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Interaction of carbohydrates and anti-inflammatory drugs with mast cells in the rat
Biochemical Pharmacology, Supplement, pp. 309-314. Pergamon Press. 1968. Printed in Great Britain
INTERACTION OF CARBOHYDRATES A N D ANTI-INFLAMMATORY D R U G S WITH MAST CELLS IN THE RAT ANDRES GOTH with the technical assistance of Mary Knoohuizen Department of Pharmacology, The University of Texas Southwestern Medical School, Dallas, Texas Abstract--Certain sugars, especially 2-deoxyglucose, can block inflammation produced by dextran or ovomucoid in the rat. This anti-inflammatory actions of sugars is well correlated with their ability to inhibit degranulation and histamine release of mast cells by dextran or ovomucoid in vitro or in vivo. Because the same sugars do not inhibit the action of compound 48/80, special receptors have been postulated for the polysaccharide. In an extension of this work, a number of anti-inflammatory drugs were tested against the effects of dextran and compound 48/80 on rat (Wistar) peritoneal mast cells by using in hibition of histamine release as a measure of their potency. All anti-inflammatory drugs tested, such as sodium salicylate, phenylbutazone, hydrocortisone sodium succinate, and colchicine, were much more active in blocking the action of dextran than that of compound 48/80. The preferential effect of sodium salicylate on the systemic action of dextran as contrasted with that of compound 48/80 was demonstrable in vivo. The greater susceptibility of the dextran effect to the anti-inflammatory drugs points to a different mode of action of the polysaccharide as compared with compound 48/80. Since edema produced by the polysaccharidecarrageenin is also highly susceptible to inhibition by anti-inflammatory drugs, this relationship may reflect a similarity in the mode of action of polysaccharides.
THE MODE o f a c t i o n o f a n t i - i n f l a m m a t o r y drugs is n o t well u n d e r s t o o d despite extensive studies on the subject. Some o f the best e x p e r i m e n t a l m o d e l s used in these studies, such as the c a r r a g e e n i n edema, 1 have failed to c o n t r i b u t e m u c h to an u n d e r s t a n d i n g o f the m e c h a n i s m s involved in a n t i - i n f l a m m a t o r y activity. It is difficult to know, in fact, w h a t b i o c h e m i c a l m e c h a n i s m s or even w h a t cellular elements p a r t i c i p a t e in these interactions. There are, on the other hand, p r o - i n f l a m m a t o r y agents, such as dextran, o v o m u c o i d , a n d yeast, which release histamine f r o m rat p e r i t o n e a l cells in vitro. 2, 3 A study o f a n t i - i n f l a m m a t o r y d r u g effects on this type o f experimental m o d e l was u n d e r t a k e n in an a t t e m p t at learning a b o u t the m e c h a n i s m s involved. In previous studies in the rat, 4, ~ the a n t i - i n f l a m m a t o r y activity o f 2-deoxyglucose a n d s o m e o t h e r sugars could be c o r r e l a t e d with their ability to block histamine release f r o m p e r i t o n e a l cells b y dextran. I n these studies there was a striking dissociation between the effectiveness o f 2-deoxyglucose against d e x t r a n and its complete ineffectiveness against c o m p o u n d 48/80 in vitro or in vivo. O n the basis o f these and related studies 6 the existence o f d e x t r a n receptors has been postulated. It is i m p o r t a n t to p o i n t o u t that, because all o f these studies were d o n e on m i x e d p e r i t o n e a l cells, the d e x t r a n receptors are n o t necessarily on m a s t cells; an interaction with other c o m p o n e n t s o f the p e r i t o n e a l washings could indirectly lead to histamine release. 309
310
ANDRESGOTH
In the present study, various anti-inflammatory drugs behaved qualitatively like 2-deoxyglucose in that they acted preferentially or exclusively on histamine release by dextran as compared with compound 48/80. These striking differences between dextran and compound 48/80 raised the possibility that the two pro-inflammatory agents may not only act on different receptors but perhaps on different cells. In an extension of this work it was found that peritoneal cell suspensions from nitrogen-mustardpretreated rats released little histamine when exposed to dextran, but their total histamine content was normal and their response to compound 48/80 was quite vigorous. These experiments suggest that pro-inflammatory agents of the polysaccharide type exert their effect by some mechanism which is basically more susceptible to various anti-inflammatory drugs. The findings with nitrogen mustard suggest that some leucocyte function may be involved in the dextran effect. MATERIALS AND METHODS Wistar rats were used in all experiments in which peritoneal cell suspensions were studied. Both Wistar and Holtzman rats were used for obtaining the in vivo data. Peritoneal cell suspensions were obtained as described in a previous paper. 5 Glucosefree Tyrode's solution with 5/zg/ml of heparin was used throughout because glucose tends to interfere with the action of dextran. All experiments were performed at 37 °. The final volume of the incubation mixtures was 3 ml. The tubes containing 2.1 ml of the peritoneal cell suspensions plus the various additions were allowed to stand for 20 min with occasional shaking. At the end of this period the tubes were centrifuged and histamine was determined on aliquots of the supernate by a fluorometric method.7 In addition, total histamine was determined on samples obtained prior to centrifugation. Results were expressed in terms of per cent of total histamine released. For in rico studies, all injections were by the i.v. route. Sodium salicylate or saline was injected 10 min before dextran or compound 48/80. Injection volumes were 0.5 ml/100 g. Capillary hematocrit determinations were done immediately before and 15 min after the injection of dextran or compound 48/80. Nitrogen mustard (2 mg/kg) was injected 5 days prior to the relevant experiments. The following drugs were used. Dextran (average molecular weight, 40,000) was purchased from Mann Laboratories; compound 48/80 was kindly supplied by Burroughs Wellcome; sodium salicylate was USP grade; phenylbutazone powder was kindly supplied by the Geigy Co. ; colchicine was USP grade. In some experiments, hydrocortisone sodium succinate (Solu-Cortef) and sodium benzoate USP were also tested. The nitrogen mustard used was sterile mechlorethamine hydrochloride trituration N F (Mustargen). RESULTS Effect of anti-inflammatory drugs on histamine release by dextran or compound 48/80 in vitro As shown in Table l, sodium salicylate, phenylbutazone, and colchicine had an inhibitory effect on histamine release. This effect was particularly marked against dextran. It is noteworthy that essentially complete inhibition could be achieved with these drugs against dextran at concentrations which had little effect on the histamine release induced by compound 48/80. Preliminary data indicate that dinitrophenol
Chemistry and biology of inflammation--l[
311
shows the same preferential effect on d e x t r a n - i n d u c e d histamine release. Sodium b e n z o a t e has similar effects but requires higher c o n c e n t r a t i o n s t h a n does s o d i u m salicylate for b l o c k i n g the a c t i o n o f dextran. H y d r o c o r t i s o n e s o d i u m succinate showed a similar preferential effect against d e x t r a n - i n d u c e d histamine release but, because the p r e p a r a t i o n c o n t a i n e d preservatives a n d no a d e q u a t e controls could be p e r f o r m e d on the possible effect o f these, these results are m e n t i o n e d with reservations. TABLE 1. EFFECT OF ANTI-INFLAMMATORYDRUGS ON HISTAMINE RELEASE FROM RAT PERITONEAL CELLS Histamine released ( ~ of total)* By dextrant By compound 48/80t
Drug None Sodium salicylate 0.67 mM Sodium salicylate 1 mM Sodium salicyte 2 mM Sodium salicylate 6"7 mM Phenylbutazone 0"33 mM Phenylbutazone 3.3 mM Colchicine 0"5 mM
13 + 4 4 0-9 3.5 3-4 0
(13)
-3 0-6 0 2 1-2
27 ± 4
(3)
--
(2)
--
(2)
(2) (2) (3)
(10)
27 24-29 33 29-37 16 12-19 7-19 15 9-17
(2) (2) (2) (2) (3)
* Mean values, ranges, and numbers of experiments (in parentheses). t Concentration of dextran: 1 mg/ml. Concentration of compound 48/'80: 0.25/zg/ml.
Effect of sodium salicylate on reaction of rats to dextran and compound 48/80 I n T a b l e 2 it is shown that, in a g r e e m e n t with the previous in vitro data, s o d i u m salicylate blocks the effect o f d e x t r a n in the whole a n i m a l m e a s u r e d b y changes in h e m a t o c r i t value. A t the same time it h a d no effect on the action o f c o m p o u n d 48/80. These changes c o r r e l a t e d well with the visible systemic reaction o f the animals to the injected p r o - i n f l a m m a t o r y agent. TABLE 2. EFFECT OF SODIUM SALICYLATEON SYSTEMIC REACTION TO DEXTRAN AND COMPOUND 48/80 IN RATS Sodium salicylate (mg/kg, i.v.) 0 100 500
Hematocrit increase* after: Dextrant Compound 48/80~ 14 zk 3 (6) 8 i 5 (6) 0 :k I (7)
22 :k 4 (5) -21 zk 1 (6)
* Mean values, S.D. and number of animals (in parentheses). t Dose of dextran: 60 mg/kg. Dose of compound 48/80: 300/zg/kg.
312
ANDRES GOTH
h~uence of nitrogen mustard pretreatment on histamhte release from peritoneal cells The differences observed between the actions of dextran and compound 48/80 shown in the previous studies suggested that either different receptors or perhaps even different cells are involved in these interactions. Because nitrogen mustard can cause a virtual disappearance of leucocytes from rat peritoneal cell suspensions without influencing their mast cell content, 8 it seemed of interest to use it as a tool for extension of the present findings. The results of nitrogen mustard pretreatment on the reaction of rat peritoneal cells to dextran and compound 48/80 are shown in Table 3.
TABLE 3. EFFECT OF NITROGENMUSTARDPRETREATMENTON RESPONSE OF PERITONEAL CELLS TO DEXTRANAND COMPOUND 48/80 Histamine release ( ~o of total) Pretreatment None Nitrogen mustard,~
By dextran* 13 -k_4(13) 2 ± 1 (6)
By compound 48/80t 27 ± 4(10) 25 ± 4 (6)
* Dextran concentration: 1 mg/ml. Compound 48]80 concentration: 0.25 txg/ml. Nitrogen mustard (mechlorethamine), 2 mg/kg, was injected 5 days prior to the experiment, by the i.v. route. Such pretreatment caused a marked inhibition of the response of the cell suspensions to dextran while it had no influence on the response to compound 48/80. It also had no effect on the total histamine content of the suspensions. DISCUSSION The experiments reported in this paper, along with previous publications, 4, (~ establish three sets of facts which may contribute to the understanding of antiinflammatory mechanisms. (1) A variety of anti-inflammatory drugs, as well as metabolic inhibitors, have a much greater effect on the response of rats to the polysaccharide dextran than to the polyamine compound 48/80. (2) This preferential effect is correlated in several instances with the relative susceptibility of peritoneal cell suspensions to the two pro-inflammatory agents, as determined by histamine release in vitro. (3) Nitrogen mustard pretreatment blocks the effect of dextran without influencing the action of compound 48/80 on peritoneal cell suspensions in vitro. The ability of anti-inflammatory drugs to block the release of histamine has been noted before. Salicylate in very high concentrations (10-25 mM) can block histamine release by compound 48/80 from rat diaphragm in vitro 9, lo and anaphylactic histamine release from guinea pig lung 11 or rabbit blood cells, 12 also in vitro. The significance of these observations is not clear because, at the very high concentrations used, salicylate acted like anoxia and certain metabolic inhibitors in that its action was reversed by glucose. Since the concentrations used would be highly toxic or lethal under in vivo conditions and since glucose is present under these same conditions, it does not seem likely that the above observations are related to what salicylates may do in vivo as anti-inflammatory drugs. In the present study, salicylates were shown to be much more active against dextran than against compound 48/80, not only in the test tube but also in preventing systemic
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effects in the rat. It would be reasonable to assume from these experiments that the anti-inflammatory action of salicylates against dextran may be related to the ability of the drug to block, directly or indirectly, some process which eventually leads to the release of histamine. The results do not imply that dextran edema is caused by histamine, because other mast cell products--serotonin and proteases--could well play a major part. The important point is that salicylate in these experiments blocks some step in the process which eventually leads to the release of histamine presumably from mast cells. Colchicine has been shown recently to block histamine release from rat peritoneal cells induced by compound 48/80.13 The findings in the present experiments (Table I) tend to confirm this but suggest that colchicine is even more potent against dextraninduced histamine release. Since compound 48/80 acts directly on mast cells, there can be little doubt that colchicine must have an effect on such cells. There is some earlier morphological evidence for an action of colchicine on mast cells. 14 The greater effect of the alkaloid on the dextran effect is difficult to evaluate on the basis of the present experiments but could mean some additional action. The results of the experiments with nitrogen mustard pretreatment are quite surprising and may require an entirely new interpretation of the dextran reaction in the rat. The great differences between dextran and compound 48/80 as regards the influence of various inhibitors on their actions have led to the suggestion 6 that the two compounds act on two different receptors, presumably both on the mast cell. The direct effect of compound 48/80 on the mast cell is well documented. 15, 16 On the other hand, the present experiments with nitrogen mustard suggest that perhaps the primary interaction of dextran is with leucocytes, because the best known influence of the alkylating agent is to cause a virtual disappearance of leucocytes with no effect on mast cells. It is too early to speculate on the mechanism whereby a primary interaction with leucocytes could lead to histamine release from mast cells, but it is interesting to note that lysosomal polypeptides have been described in rabbit leucocytes, which can cause histamine release from rat peritoneal cells. 17, a8 Although it is tempting to speculate about the mode of action of nitrogen mustard in these experiments, it is important to remember that the data are not sufficient to prove this point. Nitrogen mustard could deplete or inactivate some substance or mechanism which is necessary for the dextran reaction. REFERENCES 1. C. A. WINTER, E. A. RISLEYand G. W. Nuss, Proc. Soc. exp. Biol. Med. 111, 544 (1962). 2. W. DIASDA SILVAand A. D. LEMOSFERNANDES, Experientia 21, 96 (1965). 3. C. G. VAN ARMAN, A. J. BEGANY, L. M, MILLER and H. H. PLESS, J. Pharmac. exp. Ther. 150,
328 0965). 4. A. GOTH,Am. J. Physiol. 197, 1056 (1959). 5. A. GOTHand M. KNOOHUIZEN,Life Sci. 1,459 (1962). 6. A. GO'm, Fedn Proc. 26, 222 (1967). 7. P. A. SHORE,A. BURKHALTERand V. H. COHN,J. Pharmac. exp. Ther. 127, 182 0959). 8. F. K. AUSTEN,Fedn Proc. in press. 9. A. M. ROTHSCHILD,L VUGMANand ROCHAE SILVA, Biochem. Pharmac. 7, 248 (1961). 10. M. ROCHAE SILVA,Int. Syrup. on Injury, Inflammation and Immunity, p. 220. Williams & Wilkins Co., Baltimore (1964). 11. E. R. TRETHEWlE,Aust. J. exp. Biol. reed. Sci. 29, 443 (1951). 12. C. G. HAINING,Br. J. Pharmac. 11,357 (1956).
314 13. 14. 15. 16. 17. 18.
ANDRES GOa-H E. GILLESPIE, S. E. MALAWISTAand R. J. LEVINE, Fedn Proc. 26, 786 (1967). J. PADAWERand A. S. GORDON, Proc. Soc. exp. Biol. Med. 88, 522 (1955). D. W. FAWCETT, d. exp. Med. 100, 217 (1954). D. LAGUNOFFand E. P. BENDITT, J. exp. Med. 112, 571 (1960). A. JANOFF and B. W. ZWEIFACH,Jr. exp. Med. 120, 747 (1964). A. JANOFF, M. A. BEAN and E. SCHULLER,Life Sci. 4, 2361 0965).
COMMENTS DR. MALAWISTA: Did you test the effect of nitrogen mustard on the mast cells to see whether it might inhibit histamine release directly ? DR. GOTH: No. [ doubt that the effect could be on the release process since the cells respond in the usual manner to compound 48/80. DR. MELMON: Can you tell me why the hematocrit value increased after administration of dextran i n the rat ? DR. GOTH: This is undoubtedly a consequence of increased capillary permeability and loss of plasma from the circulation. In the rat, dextran causes increased capillary permeability, probably because it acts directly or indirectly as a mast cell discharger. In other species or in a diabetic rat, dextran would produce a decrease rather than an increase in the hematocrit value. DR. WHITEHOUSE: I could not help but be impressed by the possible connection between what Dr. Glenn was talking about and your cell system, which may have something to do with stabilization. DR. GOTH: Stabilization of some membrane may be involved in our results, but the difficulty is that [ do not know what cell it is whose membrane is being stabilized by the anti-inflammatory drugs.
INTERACTION OF CARBOHYDRATES A N D ANTI-INFLAMMATORY D R U G S WITH MAST CELLS IN THE RAT ANDRES GOTH with the technical assistance of Mary Knoohuizen Department of Pharmacology, The University of Texas Southwestern Medical School, Dallas, Texas Abstract--Certain sugars, especially 2-deoxyglucose, can block inflammation produced by dextran or ovomucoid in the rat. This anti-inflammatory actions of sugars is well correlated with their ability to inhibit degranulation and histamine release of mast cells by dextran or ovomucoid in vitro or in vivo. Because the same sugars do not inhibit the action of compound 48/80, special receptors have been postulated for the polysaccharide. In an extension of this work, a number of anti-inflammatory drugs were tested against the effects of dextran and compound 48/80 on rat (Wistar) peritoneal mast cells by using in hibition of histamine release as a measure of their potency. All anti-inflammatory drugs tested, such as sodium salicylate, phenylbutazone, hydrocortisone sodium succinate, and colchicine, were much more active in blocking the action of dextran than that of compound 48/80. The preferential effect of sodium salicylate on the systemic action of dextran as contrasted with that of compound 48/80 was demonstrable in vivo. The greater susceptibility of the dextran effect to the anti-inflammatory drugs points to a different mode of action of the polysaccharide as compared with compound 48/80. Since edema produced by the polysaccharidecarrageenin is also highly susceptible to inhibition by anti-inflammatory drugs, this relationship may reflect a similarity in the mode of action of polysaccharides.
THE MODE o f a c t i o n o f a n t i - i n f l a m m a t o r y drugs is n o t well u n d e r s t o o d despite extensive studies on the subject. Some o f the best e x p e r i m e n t a l m o d e l s used in these studies, such as the c a r r a g e e n i n edema, 1 have failed to c o n t r i b u t e m u c h to an u n d e r s t a n d i n g o f the m e c h a n i s m s involved in a n t i - i n f l a m m a t o r y activity. It is difficult to know, in fact, w h a t b i o c h e m i c a l m e c h a n i s m s or even w h a t cellular elements p a r t i c i p a t e in these interactions. There are, on the other hand, p r o - i n f l a m m a t o r y agents, such as dextran, o v o m u c o i d , a n d yeast, which release histamine f r o m rat p e r i t o n e a l cells in vitro. 2, 3 A study o f a n t i - i n f l a m m a t o r y d r u g effects on this type o f experimental m o d e l was u n d e r t a k e n in an a t t e m p t at learning a b o u t the m e c h a n i s m s involved. In previous studies in the rat, 4, ~ the a n t i - i n f l a m m a t o r y activity o f 2-deoxyglucose a n d s o m e o t h e r sugars could be c o r r e l a t e d with their ability to block histamine release f r o m p e r i t o n e a l cells b y dextran. I n these studies there was a striking dissociation between the effectiveness o f 2-deoxyglucose against d e x t r a n and its complete ineffectiveness against c o m p o u n d 48/80 in vitro or in vivo. O n the basis o f these and related studies 6 the existence o f d e x t r a n receptors has been postulated. It is i m p o r t a n t to p o i n t o u t that, because all o f these studies were d o n e on m i x e d p e r i t o n e a l cells, the d e x t r a n receptors are n o t necessarily on m a s t cells; an interaction with other c o m p o n e n t s o f the p e r i t o n e a l washings could indirectly lead to histamine release. 309
310
ANDRESGOTH
In the present study, various anti-inflammatory drugs behaved qualitatively like 2-deoxyglucose in that they acted preferentially or exclusively on histamine release by dextran as compared with compound 48/80. These striking differences between dextran and compound 48/80 raised the possibility that the two pro-inflammatory agents may not only act on different receptors but perhaps on different cells. In an extension of this work it was found that peritoneal cell suspensions from nitrogen-mustardpretreated rats released little histamine when exposed to dextran, but their total histamine content was normal and their response to compound 48/80 was quite vigorous. These experiments suggest that pro-inflammatory agents of the polysaccharide type exert their effect by some mechanism which is basically more susceptible to various anti-inflammatory drugs. The findings with nitrogen mustard suggest that some leucocyte function may be involved in the dextran effect. MATERIALS AND METHODS Wistar rats were used in all experiments in which peritoneal cell suspensions were studied. Both Wistar and Holtzman rats were used for obtaining the in vivo data. Peritoneal cell suspensions were obtained as described in a previous paper. 5 Glucosefree Tyrode's solution with 5/zg/ml of heparin was used throughout because glucose tends to interfere with the action of dextran. All experiments were performed at 37 °. The final volume of the incubation mixtures was 3 ml. The tubes containing 2.1 ml of the peritoneal cell suspensions plus the various additions were allowed to stand for 20 min with occasional shaking. At the end of this period the tubes were centrifuged and histamine was determined on aliquots of the supernate by a fluorometric method.7 In addition, total histamine was determined on samples obtained prior to centrifugation. Results were expressed in terms of per cent of total histamine released. For in rico studies, all injections were by the i.v. route. Sodium salicylate or saline was injected 10 min before dextran or compound 48/80. Injection volumes were 0.5 ml/100 g. Capillary hematocrit determinations were done immediately before and 15 min after the injection of dextran or compound 48/80. Nitrogen mustard (2 mg/kg) was injected 5 days prior to the relevant experiments. The following drugs were used. Dextran (average molecular weight, 40,000) was purchased from Mann Laboratories; compound 48/80 was kindly supplied by Burroughs Wellcome; sodium salicylate was USP grade; phenylbutazone powder was kindly supplied by the Geigy Co. ; colchicine was USP grade. In some experiments, hydrocortisone sodium succinate (Solu-Cortef) and sodium benzoate USP were also tested. The nitrogen mustard used was sterile mechlorethamine hydrochloride trituration N F (Mustargen). RESULTS Effect of anti-inflammatory drugs on histamine release by dextran or compound 48/80 in vitro As shown in Table l, sodium salicylate, phenylbutazone, and colchicine had an inhibitory effect on histamine release. This effect was particularly marked against dextran. It is noteworthy that essentially complete inhibition could be achieved with these drugs against dextran at concentrations which had little effect on the histamine release induced by compound 48/80. Preliminary data indicate that dinitrophenol
Chemistry and biology of inflammation--l[
311
shows the same preferential effect on d e x t r a n - i n d u c e d histamine release. Sodium b e n z o a t e has similar effects but requires higher c o n c e n t r a t i o n s t h a n does s o d i u m salicylate for b l o c k i n g the a c t i o n o f dextran. H y d r o c o r t i s o n e s o d i u m succinate showed a similar preferential effect against d e x t r a n - i n d u c e d histamine release but, because the p r e p a r a t i o n c o n t a i n e d preservatives a n d no a d e q u a t e controls could be p e r f o r m e d on the possible effect o f these, these results are m e n t i o n e d with reservations. TABLE 1. EFFECT OF ANTI-INFLAMMATORYDRUGS ON HISTAMINE RELEASE FROM RAT PERITONEAL CELLS Histamine released ( ~ of total)* By dextrant By compound 48/80t
Drug None Sodium salicylate 0.67 mM Sodium salicylate 1 mM Sodium salicyte 2 mM Sodium salicylate 6"7 mM Phenylbutazone 0"33 mM Phenylbutazone 3.3 mM Colchicine 0"5 mM
13 + 4 4 0-9 3.5 3-4 0
(13)
-3 0-6 0 2 1-2
27 ± 4
(3)
--
(2)
--
(2)
(2) (2) (3)
(10)
27 24-29 33 29-37 16 12-19 7-19 15 9-17
(2) (2) (2) (2) (3)
* Mean values, ranges, and numbers of experiments (in parentheses). t Concentration of dextran: 1 mg/ml. Concentration of compound 48/'80: 0.25/zg/ml.
Effect of sodium salicylate on reaction of rats to dextran and compound 48/80 I n T a b l e 2 it is shown that, in a g r e e m e n t with the previous in vitro data, s o d i u m salicylate blocks the effect o f d e x t r a n in the whole a n i m a l m e a s u r e d b y changes in h e m a t o c r i t value. A t the same time it h a d no effect on the action o f c o m p o u n d 48/80. These changes c o r r e l a t e d well with the visible systemic reaction o f the animals to the injected p r o - i n f l a m m a t o r y agent. TABLE 2. EFFECT OF SODIUM SALICYLATEON SYSTEMIC REACTION TO DEXTRAN AND COMPOUND 48/80 IN RATS Sodium salicylate (mg/kg, i.v.) 0 100 500
Hematocrit increase* after: Dextrant Compound 48/80~ 14 zk 3 (6) 8 i 5 (6) 0 :k I (7)
22 :k 4 (5) -21 zk 1 (6)
* Mean values, S.D. and number of animals (in parentheses). t Dose of dextran: 60 mg/kg. Dose of compound 48/80: 300/zg/kg.
312
ANDRES GOTH
h~uence of nitrogen mustard pretreatment on histamhte release from peritoneal cells The differences observed between the actions of dextran and compound 48/80 shown in the previous studies suggested that either different receptors or perhaps even different cells are involved in these interactions. Because nitrogen mustard can cause a virtual disappearance of leucocytes from rat peritoneal cell suspensions without influencing their mast cell content, 8 it seemed of interest to use it as a tool for extension of the present findings. The results of nitrogen mustard pretreatment on the reaction of rat peritoneal cells to dextran and compound 48/80 are shown in Table 3.
TABLE 3. EFFECT OF NITROGENMUSTARDPRETREATMENTON RESPONSE OF PERITONEAL CELLS TO DEXTRANAND COMPOUND 48/80 Histamine release ( ~o of total) Pretreatment None Nitrogen mustard,~
By dextran* 13 -k_4(13) 2 ± 1 (6)
By compound 48/80t 27 ± 4(10) 25 ± 4 (6)
* Dextran concentration: 1 mg/ml. Compound 48]80 concentration: 0.25 txg/ml. Nitrogen mustard (mechlorethamine), 2 mg/kg, was injected 5 days prior to the experiment, by the i.v. route. Such pretreatment caused a marked inhibition of the response of the cell suspensions to dextran while it had no influence on the response to compound 48/80. It also had no effect on the total histamine content of the suspensions. DISCUSSION The experiments reported in this paper, along with previous publications, 4, (~ establish three sets of facts which may contribute to the understanding of antiinflammatory mechanisms. (1) A variety of anti-inflammatory drugs, as well as metabolic inhibitors, have a much greater effect on the response of rats to the polysaccharide dextran than to the polyamine compound 48/80. (2) This preferential effect is correlated in several instances with the relative susceptibility of peritoneal cell suspensions to the two pro-inflammatory agents, as determined by histamine release in vitro. (3) Nitrogen mustard pretreatment blocks the effect of dextran without influencing the action of compound 48/80 on peritoneal cell suspensions in vitro. The ability of anti-inflammatory drugs to block the release of histamine has been noted before. Salicylate in very high concentrations (10-25 mM) can block histamine release by compound 48/80 from rat diaphragm in vitro 9, lo and anaphylactic histamine release from guinea pig lung 11 or rabbit blood cells, 12 also in vitro. The significance of these observations is not clear because, at the very high concentrations used, salicylate acted like anoxia and certain metabolic inhibitors in that its action was reversed by glucose. Since the concentrations used would be highly toxic or lethal under in vivo conditions and since glucose is present under these same conditions, it does not seem likely that the above observations are related to what salicylates may do in vivo as anti-inflammatory drugs. In the present study, salicylates were shown to be much more active against dextran than against compound 48/80, not only in the test tube but also in preventing systemic
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effects in the rat. It would be reasonable to assume from these experiments that the anti-inflammatory action of salicylates against dextran may be related to the ability of the drug to block, directly or indirectly, some process which eventually leads to the release of histamine. The results do not imply that dextran edema is caused by histamine, because other mast cell products--serotonin and proteases--could well play a major part. The important point is that salicylate in these experiments blocks some step in the process which eventually leads to the release of histamine presumably from mast cells. Colchicine has been shown recently to block histamine release from rat peritoneal cells induced by compound 48/80.13 The findings in the present experiments (Table I) tend to confirm this but suggest that colchicine is even more potent against dextraninduced histamine release. Since compound 48/80 acts directly on mast cells, there can be little doubt that colchicine must have an effect on such cells. There is some earlier morphological evidence for an action of colchicine on mast cells. 14 The greater effect of the alkaloid on the dextran effect is difficult to evaluate on the basis of the present experiments but could mean some additional action. The results of the experiments with nitrogen mustard pretreatment are quite surprising and may require an entirely new interpretation of the dextran reaction in the rat. The great differences between dextran and compound 48/80 as regards the influence of various inhibitors on their actions have led to the suggestion 6 that the two compounds act on two different receptors, presumably both on the mast cell. The direct effect of compound 48/80 on the mast cell is well documented. 15, 16 On the other hand, the present experiments with nitrogen mustard suggest that perhaps the primary interaction of dextran is with leucocytes, because the best known influence of the alkylating agent is to cause a virtual disappearance of leucocytes with no effect on mast cells. It is too early to speculate on the mechanism whereby a primary interaction with leucocytes could lead to histamine release from mast cells, but it is interesting to note that lysosomal polypeptides have been described in rabbit leucocytes, which can cause histamine release from rat peritoneal cells. 17, a8 Although it is tempting to speculate about the mode of action of nitrogen mustard in these experiments, it is important to remember that the data are not sufficient to prove this point. Nitrogen mustard could deplete or inactivate some substance or mechanism which is necessary for the dextran reaction. REFERENCES 1. C. A. WINTER, E. A. RISLEYand G. W. Nuss, Proc. Soc. exp. Biol. Med. 111, 544 (1962). 2. W. DIASDA SILVAand A. D. LEMOSFERNANDES, Experientia 21, 96 (1965). 3. C. G. VAN ARMAN, A. J. BEGANY, L. M, MILLER and H. H. PLESS, J. Pharmac. exp. Ther. 150,
328 0965). 4. A. GOTH,Am. J. Physiol. 197, 1056 (1959). 5. A. GOTHand M. KNOOHUIZEN,Life Sci. 1,459 (1962). 6. A. GO'm, Fedn Proc. 26, 222 (1967). 7. P. A. SHORE,A. BURKHALTERand V. H. COHN,J. Pharmac. exp. Ther. 127, 182 0959). 8. F. K. AUSTEN,Fedn Proc. in press. 9. A. M. ROTHSCHILD,L VUGMANand ROCHAE SILVA, Biochem. Pharmac. 7, 248 (1961). 10. M. ROCHAE SILVA,Int. Syrup. on Injury, Inflammation and Immunity, p. 220. Williams & Wilkins Co., Baltimore (1964). 11. E. R. TRETHEWlE,Aust. J. exp. Biol. reed. Sci. 29, 443 (1951). 12. C. G. HAINING,Br. J. Pharmac. 11,357 (1956).
314 13. 14. 15. 16. 17. 18.
ANDRES GOa-H E. GILLESPIE, S. E. MALAWISTAand R. J. LEVINE, Fedn Proc. 26, 786 (1967). J. PADAWERand A. S. GORDON, Proc. Soc. exp. Biol. Med. 88, 522 (1955). D. W. FAWCETT, d. exp. Med. 100, 217 (1954). D. LAGUNOFFand E. P. BENDITT, J. exp. Med. 112, 571 (1960). A. JANOFF and B. W. ZWEIFACH,Jr. exp. Med. 120, 747 (1964). A. JANOFF, M. A. BEAN and E. SCHULLER,Life Sci. 4, 2361 0965).
COMMENTS DR. MALAWISTA: Did you test the effect of nitrogen mustard on the mast cells to see whether it might inhibit histamine release directly ? DR. GOTH: No. [ doubt that the effect could be on the release process since the cells respond in the usual manner to compound 48/80. DR. MELMON: Can you tell me why the hematocrit value increased after administration of dextran i n the rat ? DR. GOTH: This is undoubtedly a consequence of increased capillary permeability and loss of plasma from the circulation. In the rat, dextran causes increased capillary permeability, probably because it acts directly or indirectly as a mast cell discharger. In other species or in a diabetic rat, dextran would produce a decrease rather than an increase in the hematocrit value. DR. WHITEHOUSE: I could not help but be impressed by the possible connection between what Dr. Glenn was talking about and your cell system, which may have something to do with stabilization. DR. GOTH: Stabilization of some membrane may be involved in our results, but the difficulty is that [ do not know what cell it is whose membrane is being stabilized by the anti-inflammatory drugs.