Agents and Actions vol. 5/1 (1975) Birkhfiuser Verlag, Basel
31
Are Platelets Important in Inflammation? by F.B. UBATUBA, E.A. HARVEY and S.H. FERREIRA. The Wellcome Research Laboratories, Langley Court, Beckenham, Kent BR3 3BS, England
Abstract The participation of platelets in acute inflammation was tested by three different traumas in rats rendered thrombocytopenic with anti-platelet serum. Thrombocytopenie rats showed normal oedema response to carrageenin, anti-platelet serum and passive cutaneous anaphylaxis.
The involvement of platelets in inflammation has been suggested by many authors [1-6]. Their role in causing abnormal endothelial permeability was supposed to be due to a focal release of 5-hydroxytryptamine, histamine and/ or other vasoactive substances by local aggregation of circulating platelets. This aggregation is thought to be induced by the basement membrane collagen which is exposed during endothelial contraction or damage, caused by the released inflammatory mediators or by the initial trauma [3]. It is now well established that non-steroidal anti-inflammatory agents inhibit aggregation of platelets [7] and their formation of prostaglandins [8] and rabbit aorta contracting substance [9]. Thus it is possible that (at least in part) the anti-inflammatory activity could derive from an effect on platelet prostaglandin production. We have tested the possible participation of platelets in inflammation induced by three different traumas in rats which have been rendered thrombocytopenic by anti-platelet serum. The rat paw oedema was induced with either carrageenin, anti-platelet serum or passive cutaneous anaphylaxis. Materials and methods (a) Rat paw oedema Albino female Wistar (Charles River) rats weighing 130-200 g were used. The phlogistic agent was injected
into the subplantar region of the hind foot. The increase in rat paw volume was measured by the mercury displacement method [10] by comparing the inflamed with the contralateral paw. The time chosen for the measurements of the oedema corresponded to the maximum effect observed. Carrageenin was dissolved in saline ( 2 ~ W/V) and the increase in paw volume measured 4 hours after injection of 0.1 ml. Anti-rat platelet serum (APS) was diluted in saline and the oedema was measured 1 hour after injection of 0.1 ml. The contralateral paw received diluted normal rabbit serum (see below). Passive cutaneous anaphylaxis [11 ] was induced by subplantar injection of ovalbumin antibody [12] diluted in saline, 24 hours prior to the intravenous injection of chick egg albumin (10 mg/kg). Saline was injected in the control paw. (b) Anti-platelet serum Platelets were separated from citrated rat plasma by repeated differential centrifugation and washings in cold Tris-buffered saline (pH 7.4) containing 1 m M EDTA, until microscopically free of contaminating red blood cells and leucocytes. The antibodies against rat platelets were raised in rabbits by injecting the fresh platelet suspension in complete Freund's adjuvant into both hind paws. The rabbits were injected weekly with a fresh platelet suspension for a period of 2 months, commencing two weeks after the initial injection. For each treatment, the platelets from 4-5 rats were given subcutaneously to each rabbit, at different sites in the back and the hind legs. To harvest the antibody the rabbits were bled by heart puncture under ether anaesthesia. The serum was separated by centrifugation, inactivated at 56~ for 30 minutes distributed in small vials and stored frozen at 20 ~ The anti-platelet activity of the rabbit serum was evaluated on rats, by the following criteria: (a) lysis of platelets in vitro [13], (b) cutaneous erythema [14], (c) blood pressure fall in anaesthetised animals and (d) thrombocytopenia.
32
Platelets in Inflammation
(c) Platelet count Blood samples collected in vials with EDTA were diluted in haematologic pipettes with I ~ ammonium oxalate and counted in RBC counting chambers by phase contrast microscopy [15]. (d) Thrombocytopenic rats Thrombocytopenia was induced by injecting intravenously anti-platelet serum diluted sufficiently in saline to reduce the platelet count to less than 2 ~ . The animals were left to recover for at least 24 hours before inducing paw inflammation. The platelet counts shown in the results were made just after the oedema measurements. Chemicals and drugs. The following chemicals and drugs were used: Carrageenin (Sigma Chemical Co.); Chick egg albumin(Koch Light Laboratories); Indomethacin (Merck, Sharp and Dohme); Mepyramine maleate (May and Baker); Methysergide (Sandoz/Wander); Dexamethasone (Merck, Sharp and Dohme). I
,ZOO
PLATELETS
F - ] PA~ ~ O L ~ E 1
~--~
INDOMETHACIN
Statistics: All comparison between means were based on Student's t-test (two tailed). The level of significance taken was 5~. Values given in the figures are Mean • S.E.M. Results T r e a t m e n t of rats with anti-platelet serum i n d u c e d a fall in circulating platelets to 1 - 3 % of n o r m a l levels w i t h o u t significant modification of the i n f l a m m a t o r y oedema in a n y of the three models studied (Fig. 1, 2, 3). The t h r o m b o cytopenia at these low levels lasted for 3-4 days. W h e n challenged with i n t r a v e n o u s serum, t h r o m b o c y t o p e n i c animals exhibited a t r a n s i e n t hypotension. This contrasts m a r k e d l y with n o r m a l animals in which a n intense a n d m u c h p r o l o n g e d h y p o t e n s i o n is o b t a i n e d in response to the same doses of anti-platelet serum. I n d o m e t h a c i n (5 mg/kg) was equally effective in reducing the carrageenin oedema p r o d u c e d i n b o t h n o r m a l a n d t h r o m b o c y t o p e n i c rats (Fig. 1). The oedema i n d u c e d by the passive
TREATED
I
-']PAW
3OO
i0.0
PL^,ELErs VOLUME
"R
m
o x
8.0
I0.0 ~a -1
z
6.0
-2 % 8.0,
L)
z
o s
>
100
6.0
z
I
m ~a m
2.0
200
z.o
CAPS
CAPS
Figure 1 Carrageenin induced oedema in normal (C) and thrombocytopenic rats. The full bars shows the reduction in the platelet count by treatment with anti-platelet serum (APS). There was no significant difference in the carrageenin oedema in control and thrombocytopenic rats and indomethacin reduced the oedema in both groups. The measurements were made 4 hours after the injection of carrageenin.
2.0
i
e
APS
C
i
o
APS
Figure 2 Passive cutaneous anaphylactic reaction in normal and thrombocytopenic rats. The measurements were made 1 hour after the intravenous injection of ovalbumin.
I?latelets in Inflammation
33
0 - - - - ' 0 T H RO MBO CY T O PE N IC ( A P S ) 4oo.
:
300
$ C O N T R O L S (C)
-
3 m
z
200" "7
x
6-
portant for the development o f the oedema. There is then an apparent contradiction in the fact that platelet depleted rats responded similarly to control rats to APS. However, it is admitted that this serum cross reacts with a vascular endothelial antigen [16, 17]. It m a y well be that the small hypotensive response to an intravenous injection o f APS (at the time o f the measurements of inflammation) is a direct vascular effect o f the serum. I f this interpretation is correct, and as no difference was f o u n d in the oedema induced by APS in normal and thrombocytopenic rats, the implication is that platelets do not contribute substantially to the oedema formation, even when there is vascular damage. Platelets, however, m a y be important in models like the Arthus reaction, in which t h r o m bosis is a relevant fact o f the inflammatory process, but this is yet to be experimentally demonstrated. Acknowledgment
The authors wish to thank Miss Jenny Fitzgerald for her skilled technical assistance. C
APS
Received 14 January 1975. Figure 3
Time course of the oedema induced by intraplantar injection of anti-platelet serum in normal (c) and thrombocytopenic animals (APS). cutaneous anaphylaxis (Fig. 2) was not reduced by indomethacin (5 mg/kg, n = 5) but methysergide (1 mg/kg, n = 5) and a combination o f methysergide and mepyramine (5 mg/kg, n = 5) p r o d u c e d an inhibition o f 59 and 83 % respectively (p < 0.05). The oedema induced by anti-platelet serum (Fig. 3), however, was not modified by similar treatment with indomethacin (n = 6), mepyramine (n = 5), methysergide (n = 5) a mixture o f methysergide and mepyramine (n = 5) or by dexamethasone (0.1 mg/kg, n = 5). Discussion
O u r results show that carrageenin and P C A oedema, both o f which can be reduced by drugs, were not modified by thrombocytopenia. The APS oedema which was resistant to anti-histamines, anti-serotonin, corticoids and nonsteroidal anti-inflammatory agents was also unaffected by m a r k e d platelet depletion. Thus in these three models o f acute experimental inflammation, it seems that platelets are unim-
References
[1] J.R. O'BRIEN,Effect of Anti-inflammatory Agents on Platelets. Lancet, 2, 894-895, 1968. [2] J.R. O'BRIEN, Effects of Salicylates on Human Platelets, Lancet, 1, 779-783, 1968. [3] A.L. ROBERTSONand P.A. KHAIRALLAH,Effects of Angiotensin H on the Permeability of the Vascular Wall; in: Angiotensin, p. 500-510, (Eds. I.H. Page &
F.M. Bumpus; Springer Verlag, 1974.) [4] B.B. VARGAFTIG,Search .[or Common Mechanisms Underlying the Various Effects of Putative Inflammatory Mediators," in: The Prostaglandins, vol. 2,
205-276, (Ed. P.W. Ramwell, Plenum Press, New York-London, 1974.) [5] A. REDEI and E. KELEMEN,Presence of Platelets in Acute Experimental Inflammatory Oedema Inhibited by Salicylate or Cortisone; in: Inflammation Biochemistry and Drug Interaction. (Eds. A. Bertelli &
J.C. Houck; Excerpta Medical Fondation, Amsterdam, 1969), p. 261-265. 16] P. GOROG and I.B. KOVACS, The Alteration of Platelet Behaviour During Various Conditions and the Effect of Anti-inflammatory Agents on Platelet Aggregation and Thrombus Formation, in: Inflammation Biochemistry and Drug Interaction. (Eds.
A. Bertelli & J.C. Houck; Excerpta Medical Foundation, Amsterdam, 1969), p. 197-203. [7] H.J. WEiss and L.M. ALEDORT,Impaired PlateletConnective Tissue Reaction in Man after Aspirin Injection. Lancet, 2, 495497, 1967.
34 [8] J.B. SMITH and A.L. WILLIS, Aspirin Selectively Inhibits Prostaglandin Production in Human Platelets. Nature, New Biol. 231, 235-237, 1971. [9] B.B. VARGAETIGand P. ZmINIS, Arachidonic AcidInduced Platelet Aggregation Accompanied by Release of Potential Inflammatory Mediators Distinct from PGE2 and PGF2~ Nature New Biol. 244, 114116, 1973. [10] G.G. VAN ARMAN, A.J. BEGANY, L.M. MILLER and H.H. PLESS,Some Details of the Inflammation Caused by Yeast and Carrageenin. J. Pharmac. exp. Therap., 150, 328-334, 1965. [11] T.S.C. ORR, P. RILEY and J.E. DOE, Potentiated Reagin Response to Egg Albumin in Ippostrongylus Braziliensis Infected Rats; II Time-Course of the Reagin Response. Immunology, 20, 185-189, 1969. [12] J. GOOSE and A . M . J . N . BLAIR, Passive Cutaneous Anaphylaxis in the Rat Induced with Two Homologous
Platelets in Inflammation
Reagin-like Antibodies and its Specific Inhibition with Disodium Cromoglyeate. Immunology, 16, 749-760, 1969. [13] W.O. CRUZ, Quantitative Method of Titrating AntiPlatelet Serum in vitro. J. Immunol. 71, 346-451, 1953. [14] L.M. TOCANTINS, Experimental Thrombopenic Purpura in the Dog. Arch. Path. 21, 69-78, 1936. [15] G. BRECHER and E.P. CRONKITE,Morphology and Enumeration of Human Blood Platelets. J. Appl. Physiol. 3, 365-377, 1950. [16] R. H.E. ELLIOTTjr. and M. A. WHIPPLE, Observations on the Interrelationship of Capillary, Platelet and Splenic Factors in Thrombocytopenic Purpura. J. Lab. Clin. Med. 26, 489-498, 1940. [17] J.F. ACKROYD,Allergic Purpuras, Including Purpuras Due to Foods, Drugs and Infections. Amer. J. Med. 14, 605-632, 1953.
31
Are Platelets Important in Inflammation? by F.B. UBATUBA, E.A. HARVEY and S.H. FERREIRA. The Wellcome Research Laboratories, Langley Court, Beckenham, Kent BR3 3BS, England
Abstract The participation of platelets in acute inflammation was tested by three different traumas in rats rendered thrombocytopenic with anti-platelet serum. Thrombocytopenie rats showed normal oedema response to carrageenin, anti-platelet serum and passive cutaneous anaphylaxis.
The involvement of platelets in inflammation has been suggested by many authors [1-6]. Their role in causing abnormal endothelial permeability was supposed to be due to a focal release of 5-hydroxytryptamine, histamine and/ or other vasoactive substances by local aggregation of circulating platelets. This aggregation is thought to be induced by the basement membrane collagen which is exposed during endothelial contraction or damage, caused by the released inflammatory mediators or by the initial trauma [3]. It is now well established that non-steroidal anti-inflammatory agents inhibit aggregation of platelets [7] and their formation of prostaglandins [8] and rabbit aorta contracting substance [9]. Thus it is possible that (at least in part) the anti-inflammatory activity could derive from an effect on platelet prostaglandin production. We have tested the possible participation of platelets in inflammation induced by three different traumas in rats which have been rendered thrombocytopenic by anti-platelet serum. The rat paw oedema was induced with either carrageenin, anti-platelet serum or passive cutaneous anaphylaxis. Materials and methods (a) Rat paw oedema Albino female Wistar (Charles River) rats weighing 130-200 g were used. The phlogistic agent was injected
into the subplantar region of the hind foot. The increase in rat paw volume was measured by the mercury displacement method [10] by comparing the inflamed with the contralateral paw. The time chosen for the measurements of the oedema corresponded to the maximum effect observed. Carrageenin was dissolved in saline ( 2 ~ W/V) and the increase in paw volume measured 4 hours after injection of 0.1 ml. Anti-rat platelet serum (APS) was diluted in saline and the oedema was measured 1 hour after injection of 0.1 ml. The contralateral paw received diluted normal rabbit serum (see below). Passive cutaneous anaphylaxis [11 ] was induced by subplantar injection of ovalbumin antibody [12] diluted in saline, 24 hours prior to the intravenous injection of chick egg albumin (10 mg/kg). Saline was injected in the control paw. (b) Anti-platelet serum Platelets were separated from citrated rat plasma by repeated differential centrifugation and washings in cold Tris-buffered saline (pH 7.4) containing 1 m M EDTA, until microscopically free of contaminating red blood cells and leucocytes. The antibodies against rat platelets were raised in rabbits by injecting the fresh platelet suspension in complete Freund's adjuvant into both hind paws. The rabbits were injected weekly with a fresh platelet suspension for a period of 2 months, commencing two weeks after the initial injection. For each treatment, the platelets from 4-5 rats were given subcutaneously to each rabbit, at different sites in the back and the hind legs. To harvest the antibody the rabbits were bled by heart puncture under ether anaesthesia. The serum was separated by centrifugation, inactivated at 56~ for 30 minutes distributed in small vials and stored frozen at 20 ~ The anti-platelet activity of the rabbit serum was evaluated on rats, by the following criteria: (a) lysis of platelets in vitro [13], (b) cutaneous erythema [14], (c) blood pressure fall in anaesthetised animals and (d) thrombocytopenia.
32
Platelets in Inflammation
(c) Platelet count Blood samples collected in vials with EDTA were diluted in haematologic pipettes with I ~ ammonium oxalate and counted in RBC counting chambers by phase contrast microscopy [15]. (d) Thrombocytopenic rats Thrombocytopenia was induced by injecting intravenously anti-platelet serum diluted sufficiently in saline to reduce the platelet count to less than 2 ~ . The animals were left to recover for at least 24 hours before inducing paw inflammation. The platelet counts shown in the results were made just after the oedema measurements. Chemicals and drugs. The following chemicals and drugs were used: Carrageenin (Sigma Chemical Co.); Chick egg albumin(Koch Light Laboratories); Indomethacin (Merck, Sharp and Dohme); Mepyramine maleate (May and Baker); Methysergide (Sandoz/Wander); Dexamethasone (Merck, Sharp and Dohme). I
,ZOO
PLATELETS
F - ] PA~ ~ O L ~ E 1
~--~
INDOMETHACIN
Statistics: All comparison between means were based on Student's t-test (two tailed). The level of significance taken was 5~. Values given in the figures are Mean • S.E.M. Results T r e a t m e n t of rats with anti-platelet serum i n d u c e d a fall in circulating platelets to 1 - 3 % of n o r m a l levels w i t h o u t significant modification of the i n f l a m m a t o r y oedema in a n y of the three models studied (Fig. 1, 2, 3). The t h r o m b o cytopenia at these low levels lasted for 3-4 days. W h e n challenged with i n t r a v e n o u s serum, t h r o m b o c y t o p e n i c animals exhibited a t r a n s i e n t hypotension. This contrasts m a r k e d l y with n o r m a l animals in which a n intense a n d m u c h p r o l o n g e d h y p o t e n s i o n is o b t a i n e d in response to the same doses of anti-platelet serum. I n d o m e t h a c i n (5 mg/kg) was equally effective in reducing the carrageenin oedema p r o d u c e d i n b o t h n o r m a l a n d t h r o m b o c y t o p e n i c rats (Fig. 1). The oedema i n d u c e d by the passive
TREATED
I
-']PAW
3OO
i0.0
PL^,ELErs VOLUME
"R
m
o x
8.0
I0.0 ~a -1
z
6.0
-2 % 8.0,
L)
z
o s
>
100
6.0
z
I
m ~a m
2.0
200
z.o
CAPS
CAPS
Figure 1 Carrageenin induced oedema in normal (C) and thrombocytopenic rats. The full bars shows the reduction in the platelet count by treatment with anti-platelet serum (APS). There was no significant difference in the carrageenin oedema in control and thrombocytopenic rats and indomethacin reduced the oedema in both groups. The measurements were made 4 hours after the injection of carrageenin.
2.0
i
e
APS
C
i
o
APS
Figure 2 Passive cutaneous anaphylactic reaction in normal and thrombocytopenic rats. The measurements were made 1 hour after the intravenous injection of ovalbumin.
I?latelets in Inflammation
33
0 - - - - ' 0 T H RO MBO CY T O PE N IC ( A P S ) 4oo.
:
300
$ C O N T R O L S (C)
-
3 m
z
200" "7
x
6-
portant for the development o f the oedema. There is then an apparent contradiction in the fact that platelet depleted rats responded similarly to control rats to APS. However, it is admitted that this serum cross reacts with a vascular endothelial antigen [16, 17]. It m a y well be that the small hypotensive response to an intravenous injection o f APS (at the time o f the measurements of inflammation) is a direct vascular effect o f the serum. I f this interpretation is correct, and as no difference was f o u n d in the oedema induced by APS in normal and thrombocytopenic rats, the implication is that platelets do not contribute substantially to the oedema formation, even when there is vascular damage. Platelets, however, m a y be important in models like the Arthus reaction, in which t h r o m bosis is a relevant fact o f the inflammatory process, but this is yet to be experimentally demonstrated. Acknowledgment
The authors wish to thank Miss Jenny Fitzgerald for her skilled technical assistance. C
APS
Received 14 January 1975. Figure 3
Time course of the oedema induced by intraplantar injection of anti-platelet serum in normal (c) and thrombocytopenic animals (APS). cutaneous anaphylaxis (Fig. 2) was not reduced by indomethacin (5 mg/kg, n = 5) but methysergide (1 mg/kg, n = 5) and a combination o f methysergide and mepyramine (5 mg/kg, n = 5) p r o d u c e d an inhibition o f 59 and 83 % respectively (p < 0.05). The oedema induced by anti-platelet serum (Fig. 3), however, was not modified by similar treatment with indomethacin (n = 6), mepyramine (n = 5), methysergide (n = 5) a mixture o f methysergide and mepyramine (n = 5) or by dexamethasone (0.1 mg/kg, n = 5). Discussion
O u r results show that carrageenin and P C A oedema, both o f which can be reduced by drugs, were not modified by thrombocytopenia. The APS oedema which was resistant to anti-histamines, anti-serotonin, corticoids and nonsteroidal anti-inflammatory agents was also unaffected by m a r k e d platelet depletion. Thus in these three models o f acute experimental inflammation, it seems that platelets are unim-
References
[1] J.R. O'BRIEN,Effect of Anti-inflammatory Agents on Platelets. Lancet, 2, 894-895, 1968. [2] J.R. O'BRIEN, Effects of Salicylates on Human Platelets, Lancet, 1, 779-783, 1968. [3] A.L. ROBERTSONand P.A. KHAIRALLAH,Effects of Angiotensin H on the Permeability of the Vascular Wall; in: Angiotensin, p. 500-510, (Eds. I.H. Page &
F.M. Bumpus; Springer Verlag, 1974.) [4] B.B. VARGAFTIG,Search .[or Common Mechanisms Underlying the Various Effects of Putative Inflammatory Mediators," in: The Prostaglandins, vol. 2,
205-276, (Ed. P.W. Ramwell, Plenum Press, New York-London, 1974.) [5] A. REDEI and E. KELEMEN,Presence of Platelets in Acute Experimental Inflammatory Oedema Inhibited by Salicylate or Cortisone; in: Inflammation Biochemistry and Drug Interaction. (Eds. A. Bertelli &
J.C. Houck; Excerpta Medical Fondation, Amsterdam, 1969), p. 261-265. 16] P. GOROG and I.B. KOVACS, The Alteration of Platelet Behaviour During Various Conditions and the Effect of Anti-inflammatory Agents on Platelet Aggregation and Thrombus Formation, in: Inflammation Biochemistry and Drug Interaction. (Eds.
A. Bertelli & J.C. Houck; Excerpta Medical Foundation, Amsterdam, 1969), p. 197-203. [7] H.J. WEiss and L.M. ALEDORT,Impaired PlateletConnective Tissue Reaction in Man after Aspirin Injection. Lancet, 2, 495497, 1967.
34 [8] J.B. SMITH and A.L. WILLIS, Aspirin Selectively Inhibits Prostaglandin Production in Human Platelets. Nature, New Biol. 231, 235-237, 1971. [9] B.B. VARGAETIGand P. ZmINIS, Arachidonic AcidInduced Platelet Aggregation Accompanied by Release of Potential Inflammatory Mediators Distinct from PGE2 and PGF2~ Nature New Biol. 244, 114116, 1973. [10] G.G. VAN ARMAN, A.J. BEGANY, L.M. MILLER and H.H. PLESS,Some Details of the Inflammation Caused by Yeast and Carrageenin. J. Pharmac. exp. Therap., 150, 328-334, 1965. [11] T.S.C. ORR, P. RILEY and J.E. DOE, Potentiated Reagin Response to Egg Albumin in Ippostrongylus Braziliensis Infected Rats; II Time-Course of the Reagin Response. Immunology, 20, 185-189, 1969. [12] J. GOOSE and A . M . J . N . BLAIR, Passive Cutaneous Anaphylaxis in the Rat Induced with Two Homologous
Platelets in Inflammation
Reagin-like Antibodies and its Specific Inhibition with Disodium Cromoglyeate. Immunology, 16, 749-760, 1969. [13] W.O. CRUZ, Quantitative Method of Titrating AntiPlatelet Serum in vitro. J. Immunol. 71, 346-451, 1953. [14] L.M. TOCANTINS, Experimental Thrombopenic Purpura in the Dog. Arch. Path. 21, 69-78, 1936. [15] G. BRECHER and E.P. CRONKITE,Morphology and Enumeration of Human Blood Platelets. J. Appl. Physiol. 3, 365-377, 1950. [16] R. H.E. ELLIOTTjr. and M. A. WHIPPLE, Observations on the Interrelationship of Capillary, Platelet and Splenic Factors in Thrombocytopenic Purpura. J. Lab. Clin. Med. 26, 489-498, 1940. [17] J.F. ACKROYD,Allergic Purpuras, Including Purpuras Due to Foods, Drugs and Infections. Amer. J. Med. 14, 605-632, 1953.
