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Importance of platelets in increased vascular permeability evoked by experimental haemarthrosis in synovium of the rat
Pathology
ISSN: 0031-3025 (Print) 1465-3931 (Online) Journal homepage: https://www.tandfonline.com/loi/ipat20
Importance Of Platelets In Increased Vascular Permeability Evoked By Experimental Haemarthrosis In Synovium Of The Rat Leon P. Bignold To cite this article: Leon P. Bignold (1980) Importance Of Platelets In Increased Vascular Permeability Evoked By Experimental Haemarthrosis In Synovium Of The Rat, Pathology, 12:2, 169-179 To link to this article: https://doi.org/10.3109/00313028009060070
Published online: 06 Jul 2009.
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Pathology (1980), 12, pp. 169-79
IMPORTANCE OF PLATELETS IN INCREASED VASCULAR PERMEABILITY EVOKED BY EXPERIMENTAL HAEMARTHROSIS IN SYNOVIUM OF THE RAT LEONP. BIGNOLD School of Pathology, University of New South Wales, Sydney
Summary Increased vascular permeability of synovium induced by experimental haemarthrosis was studied in the stifle joint of the rat. Abnormal permeability was detected by injecting animals intravenously with colloidal carbon and examining the synovial vessels for intramural deposits of carbon. Both fresh and heparinized whole blood injected into the joint induced a marked permeability response of synovial venules which persisted for 18 hours. Platelets suspended in heparinized Tyrode solution induced a similar response, but one lasting only 12 hours. The permeability effects of suspensions of leucocytes and erythrocytes on the synovial vasculature were relatively mild and were maximal 5 and 12 h respectively after intra-articular injection. Heparinized platelet-free plasma had no significant permeability effects. These results indicate that platelets may play an important role in the pathogenesis of synovial inflammation in haemarthrosis.
Key Words: platelet, rat, haemarthrosis, experimental, blood vessels, permeability
Haemorrhage into the cavity of a synovial joint, whether due to trauma or haemophilia, evokes an acute inflammatory response in the synovial membrane.'-3After single episodes of haemarthrosis, acute inflammation of synovial membrane lasts 2 4 4 8 h and resolves without sequelae. When associated with repeated haemarthroses, however, synovial inflammation is followed by synovial proliferation and fibrosis as well as destruction of cartilage.2. The histopathological features of synovial inflammation in haemarthrosis, viz., vasodilatation, oedema, and leucocytic infiltration have been described for both man2. and experimental animal^.^. Nevertheless, little is known of the mechanisms by which intraarticular haemorrhage evokes this response. Storti & Ascari6 consider that the iron contained in the haemoglobin of the intra-articular blood might be responsible for the synovial inflammation of haemarthrosis, while Arnold and Hilgartner7 have suggested that this inflammation may result from an immune reaction to antigens on the surface of the intra-articular erythrocytes. Experimental studies of inflammation induced by haemarthrosis in synovial membrane of the rat' have shown that introduction of autologous
170 BIGNOLD Pathology (1980), 12, April blood into the cavity of the stifle is followed by an immediate, marked increase in permeability of synovial blood vessels, which lasts up to 24 hours. The present paper describes an investigation of the relative importance of plasma and cellular components of blood in this aspect of synovial inflammation induced by haemarthrosis. The preparations tested for their permeability effects were fresh autologous whole blood, isologous whole blood anticoagulated with heparin, heparinized platelet-free plasma, and suspensions of erythrocytes, leucocytes and platelets in heparinized Tyrode solution. Synovial membranes were prepared for electron microscopy at times at which a significant permeability response was induced by each preparation. MATERIALS AND METHODS Animals Wistar rats (body-weight 250400 g) of both sexes were used. and were lightly anaesthetized with ether for all injections. Blood preparaiions Siliconized glassware or plastic-ware was used throughout. Fresh autologous whole blood was obtained from rats by drawing blood from the lateral tail vein into a sterile plastic disposable syringe. After being drawn, fresh blood was injected into test joints before coagulation occurred. Isologous heparinized whole blood was obtained by cardiac puncture of anaesthetized rats, 10 ml being drawn from each animal into a plastic syringe containing 10 units of heparin (Commonwealth Serum Laboratories, Melbourne). Heparinized platelet-free plasma was prepared by a method similar to that of Haslam & Mills9 Heparinized whole blood (as above) was centrifuged at 220 G for 10 min. The platelet rich plasma was removed and centrifuged twice at 10,000 G for 15 min. The final supernatant was retained as the platelet-free plasma. A suspension of erythrocytes in heparinized Tyrode solution was obtained by centrifugation of 10 ml heparinized whole blood (as above) at 220 G for 10 min. The bottom halfof the sediment was removed and made up to 5 ml with Tyrode solution containing 1 unit heparin per ml at pH 7 . 3 . This suspension was centrifuged at 220 G for 10 min; the bottom half of the sediment was again removed and made up to 2.5 ml with heparinized Tyrode solution as before. The process was repeated to give 1.2 ml of the final suspension of erythrocytes. The cells of this suspension were counted in an Improved Neubauer chamber and numbered 5.37 x 1012erythrocytes, 0.28 x lo9 leucocytes and fewer than 1 x lo9 platelets per 1. The number of erythrocytes in the suspension therefore approximated the number in the peripheral blood of normal rats." A suspension of leucocytes was prepared by a method adapted from that of Killmann." Fresh blood was obtained by cardiac puncture and defibrinated by agitation with glass beads. Nine ml of defibrinated blood was gently mixed with 4.5 ml of 6% dextran (average m.w. 150,000)in 0.9% saline ('Dextraven', Fisons, Loughborough, Leicestershire, England) and allowed to stand at room temperature for 1 h. Seven ml of the supernatant was removed and centrifuged at 220 G for 10 min; the cells in the pellet so obtained were then washed twice with heparinized Tyrode solution and finally suspended in 4.5 ml of the same solution. Total and differential leucocyte counts were comparable for the defibrinated blood and the final suspension: total, 14.8 x 109/l;lymphocytes 767:. monocytes 6% and polymorphonuclear leucocytes 18%. These numbers of leucocytes approximated those in the peripheral blood of normal rats.'* When the leucocytes of the suspension were tested with Trypan blue, more than 90% excluded the dye. Further evidence of viability of the leucocytes was obtained by incubating them with sodium uratecrystals by the method of Rajan." Phagocytosis of the crystals by polymorphonuclear cells was observed. The erythrocytes and platelets in the final suspension numbered 36 x 109/1and fewer than I x 109/1respectively. A suspension of platelets in heparinized Tyrode solution was prepared by a method similar to that of Valone et al.13 Heparinized whole blood (see above) was centrifuged at 220 G for 10 min and the platelet rich plasma removed. This plasma was centrifuged at 880 G for I5 min, the supernatant discarded, and the sediment resuspended in heparinized Tyrode solution. The final suspension contained fewer than 10 x lo9 erythrocytes. fewer than 0.05 x lo9 leucocytes and 451 x lo9 platelets per 1 when counted as before. The number of platelets in this suspension therefore approximated the number in the peripheral blood of normal rats.'" Increased vascular permeability Animals were injected intravenously with a suspension of colloidal carbon (Pelikan, CI 1/143 la, Gunther Wagner, Hanover) 0. I m1/100 g body-weight, 0,2,5, 12,18 or 24 h after injection of the joints. Within an hour of intravenous injection, circulating colloidal carbon is removed from the blood by the
SYNOVIAL VASCULAR PERMEABILITY I N HAEMARTHKOSIS
I71
reticulo-endothelial system, but accumulates during that period in the walls of highly permeable blood vessel^.^" Seventy-five min after intravenous injection of the colloidal carbon, each animal was anaesthetized and killed by exsanguination, and the stifle joints opened to expose the synovium. The synovial membranes were fixed in lo",,, formaldehyde, rendered translucent by immersion in glycerol, and examined with a dissecting microscope. The amount of carbon deposited in the walls of the synovial vessel (referred to as 'labelling' of the vessels) of each joint served as an index of the increased vascular permeability induced in the synovium. The intensity of labelling of the vessels of each synovium was scored 'blind' by a single observer on an arbitrary scale 0 to 5; 0 = less than 2 labelled vessels, and 5 = more than one hundred vessels per aynoviuni. Design ofexperin?en/s The effects of fresh autologous blood and heparinized isologous blood o n the vascular permeability of synovium were tested in 6 groups o f 4 rats. 0.05 ml ofeach preparation being injected into the right and left stiflejointsrespectively ofeach animal. The right and left stiflejoints o f a further 6 pairs ofrats, injected with Tyrode solution alone, served as controls. Groups of 4 test rats and pairs of control rats were given intravenous colloidal carbon just prior to (referred to as ' 0 ' )as well as 2, 5, 12, 18 and 24 h respectively after intra-articular injection of test substances. The permeability effects of plasma, as well as or suspensions of' erythrocytes. leucocytes and platelets in heparinized Tyrode solution were each tested in batches of 12 rats by injecting 0.05 ml of the appropriate preparation into the animals' right and left stifle joints. The joints of a further 12 rats, injected with 0.05 ml heparinized Tyrode solution alone. served as controls. Pairs of test and control animals were given colloidal carbon intravenously after the same time intervals as in the previous experiment. Electron microscopj. Synovial membranes were prepared for electron microscopy at times of significant vascular labelling induced by each preparation, viz.. 15 min and 12 h after intra-articular injection ofplatelet suspension, 4 h after injection of leucocyte suspension and 10 h after injection of erythrocyte suspension. The synovium of each joint was fixed by injecting 0.1 ml of combined aldehyde tixative" into the articular cavity while the animal was anaesthetized. The synovium was then removed without delay and cut into blocks 0.5 mm x 1.0 mm x 3.0 nim. Fixation was continued in aldehydes for 4 h and then in 2", osmium tetroxide for a further 4 h. The blocks of synovium were then dehydrated in ethanol and acetone. and embedded in Spurr's epoxy resin. The preparations were sectioned on an LKB ultramicrotome, stained with uranyl acetate and lead citrate. and examined with a Philips 300 electron microscope.
RESULTS Fresh und unticouguluted whole blood Gross uppeurunce ofthe joints Similar changes were induced in the test joints by fresh and heparinized preparations of blood. The cavities of joints after each interval contained red glairy fluid and no intra-articular clotting of either the fresh or heparinized blood was observed. The synovial membranes of the test joints were moderately oedematous at 2,6 and 12 h, but only slightly oedematous at 0 and 24 h. Examination of 'control' synovial membranes revealed no abnormalities.
Permeubility response A comparable increase of permeability was induced in the venules of the synovium of the stifle joint by fresh autologous whole blood and isologous whole blood anticoagulated with heparin (Fig. 1). Vascular labelling was observed in the synovial membranes of all joints of animals given intravenous carbon up to 18 h after injection of the joints. For both preparations of blood, the intensity of labelling was maximal i n the initial 2 h after injection of the joints (see Fig. 2a) and remained moderate in intensity up to 18 h (see fig. 2b). At 24 h, only 2 of 4 synovial membranes of joints injected with heparinized blood exhibited vascular labelling (scores = 2 and 1, respectively). Of the control synovial membranes, 2 of 4 showed labelling at 0 h (score = I in each case) and 1 of 4 at 18 h (score = 2). The small amount of vascular labelling in control synovial membranes may have been due to slight haemorrhage from the synovium provoked by entry of the needle into the joint cavity.
172
Pathology (1980), 12, April
BIGNOLD
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Hours FIG. 1 Increased vascular permeability (assessed by carbon labelling of venules) in synovium of the rat, evoked by intra-articular injection of fresh autologous whole blood (F),and heparinized isologous whole blood (H). C-controls, injected with heparinized Tyrode solution
Components of blood Gross appearance of joints None of the joints injected with either plasma or leucocytes showed any abnormality at dissection. Joints injected with the suspension of erythrocytes contained a small amount of haemoglobin stained fluid, but were otherwise normal. On the other hand, joints injected with the suspension of platelets contained variable amounts of clear fluid, and their synovial membranes were oedematous. These changes were marked at 6 h but were slight at other times. Permeability responses: Plasma (Fig. 3) Vascular labelling was found in only 1 of 24 joints injected with heparinized platelet-free plasma. This permeability response was not significantly different to that in control joints (Fig. 1). Erythrocytes (Fig. 3) Labelling of synovial vessels was observed in 10 of 24 joints injected with the suspension of erythrocytes in heparinized Tyrode solution. Three of the 4
173 synovial membranes showed vascular labelling at 2 h, 1/4 at 5 h, 4/4 at 12 h and 2/4 at 24 h. The amount of vascular labelling considerably exceeded that in control joints at 12 h. SYNOVIAL VASCULAR PERMEABILITY IN HAEMARTHROSIS
Leucocytes (Fig. 3) Vascular labelling was found in 9 of 24 joints injected with the suspension of leucocytes in heparinized Tyrode solution. One of the 4 synovial membranes showed vascular labelling at 0 h, 4/4 at 2 h, and 4/4 at 5 h. No labelling of vessels occurred at 12, 18 or 24 h. The amount of labelling exceeded that in controls at 2 and 5 h. Platelets (Fig. 3) The vascular labelling induced in synovial membranes by the suspension of platelets in heparinized Tyrode solution had an intensity in the initial 2 h similar to that evoked by autologous whole blood in the same period (Fig. 1). However, the duration of the permeability effect of platelets was shorter than for whole blood, being greatly reduced at 12 h and comparable in intensity to that in controls at 18 and 24 h. Electron microscopy Synovial membranes were examined in electron microscopy 15 min and 12 h after intraarticular injection of platelets, 4 h after injection of leucocytes and 10 h after injection of erythrocytes. In each case, the changes were similar. Deposits of carbon were confined to the walls of venules (Fig. 4). Synovial capillaries with either continuous or fenestrated endothelium as described by Suter & Majno16 were unaffected. The carbon particles were situated beneath the endothelial cells, in gaps between the endothelial cells (Fig. 5 ) , and in the perivascular spaces. Small collections of carbon particles were also present in vacuoles of endothelial cells (Fig. 4). The nuclei of the endothelial cells of affected venules frequently showed multiple indentations of the nuclear membrane. No morphological evidence was found of injury to endothelial cells of venules or capillaries. DISCUSSION The results of the present experiments show that single injections of blood into the stifle joints of rats evoke a marked permeability response (indicated by the accumulation of circulating colloidal carbon in the walls of blood vessels in the synovial membranes). This permeability response is intense in the first 2 h and persists to a lesser degree for 18 h. It is neither reduced in intensity nor shortened by anticoagulation of the injected blood with heparin. Some indication of the mechanism of this response is provided by the study of the effects of preparations of separated plasma, erythrocytes, leucocytes and platelets on vascular permeability of synovial membranes. Plasma (anticoagulated with heparin) has no detectable effect on the permeability of synovial blood vessels at any time up to 24 h. Erythrocytes and leucocytes in physiological solution induce only mild responses, with maximal effects occurring 12 and 5 h respectively after intra-articular injection. In contrast to these mild responses, platelets suspended in physiological solution evoke a marked response which, being maximal in the first 2 h and persisting for 12 h, closely resembles the response evoked by whole blood. The electron microscopic findings in the synovial vessels after intra-articular injection of each test preparation resemble the changes induced by histamine, serotonin and bradykinin in synovium” and skeletal muscle.’* The observed increase of vascular permeability associated with formation of gaps between venular endothelial cells, but without morphological evidence of injury to the endothelium probably results from mediation by permeability factors.l9
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Pathology (1980), 12, April
BIGNOLD
FIG. 2a FIG. 2 Synovial membranes of stifle joints of rats illustrating vascular labelling with colloidal carbon after intra-articular injection of whole blood (a) First hour after injection of joint (labelling score = 5) (b) 18th hour after injection of joint (labelling score = 2) p = patella, tc = tibia1 condyle (cleared in glycerol x 10)
SYNOVIAL VASCULAR PERMEABILITY IN HAEMARTHROSIS
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FIG. 2b
These data, therefore, suggest that platelets are the major factor in the induction of increased vascular permeability by experimental haemarthrosis, and that they may evoke this response by releasing a chemical mediator rather than by causing direct vascular injury. Several chemical factors which might be responsible for these permeability effects have been found in platelets. These substances include histamine and serotonin, prostaglandins and several proteins. Histamine and serotonin are known to induce increased vascular permeability in synovial inembrane of the rat for 10-1 5 min.” However, rat platelets contain
176
Pathology (1980), 12, April
BIGNOLD
51 4
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Hours ERYTHROCYTES
Hours PLASMA
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24 Hours PLATELETS
FIG. 3 Increased vascular permeability induced in synovium of the rat by heparinized plasma and suspensions of erythrocytes, leucocytes and platelets in heparinized Tyrode solution
less than 0.1 p g histamine and 0.2-0.4 p g serotonin per lo9 platelets.” Therefore, because the number ofplatelets injected into each joint in the present experiments was approximately 1-2 x lo5, the amounts of histamine and serotonin injected into each joint in the platelets were of the order of 1/10,00Oth of the amounts of those substances required to induce permeability responses in the synovial membrane.” Histamine and serotonin are, therefore, unlikely to be responsible for the entire permeability effect of intra-articularly injected platelets in the rat. Various prostaglandins (PG) are produced by platelets and are capable of inducing
SYNOVIAL VASCULAR PERMEABILITY IN HAEMARTHROSIS
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FIG. 4 Electron micrograph of synovial venule 1 hour after intra-articular injection of platelet suspension showing intra-mural deposits of carbon (long arrows). Small deposits of carbon are also present within endothelial cells (short arrows). x 12000
increased vascular permeability in tissues of man” and the Moreover, PGE, induces increased vascular permeability in canine synovial membrane.24 Therefore, prostaglandins may well have a role in the permeability response induced by platelets in synovial membrane. Another factor which may be involved in the permeability response evoked by platelets is the cationic protein isolated by Nachman and c o - ~ o r k e r s . ~26’ .This protein was extracted from human platelets, however, and its presence in rat platelets is not established. In addition to these various factors, a platelet lysosomal enzyme, phospholipase A, which is capable of liberating histamine from mast cells2’ has also been implicated in the synthesis of prostaglandins.28 One or several of these factors may play a role in the increased vascular permeability induced in synovium by haemarthrosis. The relatively minor permeability effects induced by the suspension of leucocytes may well have been due to permeability-enhancing substances which are known to be present in leucocytes, including small amounts of histamine and serotonin?’ certain cationic proteins29 and lysosomal enzymes capable of releasing permeability factors such as kinins from ~lasrna.~O On - ~the ~ other hand, the permeability effect .of the suspension of erythrocytes is unexplained, since no permeability factors deriving from these cells have been described.
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BIGNOLD
Pathology (1980), 12. April
FIG. 5 Escape of circulating particles of carbon (c) through a gap between two endothelial cells (e. e) of a synovial venule. r = erythrocyte in lumen. EM x 20000
It is surprising that in the present experiments, plasma failed to evoke a detectable vascular permeability response in view of the numerous permeability factors, including kinins and derivatives of the complement system which are known to originate from this component of blood (see reviews33.34). Nevertheless, a role for plasma in the total permeability response to haemarthrosis, is not necessarily excluded since the generation of permeability factors in plasma may require the simultaneous presence of the blood cells particularly leucocytes.”. 32 ACKNOWLEDGEMENTS I am grateful to Professor A. W. J. Lykke, School of Pathology, University of New South Wales, for assistance with the preparation of this paper. Drs M. C. Rozenberg and C. Grace, Division of Haematology, Department of Pathology, Prince Henry Hospital, Sydney, provided valuable advice on the various haematological techniques. The work reported in this paper was carried out while the author was a Medical Postgraduate Scholar supported by the National Health and Medical Research Council of Australia. Address f o r correspondence: L.P.B., Royal Prince Alfred Hospital, Missenden Road, Camperdown, N.S.W.,
Australia 2050
References 1. SMILLIE, I. S. (1970): Injuries of ihe Knee Joint, 4th
ed. Livingstone, Edinburgh. pp. 19-20. 2. KEY,.I. A. (1932): Hemophilic arthritis (bleeder’s joints). Ann. Surg. 95, 198-225.
4. WOLF,C. R. & MANKIN, H. J. (1965): The effect of experimental hemarthrosis on the articular cartilage of rabbit knee joints. J . Bone Joint Surg. 47A, 1203-1210.
SYNOVIAL VASCULAR PERMEABILITY IN HAEMARTHROSIS STOR OR TI, E. & ASCARI,E. (1975): On the pathogenesis of haemophilic haemarthrosis. Haemcrrologica iPavia) 60, 129-135. M . W. (1977): 7. ARNOLD,W. D. & HILGARTNER. Hemophilic arthropathy. Current concepts of pathogenesis and management. J . Bone Joint Surg. 59A, 287-305. 8. BIGNOLD,L. P. & LYKKE,A. W. J. (1975a):
Increased vascular permeability evoked by mechanical trauma and haemarthrosis in synovium of the rat. Patho/ogy 7, 263-271. 9. HASLAM,R. J. & MILLS,D. C. B. (1967): The adenylate kinase of human plasma. erythrocytes and platelets in relation to the degradation of adenosine diphosphate in plasma. Biochem. J . 103, 773-784. 10. HARDY,J. (1967): Haematology of rats and mice. In Pathology oj'Lahorarorj~Ruts and Mice. Eds E. Cotchin & F. J. C. Roe. Blackwell Scientific Publications, Oxford. pp. 501-536. 11. KILLMANN, S. A. (1959): Studies on the leucocyte agglutination test. Acta. Pathol. Microhiol. Scund. 45, 17-31. 12. RAJAN, K. T. (1966): Lysosomes and gout. Nature 210, 959-960. E. J. 13. VALONE,F. H., AUSTEN,K. F. & GOETZL, (1974): Modulation of the random migration of human platelets. J . Clin. Invest. 54, 1100-1 106. R. S., SUTER,E. R. & MAJNO,G . (1967): 14. COTRAN, The use of colloidal carbon as a tracer for vascular injury-a review. Vasc. Dis. 4, 107-127. 15. KARNOVSKY, M . J. (1965): A formaldehydeglutaraldehyde fixative of high osmolarity for use in electron microscopy. J . Cefi. Bid. 27, 137A-138A. 16. SUTER,E. R. & MAJNO,G . (1964): Ultrastructure ofjoint capsule in the rat: presence of two kinds of capillaries. Nnrure 202, 920-92 I, I~.BIGNOLD L., P. & LYKKE.A. W. J . (1975b): Increased vascular permeability induced in synovialis of the rat by histamine, serotonin and bradykinin. Experientia 31, 671-672. 18. MAJNO, G., PALADE.G. E. & SCHOEFL,G . I . (1961): Studies on inflammation. 11. The site of action of histamine and serotonin along the vascular tree: a topographic study. J . Biophys. Biochem. Cytol. 11, 607426. 19. COTRAN,R. S. & MAJNO,G. (1964): A light and electron microscopic analysis of vascular injury. Ann. N . Y. Acad. Sci. 116, 750-764. J. H. & JAQUES,R. (1954): The ~~.HUMPHREY, histamine and serotonin content of the platelets and polymorphonuclear leucocytes of various species. J . Physiol. (Lond.) 124, 303-310.
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~ ~ . C R U N K H O RP.N .& W t m s . A. L. (1971): Cutaneous reactions to intradermal prostaglandins. Br. J . Pharmacol. 41, 49-56. G. & WEINER.R. (1968): Microcirculatory 22. KALEY, studies with prostaglandin E ( I ) . In Prostaglandin Symposium of Worcesrer Foundution j o r E.\-perimental Biology. Eds P. W. Ramwell & J . E. Shaw. J . Wiley. New York. pp. 321-328. 23. AROKA.S., LAHIRI,P. K. & SANYAL. R . K. (1970): The role of prostaglandin E, in inflammatory process in the rat. Int. Arch. Allergy Appl. Immunol. 39, 186-1 9 I . 24. GRENNAN, D. M.. MITCHELL, W., MII.I.FR, W. C I al. (1977): The effects of prostaglandin E,, bradykinin and histamine on canine synovial vascular permeability. Br. J . Pharmacol. 60, 25 1 -254. B. 25. NACHMAN,R. L.. WEKSLER.B. & FERRIS, ( 1 970): Increased vascular permeability produced by platelet granule cationic extract. J . Clrn. Invrsr. 49, 274-28 I . 26. NACHMAN. R. L. & WEKSLER,B. (1972): The platelet as an inflammatory cell. Ann. N . Y. Acad. Sci. 201, 131-137. B. (1963): Mechanism of histamine release 27. UVNAS, in mast cells. Ann. N. Y. Acad. Sci. 103, 278-284. 28. SMITH,J. B. (1974): Platelets and permeability factors. In White Cells and Inflammation. Ed. C. G . Van Arman. Charles C. Thomas. Springfield. pp, 3-14. N . S. & COCHRANE, C. G. (1968): 29. RANAIXVE, Isolation and characterisation of permeability factors from rabbit neutrophils. J . E.yp. Mrd. 128, 605422. 30. MELMON. K . L. & CLINE,M. J. (1967): Interaction of plasma kinins and granulocytes. Noturr 213, 90-92. 31. WARD,P. A. & HILL,J . H. (1970): C 5 chemotactic fragments produced by an enzyme in lysosomal granules of neutrophils. J. Immunol. 104,535-543. L. M. (1972): Leukocyte kinino32. GREENBAUM, genases and leukokinins from normal and malignant cells. Am. J . Pafhol. 68, 61 3 4 2 4 . 33. WILHELM,D. L. (1973): Chemical mediators. In The 1nflammator.y Process, 2nd ed.. Vol. 2. Eds B. W. Zweifach, L. Grant & R. J . McCluskey. Academic Press, New York. pp. 251-301. 34. RYAN,G. B. & MAJNO,G. (1977): Acute inflammation. A review. Am. J. Parhol. 86, 183-276.
ISSN: 0031-3025 (Print) 1465-3931 (Online) Journal homepage: https://www.tandfonline.com/loi/ipat20
Importance Of Platelets In Increased Vascular Permeability Evoked By Experimental Haemarthrosis In Synovium Of The Rat Leon P. Bignold To cite this article: Leon P. Bignold (1980) Importance Of Platelets In Increased Vascular Permeability Evoked By Experimental Haemarthrosis In Synovium Of The Rat, Pathology, 12:2, 169-179 To link to this article: https://doi.org/10.3109/00313028009060070
Published online: 06 Jul 2009.
Submit your article to this journal
Article views: 3
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Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=ipat20
Pathology (1980), 12, pp. 169-79
IMPORTANCE OF PLATELETS IN INCREASED VASCULAR PERMEABILITY EVOKED BY EXPERIMENTAL HAEMARTHROSIS IN SYNOVIUM OF THE RAT LEONP. BIGNOLD School of Pathology, University of New South Wales, Sydney
Summary Increased vascular permeability of synovium induced by experimental haemarthrosis was studied in the stifle joint of the rat. Abnormal permeability was detected by injecting animals intravenously with colloidal carbon and examining the synovial vessels for intramural deposits of carbon. Both fresh and heparinized whole blood injected into the joint induced a marked permeability response of synovial venules which persisted for 18 hours. Platelets suspended in heparinized Tyrode solution induced a similar response, but one lasting only 12 hours. The permeability effects of suspensions of leucocytes and erythrocytes on the synovial vasculature were relatively mild and were maximal 5 and 12 h respectively after intra-articular injection. Heparinized platelet-free plasma had no significant permeability effects. These results indicate that platelets may play an important role in the pathogenesis of synovial inflammation in haemarthrosis.
Key Words: platelet, rat, haemarthrosis, experimental, blood vessels, permeability
Haemorrhage into the cavity of a synovial joint, whether due to trauma or haemophilia, evokes an acute inflammatory response in the synovial membrane.'-3After single episodes of haemarthrosis, acute inflammation of synovial membrane lasts 2 4 4 8 h and resolves without sequelae. When associated with repeated haemarthroses, however, synovial inflammation is followed by synovial proliferation and fibrosis as well as destruction of cartilage.2. The histopathological features of synovial inflammation in haemarthrosis, viz., vasodilatation, oedema, and leucocytic infiltration have been described for both man2. and experimental animal^.^. Nevertheless, little is known of the mechanisms by which intraarticular haemorrhage evokes this response. Storti & Ascari6 consider that the iron contained in the haemoglobin of the intra-articular blood might be responsible for the synovial inflammation of haemarthrosis, while Arnold and Hilgartner7 have suggested that this inflammation may result from an immune reaction to antigens on the surface of the intra-articular erythrocytes. Experimental studies of inflammation induced by haemarthrosis in synovial membrane of the rat' have shown that introduction of autologous
170 BIGNOLD Pathology (1980), 12, April blood into the cavity of the stifle is followed by an immediate, marked increase in permeability of synovial blood vessels, which lasts up to 24 hours. The present paper describes an investigation of the relative importance of plasma and cellular components of blood in this aspect of synovial inflammation induced by haemarthrosis. The preparations tested for their permeability effects were fresh autologous whole blood, isologous whole blood anticoagulated with heparin, heparinized platelet-free plasma, and suspensions of erythrocytes, leucocytes and platelets in heparinized Tyrode solution. Synovial membranes were prepared for electron microscopy at times at which a significant permeability response was induced by each preparation. MATERIALS AND METHODS Animals Wistar rats (body-weight 250400 g) of both sexes were used. and were lightly anaesthetized with ether for all injections. Blood preparaiions Siliconized glassware or plastic-ware was used throughout. Fresh autologous whole blood was obtained from rats by drawing blood from the lateral tail vein into a sterile plastic disposable syringe. After being drawn, fresh blood was injected into test joints before coagulation occurred. Isologous heparinized whole blood was obtained by cardiac puncture of anaesthetized rats, 10 ml being drawn from each animal into a plastic syringe containing 10 units of heparin (Commonwealth Serum Laboratories, Melbourne). Heparinized platelet-free plasma was prepared by a method similar to that of Haslam & Mills9 Heparinized whole blood (as above) was centrifuged at 220 G for 10 min. The platelet rich plasma was removed and centrifuged twice at 10,000 G for 15 min. The final supernatant was retained as the platelet-free plasma. A suspension of erythrocytes in heparinized Tyrode solution was obtained by centrifugation of 10 ml heparinized whole blood (as above) at 220 G for 10 min. The bottom halfof the sediment was removed and made up to 5 ml with Tyrode solution containing 1 unit heparin per ml at pH 7 . 3 . This suspension was centrifuged at 220 G for 10 min; the bottom half of the sediment was again removed and made up to 2.5 ml with heparinized Tyrode solution as before. The process was repeated to give 1.2 ml of the final suspension of erythrocytes. The cells of this suspension were counted in an Improved Neubauer chamber and numbered 5.37 x 1012erythrocytes, 0.28 x lo9 leucocytes and fewer than 1 x lo9 platelets per 1. The number of erythrocytes in the suspension therefore approximated the number in the peripheral blood of normal rats." A suspension of leucocytes was prepared by a method adapted from that of Killmann." Fresh blood was obtained by cardiac puncture and defibrinated by agitation with glass beads. Nine ml of defibrinated blood was gently mixed with 4.5 ml of 6% dextran (average m.w. 150,000)in 0.9% saline ('Dextraven', Fisons, Loughborough, Leicestershire, England) and allowed to stand at room temperature for 1 h. Seven ml of the supernatant was removed and centrifuged at 220 G for 10 min; the cells in the pellet so obtained were then washed twice with heparinized Tyrode solution and finally suspended in 4.5 ml of the same solution. Total and differential leucocyte counts were comparable for the defibrinated blood and the final suspension: total, 14.8 x 109/l;lymphocytes 767:. monocytes 6% and polymorphonuclear leucocytes 18%. These numbers of leucocytes approximated those in the peripheral blood of normal rats.'* When the leucocytes of the suspension were tested with Trypan blue, more than 90% excluded the dye. Further evidence of viability of the leucocytes was obtained by incubating them with sodium uratecrystals by the method of Rajan." Phagocytosis of the crystals by polymorphonuclear cells was observed. The erythrocytes and platelets in the final suspension numbered 36 x 109/1and fewer than I x 109/1respectively. A suspension of platelets in heparinized Tyrode solution was prepared by a method similar to that of Valone et al.13 Heparinized whole blood (see above) was centrifuged at 220 G for 10 min and the platelet rich plasma removed. This plasma was centrifuged at 880 G for I5 min, the supernatant discarded, and the sediment resuspended in heparinized Tyrode solution. The final suspension contained fewer than 10 x lo9 erythrocytes. fewer than 0.05 x lo9 leucocytes and 451 x lo9 platelets per 1 when counted as before. The number of platelets in this suspension therefore approximated the number in the peripheral blood of normal rats.'" Increased vascular permeability Animals were injected intravenously with a suspension of colloidal carbon (Pelikan, CI 1/143 la, Gunther Wagner, Hanover) 0. I m1/100 g body-weight, 0,2,5, 12,18 or 24 h after injection of the joints. Within an hour of intravenous injection, circulating colloidal carbon is removed from the blood by the
SYNOVIAL VASCULAR PERMEABILITY I N HAEMARTHKOSIS
I71
reticulo-endothelial system, but accumulates during that period in the walls of highly permeable blood vessel^.^" Seventy-five min after intravenous injection of the colloidal carbon, each animal was anaesthetized and killed by exsanguination, and the stifle joints opened to expose the synovium. The synovial membranes were fixed in lo",,, formaldehyde, rendered translucent by immersion in glycerol, and examined with a dissecting microscope. The amount of carbon deposited in the walls of the synovial vessel (referred to as 'labelling' of the vessels) of each joint served as an index of the increased vascular permeability induced in the synovium. The intensity of labelling of the vessels of each synovium was scored 'blind' by a single observer on an arbitrary scale 0 to 5; 0 = less than 2 labelled vessels, and 5 = more than one hundred vessels per aynoviuni. Design ofexperin?en/s The effects of fresh autologous blood and heparinized isologous blood o n the vascular permeability of synovium were tested in 6 groups o f 4 rats. 0.05 ml ofeach preparation being injected into the right and left stiflejointsrespectively ofeach animal. The right and left stiflejoints o f a further 6 pairs ofrats, injected with Tyrode solution alone, served as controls. Groups of 4 test rats and pairs of control rats were given intravenous colloidal carbon just prior to (referred to as ' 0 ' )as well as 2, 5, 12, 18 and 24 h respectively after intra-articular injection of test substances. The permeability effects of plasma, as well as or suspensions of' erythrocytes. leucocytes and platelets in heparinized Tyrode solution were each tested in batches of 12 rats by injecting 0.05 ml of the appropriate preparation into the animals' right and left stifle joints. The joints of a further 12 rats, injected with 0.05 ml heparinized Tyrode solution alone. served as controls. Pairs of test and control animals were given colloidal carbon intravenously after the same time intervals as in the previous experiment. Electron microscopj. Synovial membranes were prepared for electron microscopy at times of significant vascular labelling induced by each preparation, viz.. 15 min and 12 h after intra-articular injection ofplatelet suspension, 4 h after injection of leucocyte suspension and 10 h after injection of erythrocyte suspension. The synovium of each joint was fixed by injecting 0.1 ml of combined aldehyde tixative" into the articular cavity while the animal was anaesthetized. The synovium was then removed without delay and cut into blocks 0.5 mm x 1.0 mm x 3.0 nim. Fixation was continued in aldehydes for 4 h and then in 2", osmium tetroxide for a further 4 h. The blocks of synovium were then dehydrated in ethanol and acetone. and embedded in Spurr's epoxy resin. The preparations were sectioned on an LKB ultramicrotome, stained with uranyl acetate and lead citrate. and examined with a Philips 300 electron microscope.
RESULTS Fresh und unticouguluted whole blood Gross uppeurunce ofthe joints Similar changes were induced in the test joints by fresh and heparinized preparations of blood. The cavities of joints after each interval contained red glairy fluid and no intra-articular clotting of either the fresh or heparinized blood was observed. The synovial membranes of the test joints were moderately oedematous at 2,6 and 12 h, but only slightly oedematous at 0 and 24 h. Examination of 'control' synovial membranes revealed no abnormalities.
Permeubility response A comparable increase of permeability was induced in the venules of the synovium of the stifle joint by fresh autologous whole blood and isologous whole blood anticoagulated with heparin (Fig. 1). Vascular labelling was observed in the synovial membranes of all joints of animals given intravenous carbon up to 18 h after injection of the joints. For both preparations of blood, the intensity of labelling was maximal i n the initial 2 h after injection of the joints (see Fig. 2a) and remained moderate in intensity up to 18 h (see fig. 2b). At 24 h, only 2 of 4 synovial membranes of joints injected with heparinized blood exhibited vascular labelling (scores = 2 and 1, respectively). Of the control synovial membranes, 2 of 4 showed labelling at 0 h (score = I in each case) and 1 of 4 at 18 h (score = 2). The small amount of vascular labelling in control synovial membranes may have been due to slight haemorrhage from the synovium provoked by entry of the needle into the joint cavity.
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Hours FIG. 1 Increased vascular permeability (assessed by carbon labelling of venules) in synovium of the rat, evoked by intra-articular injection of fresh autologous whole blood (F),and heparinized isologous whole blood (H). C-controls, injected with heparinized Tyrode solution
Components of blood Gross appearance of joints None of the joints injected with either plasma or leucocytes showed any abnormality at dissection. Joints injected with the suspension of erythrocytes contained a small amount of haemoglobin stained fluid, but were otherwise normal. On the other hand, joints injected with the suspension of platelets contained variable amounts of clear fluid, and their synovial membranes were oedematous. These changes were marked at 6 h but were slight at other times. Permeability responses: Plasma (Fig. 3) Vascular labelling was found in only 1 of 24 joints injected with heparinized platelet-free plasma. This permeability response was not significantly different to that in control joints (Fig. 1). Erythrocytes (Fig. 3) Labelling of synovial vessels was observed in 10 of 24 joints injected with the suspension of erythrocytes in heparinized Tyrode solution. Three of the 4
173 synovial membranes showed vascular labelling at 2 h, 1/4 at 5 h, 4/4 at 12 h and 2/4 at 24 h. The amount of vascular labelling considerably exceeded that in control joints at 12 h. SYNOVIAL VASCULAR PERMEABILITY IN HAEMARTHROSIS
Leucocytes (Fig. 3) Vascular labelling was found in 9 of 24 joints injected with the suspension of leucocytes in heparinized Tyrode solution. One of the 4 synovial membranes showed vascular labelling at 0 h, 4/4 at 2 h, and 4/4 at 5 h. No labelling of vessels occurred at 12, 18 or 24 h. The amount of labelling exceeded that in controls at 2 and 5 h. Platelets (Fig. 3) The vascular labelling induced in synovial membranes by the suspension of platelets in heparinized Tyrode solution had an intensity in the initial 2 h similar to that evoked by autologous whole blood in the same period (Fig. 1). However, the duration of the permeability effect of platelets was shorter than for whole blood, being greatly reduced at 12 h and comparable in intensity to that in controls at 18 and 24 h. Electron microscopy Synovial membranes were examined in electron microscopy 15 min and 12 h after intraarticular injection of platelets, 4 h after injection of leucocytes and 10 h after injection of erythrocytes. In each case, the changes were similar. Deposits of carbon were confined to the walls of venules (Fig. 4). Synovial capillaries with either continuous or fenestrated endothelium as described by Suter & Majno16 were unaffected. The carbon particles were situated beneath the endothelial cells, in gaps between the endothelial cells (Fig. 5 ) , and in the perivascular spaces. Small collections of carbon particles were also present in vacuoles of endothelial cells (Fig. 4). The nuclei of the endothelial cells of affected venules frequently showed multiple indentations of the nuclear membrane. No morphological evidence was found of injury to endothelial cells of venules or capillaries. DISCUSSION The results of the present experiments show that single injections of blood into the stifle joints of rats evoke a marked permeability response (indicated by the accumulation of circulating colloidal carbon in the walls of blood vessels in the synovial membranes). This permeability response is intense in the first 2 h and persists to a lesser degree for 18 h. It is neither reduced in intensity nor shortened by anticoagulation of the injected blood with heparin. Some indication of the mechanism of this response is provided by the study of the effects of preparations of separated plasma, erythrocytes, leucocytes and platelets on vascular permeability of synovial membranes. Plasma (anticoagulated with heparin) has no detectable effect on the permeability of synovial blood vessels at any time up to 24 h. Erythrocytes and leucocytes in physiological solution induce only mild responses, with maximal effects occurring 12 and 5 h respectively after intra-articular injection. In contrast to these mild responses, platelets suspended in physiological solution evoke a marked response which, being maximal in the first 2 h and persisting for 12 h, closely resembles the response evoked by whole blood. The electron microscopic findings in the synovial vessels after intra-articular injection of each test preparation resemble the changes induced by histamine, serotonin and bradykinin in synovium” and skeletal muscle.’* The observed increase of vascular permeability associated with formation of gaps between venular endothelial cells, but without morphological evidence of injury to the endothelium probably results from mediation by permeability factors.l9
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FIG. 2a FIG. 2 Synovial membranes of stifle joints of rats illustrating vascular labelling with colloidal carbon after intra-articular injection of whole blood (a) First hour after injection of joint (labelling score = 5) (b) 18th hour after injection of joint (labelling score = 2) p = patella, tc = tibia1 condyle (cleared in glycerol x 10)
SYNOVIAL VASCULAR PERMEABILITY IN HAEMARTHROSIS
175
FIG. 2b
These data, therefore, suggest that platelets are the major factor in the induction of increased vascular permeability by experimental haemarthrosis, and that they may evoke this response by releasing a chemical mediator rather than by causing direct vascular injury. Several chemical factors which might be responsible for these permeability effects have been found in platelets. These substances include histamine and serotonin, prostaglandins and several proteins. Histamine and serotonin are known to induce increased vascular permeability in synovial inembrane of the rat for 10-1 5 min.” However, rat platelets contain
176
Pathology (1980), 12, April
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Hours PLASMA
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FIG. 3 Increased vascular permeability induced in synovium of the rat by heparinized plasma and suspensions of erythrocytes, leucocytes and platelets in heparinized Tyrode solution
less than 0.1 p g histamine and 0.2-0.4 p g serotonin per lo9 platelets.” Therefore, because the number ofplatelets injected into each joint in the present experiments was approximately 1-2 x lo5, the amounts of histamine and serotonin injected into each joint in the platelets were of the order of 1/10,00Oth of the amounts of those substances required to induce permeability responses in the synovial membrane.” Histamine and serotonin are, therefore, unlikely to be responsible for the entire permeability effect of intra-articularly injected platelets in the rat. Various prostaglandins (PG) are produced by platelets and are capable of inducing
SYNOVIAL VASCULAR PERMEABILITY IN HAEMARTHROSIS
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FIG. 4 Electron micrograph of synovial venule 1 hour after intra-articular injection of platelet suspension showing intra-mural deposits of carbon (long arrows). Small deposits of carbon are also present within endothelial cells (short arrows). x 12000
increased vascular permeability in tissues of man” and the Moreover, PGE, induces increased vascular permeability in canine synovial membrane.24 Therefore, prostaglandins may well have a role in the permeability response induced by platelets in synovial membrane. Another factor which may be involved in the permeability response evoked by platelets is the cationic protein isolated by Nachman and c o - ~ o r k e r s . ~26’ .This protein was extracted from human platelets, however, and its presence in rat platelets is not established. In addition to these various factors, a platelet lysosomal enzyme, phospholipase A, which is capable of liberating histamine from mast cells2’ has also been implicated in the synthesis of prostaglandins.28 One or several of these factors may play a role in the increased vascular permeability induced in synovium by haemarthrosis. The relatively minor permeability effects induced by the suspension of leucocytes may well have been due to permeability-enhancing substances which are known to be present in leucocytes, including small amounts of histamine and serotonin?’ certain cationic proteins29 and lysosomal enzymes capable of releasing permeability factors such as kinins from ~lasrna.~O On - ~the ~ other hand, the permeability effect .of the suspension of erythrocytes is unexplained, since no permeability factors deriving from these cells have been described.
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Pathology (1980), 12. April
FIG. 5 Escape of circulating particles of carbon (c) through a gap between two endothelial cells (e. e) of a synovial venule. r = erythrocyte in lumen. EM x 20000
It is surprising that in the present experiments, plasma failed to evoke a detectable vascular permeability response in view of the numerous permeability factors, including kinins and derivatives of the complement system which are known to originate from this component of blood (see reviews33.34). Nevertheless, a role for plasma in the total permeability response to haemarthrosis, is not necessarily excluded since the generation of permeability factors in plasma may require the simultaneous presence of the blood cells particularly leucocytes.”. 32 ACKNOWLEDGEMENTS I am grateful to Professor A. W. J. Lykke, School of Pathology, University of New South Wales, for assistance with the preparation of this paper. Drs M. C. Rozenberg and C. Grace, Division of Haematology, Department of Pathology, Prince Henry Hospital, Sydney, provided valuable advice on the various haematological techniques. The work reported in this paper was carried out while the author was a Medical Postgraduate Scholar supported by the National Health and Medical Research Council of Australia. Address f o r correspondence: L.P.B., Royal Prince Alfred Hospital, Missenden Road, Camperdown, N.S.W.,
Australia 2050
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~ ~ . C R U N K H O RP.N .& W t m s . A. L. (1971): Cutaneous reactions to intradermal prostaglandins. Br. J . Pharmacol. 41, 49-56. G. & WEINER.R. (1968): Microcirculatory 22. KALEY, studies with prostaglandin E ( I ) . In Prostaglandin Symposium of Worcesrer Foundution j o r E.\-perimental Biology. Eds P. W. Ramwell & J . E. Shaw. J . Wiley. New York. pp. 321-328. 23. AROKA.S., LAHIRI,P. K. & SANYAL. R . K. (1970): The role of prostaglandin E, in inflammatory process in the rat. Int. Arch. Allergy Appl. Immunol. 39, 186-1 9 I . 24. GRENNAN, D. M.. MITCHELL, W., MII.I.FR, W. C I al. (1977): The effects of prostaglandin E,, bradykinin and histamine on canine synovial vascular permeability. Br. J . Pharmacol. 60, 25 1 -254. B. 25. NACHMAN,R. L.. WEKSLER.B. & FERRIS, ( 1 970): Increased vascular permeability produced by platelet granule cationic extract. J . Clrn. Invrsr. 49, 274-28 I . 26. NACHMAN. R. L. & WEKSLER,B. (1972): The platelet as an inflammatory cell. Ann. N . Y. Acad. Sci. 201, 131-137. B. (1963): Mechanism of histamine release 27. UVNAS, in mast cells. Ann. N. Y. Acad. Sci. 103, 278-284. 28. SMITH,J. B. (1974): Platelets and permeability factors. In White Cells and Inflammation. Ed. C. G . Van Arman. Charles C. Thomas. Springfield. pp, 3-14. N . S. & COCHRANE, C. G. (1968): 29. RANAIXVE, Isolation and characterisation of permeability factors from rabbit neutrophils. J . E.yp. Mrd. 128, 605422. 30. MELMON. K . L. & CLINE,M. J. (1967): Interaction of plasma kinins and granulocytes. Noturr 213, 90-92. 31. WARD,P. A. & HILL,J . H. (1970): C 5 chemotactic fragments produced by an enzyme in lysosomal granules of neutrophils. J. Immunol. 104,535-543. L. M. (1972): Leukocyte kinino32. GREENBAUM, genases and leukokinins from normal and malignant cells. Am. J . Pafhol. 68, 61 3 4 2 4 . 33. WILHELM,D. L. (1973): Chemical mediators. In The 1nflammator.y Process, 2nd ed.. Vol. 2. Eds B. W. Zweifach, L. Grant & R. J . McCluskey. Academic Press, New York. pp. 251-301. 34. RYAN,G. B. & MAJNO,G. (1977): Acute inflammation. A review. Am. J. Parhol. 86, 183-276.