary tube was studied with a projection microscope and measured by planimetry. The migration was studies in TPP containing different concentrations of drugs in active supernatants (AS)(from Con-A incubated lymphocytes) and control supernatants (CS). Yore details about the thrombocyte migration assay are found elsewhere (18). Assay for in vitro drug effect on leucocyte and thrombocyte migration: A migration index under drug effect in vitro of leucocytes (LMIdrug vitro) and thrombocytes TMIdrUg vitro) represented: mean migration area of cells under drug influence L?lIor TMIdrug vitro = mean migration area in drug-free medium AsSay for in vitro drug effect on leucocyte and thrombocvte migration: The ability of leucocytes/thrombocytes to migrate be ore and after treatment was studied and expressed as a migration index: mean migration area of cells after treatment LMI or TMI drug vitro = mean migration area of cells before treatment Assav for the effect of LYIA/TMIA containing supernatants on leucocvte/thrombocvte migration: The migration of leucocytes/ thrombocytesunder AS in different concentrations was compared with the-migration in similarly concentrated CS. The migration index of leucocytes/thrombocytes under AS influence was expressed as an index: mean migration area of cells under AS influence LMI or TMIAS = mean migration area of cells under CS influence ASSaY for in vitro and in vivo drug modifications of the effect of -IA on leucocytes and thrombocytes: Drugs were added to both AS and CS cell suspensions so to form the final concentrations indicated in Table 2 or given orally to patients. The areas of migration of leucocytes/thrombocytes in AS/CS containing drugs (or after drug therapy) or without drugs were measured and a migration index as described above calculated. Calculations: Each examination was repeated four times. The

TMIA OF HUMAN LY?4PIiOCYTES

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301

mean of the values observed in each group of the subjects at the same stage of treatment (in vitro or in viva) was calculated. The difference between the groups was evaluated by Wilcoxon's rank sum test. RESULTS LMIA and TWIA of supernatantsin different concentrations The LMIA containing supernatantsinhibited the migration of leucocytesin concentrationsdependent relation (Fig. 1). As CIC/9) did not significantlymodify the migration areas of leucocytesand the AS (K/27) induced a slightly but non-significant increase of the migration areas of leucocytes.Comparable results were obtained with the thrombocytemigration. However, the AS (X/27) increased the migration significantly (Fig. 1). Direct drug effect on leucocyte and thrombocytemigration The highest concentration DIPY tends to increase the MI the stimulationof employed caused significant (p ( Hi leucocytemigration (MI = 1.44 2 0.13). LASA did not significantly influence the leucocyte migration. The thrombocytes were not influenced. Effect of drug therapy (in vivo administration)on leucocyte and thrombocytemigration Treatment with DIPY and ASA or with combinationsdid not induce any significant in vivo modification of leucocyte migration and thrombocytemigration. ay& and thr~y~grat%n innibiteysctivity of supwmtants in diftwmt concmtmtions, Illgrotlon

index

13.

I 09 t 0.6 0.7 t

0.6 0.5 OL Llk

3XlC

IC s lnitiol concmtration.

cl . Lwcocyte¶. Ei . lhrombocytes

FIG. 1 Leucocyte and thrombocytemigration inhibitory activity of supernatantsin different concentrations. Vertical lines indicate + 1 SD.

302

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TXIX OF HIXkK LUPHOCYTES

Effect of drug on MIA

and T-IA production

In Vitro both DIPY and LASA in concentrations conparable with those obtained in blood during treatment caused significant inhibition of LMIA and TMIA production (Fig. 2). In viva the LMIA and TMIA production was not modified during treatment with one drug. Combined therapy for 24 hours, however, Signif-

icantly decreased the ability of lymphocytes to produce QUA and TMIA under Con-A stimulation (Fig. 3). In VI”0,tkct 0, d

onLhuAandrut* oroduct~on

12

!.tSD

It

i !

IO a9

1

:

I

0.9 07 06 05 04 I

C

UPV

IASA

In vitro effect of drugs on LMIA and TMIA production. Vertical lines indicate f SD

t Migration index

I *t SD 10 a9 08 07 06 as 0.L

‘.A!%

inhibition. IC uprmotantr .

CI Migration ASA s Acetylrolicylat9

octd

tlteton.

c

MPY * Diiidomok OtPY.ASA. Com3hd

WY

ASA

WY*As*

th9lapy thuan.

FIG. 3. In vivo effect of drugs on LMIA and TMIA production. Vertical lines indicate + SD

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303

Drug modification of leucocyte/thrombocyteresponse to LMIA/TMIA When leucocytes/thrombocytes were incubated in vitro with either DIPY or LASA, their ability to respond to a standard LMIA preparation was depressed in both cases (Fig. 4). Treatment with one of the drugs for more than 24 hours, however, did not decrease the leucocyte reactivity to LMIA, but did, however, decrease the thrombocyte response to TMIA. Only leucocytes from patients treated simultaneouslywith both DIPY and ASA were inhibited in their response to LMIA (Fig. 5).

In viva drw

modification of Ieucocyte / thrombocytc response to LMIA 1 MA.

Migration index

I

I =lSO

1.21.1 . 1.0 ag0.6 0.7 -

f

0.6 0.5 -

0.4-

c

C = Miation

rf

1

f

-L DIPY

ASA

I

inhibitian. IC supernatants.

ASA= Acetylsalicylate

acid therapy.

DIPY + Dipyridamole therapy.

DIPYeASA = Combined therapy.

FIG.

4

In vitro drug modification of leucocyte/ thrombocyte reaponse to LMIA/TMIA. Vertical lines indicate + SD

304

MIA OF HUMAN LYMPHOCYTES

In vitro drua modification of leucocyte/thrombo~yte

vo1.15,?io.3/4

response to LMIA I MA.

Migration index

t 1.2 1.11.0 0.9 0.6 0.7 -

f

0.6 cl50.1 -

c

)IpI

C s Migration inhibition obtained with IC supernatantt . DIPY = Dipyriclamde 10jJg/ml . LASA : Lysine - acetylsalicylate

FIG.

3 j&ml

5.

In vivo drug modification of leucocyte/ thrombocyte response to LMIA/TMIA. Vertical lines indicate + SD. DISCUSSION A biological activity of lymphokines is directed at the migration of thrombocytes.The thrombocytes react to lymphokines in a similar way to leucocytes by migration inhibition. The inhibition of thrombocytemigration is dependent on the concentrationof supernatantsand is antagonized by drugs known also to antagonize other lymphokine activities such as LMIA (4, 5, 81, thrombocyte aggregating activity (51, procoagulant activity (61, and granulocyte fibrinolytic releasing activity (7). The ability of thrombocytesto migrate and their reaction by migration inhibition to lymphokines could explain-the rapid accummulationand aggregationof thrombocytes in kidneys transplantedto specificallysensitized animal recipients (18). The observation is also substantiatedby the favourable effect of lymphokines inhibitors such as DIPY and ASA in the prevention

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TMIA OFHUMANLYMPHOCYTES

305

of the thromboticprocess in the inflammatorydamage caused by cell-mediatedimmune reactions (4, 5, 6, 12, 13, 14, 15). The present study showed a difference in response between leucocytesand thrombocytes: 1. The thrombocytesrespond to lower concentrationsof active supernatants. 2. In vivo they are also influenced by one-drug therapy. A satisfactoryexplanationof this difference is not possible at the moment. It is possible that the morphologicalstructure of thrombocytes:cell-fragmentswithout nucleus, with a short survival time and different enzymatic equipment is the key to this explanation. The thrombocytemigration assay offers the possibilityof studying the mechanism of the attraction of thrombocytoeto the sites of the injury. Diluted supernatantswere observed to have a stimulatingeffect upon thrombocytesmigration which corresponds in vivo with the nattractionnand in higher concentrationan inhibitory effect which no longer allows the thrombocytesonce in the injured area to leave. In this way the thrombocytesare accummulatedand produce the secondary thrombosis. It is easy to understand that the inhibition of "cellsensitivityof lymphokines"is a factor which prevents thrombosis, inflammationand finally tissue damage. Until now we accummulateda conclusive series of data about the efficiency of DIPY and ASA in such situation. These experimentalaspects have their clinical correspondence by in vivo reproducibilityin our same studies as well as by therapeuticalresults of different authors (1, 12, 13, 14, 151. The thrombocytemigration assay is a good method for innnunological '(12)and pharmacologicalstudies and explores a new function of thrombocytes.It seems that at least, as far as DIPY and ASA are concerned, drugs active in other tests (aggregation,adhesion) also influence the migration of thrombocytes. In our opinion the test has a place in clinical studies of "thrombocyteactive drugs", antiinflammatorydrugs or in cellmediated immune reactions. ACKNOWLEDGEMENTS I am grateful to Prof. Gunnar Bendixen, University Hospital Rigshospitalet,Copenhagen, and Mr. Finn MBlgaard, Boehringer Iagelheim A/S, Copenhagen, for valuable advice and help.

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2.

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3.

BENDTZEN, K., ANDERSEN, V. and BENDIXEN, G. An in vitro assay of leucocyte migration inhibitory activity from human lymphocytesstimulated with Concanavalin-A. Acta Allergol. 30, 133, 1975.

4.

COEUGNIET, E., BENDTZEN, K. and BENDIXEN, G. Leucocyte migration inhibitory activity of concanavalin-A-stimulated human lymphocytes. Modification by dipyridamole, lysineacetylsalicylate and heparin. Acta Med. Stand. 199, 99, 1976.

5.

CGEUGNIET, E. and BENDIXEN, G. Lymphokines and thrombosis I: Thrombocyte aggregating activity released by human lymphocytes stimulated with Concanavalin-A. Acta Allergol. 31, 94, 1976.

6.

COEUGNIET, E. and BENDIXEN, G. Lymphokines and thrombosis II: Procoagulant activity produced by human lymphocytes stimulated with Concanavalin-A. Acta Allergol. 31, 107, 1976.

7.

COEUGNIET, E. and BENDIXEN, G. Lymphokines and thrombosis III: Influence on fibrinolysis of culture supernatants of human lymphocytes stimulated with Concanavalin-A. In preparation.

8.

COEUGNIET, E., BENDTZEN, K., Soeberg, B. and BENDIXEN, G. Leucocyte migration inhibitory activity of Concanavalin-Astimulated lymphocytes. In vivo and in vitro modifications with dipyridamole and acetylsalicylic acid. Acta Med. Stand. 201, 197, 1977.

9.

COEUGNIET, E, and Bendixen, G. Leucocyte migration inhibitory activity of Concanavalin-A-stimulated human lymphocytes. Comparison of leucocyte migration capillary technique, leucocyte migration agarose technique. Acta Path. Microbial. Stand., Sect. C, 84, 485, 1976.

10.

COEUGNIET, E. and BENDIXEN, G. Leucocyte migration fibrinolysis technique: Description of a method. Biomedicine, 25, 1976.

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COLVIN, R. and DVORAK, H. Role of clotting system in cell-mediated hypersensitivity. J. Immunol. 114, 377, 1975.

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DUQUESNOY, R., LORENTZEN, D. and ASTER, 4. Olatelet migration inhibition: A new method for detection of platelet antibodies. Blood 45, 741, 1975.

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GRIEP, R., WEXLER, L., STIMSON, E., DONG, E. and SHUXWAY, N. Coronaq arteriography following cardiac transplantation. J. Amer. Med. Ass. 221, 147, 1972.

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15.

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KfiBLER,W. Die Bindung des Koronarodilatators Dipyridamol und die Plasmaeiweissk6rper des Menschen. Arch. Kreislaufforschg. 64, 115, 1971.

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LAGRUE, G., XHENEUMONT, s., BRANBLLEC, A., mu3Ec, G. and WEIL, B. A vascular permeability factor elaborated from lymphocytes. I. Demonstration in patients with nephrotic syndrome. Biomedicine 23, 37, 1975.

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LOWENHAIJPT, R., MILLER, M. and GLUECK, H. Platelet migration and chemotaxis demonstrated in vitro. Thromb. Res. 3, 477, 1973.

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POBORAN, V.) PETRUSCA, A. and COEUGNIET, E. Therapeutical principles in the renal determinations of collagen diseases. The IIIrd National Congress of Pediatrics, Bucharest 1974.

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ROCKLIN, R.E. Clinical applications of in vitro lymphocyte test. In: Progress in clinical immunology. R.S. Schwartz (Ed.). New York: Grune and Stratton, 1974, p. 21.

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VALONE, F., AUSTEN, K. and GOETZL, E. Inhibition by aspirin of the enhanced migration of human platelets in response to prostaglandin precursors. Immunological Communications 4, 139, 1975.