THE EFFECT OF STREPTOCOCCAL ENZYMES AND OTHER AGENTS ON EXPERIMENTAL INFLAMMATION By Samuel Gordon and Frank B. Ablondi Biochemical Research Section, Research Division, American Cyanamid Co., Pearl River, N. Y. The role of proteolytic enzymes in the inflammatory process has been considered by many investigators. Menkin's studies' on the cellular response in inflammation have shown that tissue breakdown products pro- duce inflammatory reactions. Menkin has suggested that some of the characteristics of inflammation-for example, emigration of leukocytes from blood vessels, their accumulation at a site of injury, and increased capillary permeability-are due to a substance that is polypeptide in nature. He also showed2that prolonged incubation of serum or of crystalline serum albumin with trypsin yielded products that increased capillary permeability and caused emigration of leukocytes. Duthie and Chains have demonstrated that substances with similar inflammation-inducing properties could be obtained by digesting different proteins with proteolytic enzymes. These substances were obtained from serum albumin, egg albumin, casein, fibrin, serum globulin, gelatin, and edestin. These authors concluded that the active principle was a polypeptide, and made attempts to purify it. Cullum- bine and Rydon4 also investigated protein digests and came to the same conclusion. By careful analysis of the polypeptides, Spectors was able to establish that, in the case of a peptic digest of fibrin, a number of peptides of average chain length of from 8 to 14 amino acid residues could cause leukocyte emigration and increased capillary permeability. The evidence presented above suggested that polypeptide or other protein breakdown materials were responsible, at least in part, for the events occur- ring during the inflammatory process. If such were the case, it seemed conceivable that modification of the proteolytic status of the organism might influence the course of inflammation. Indeed, the work of Innerfields in the study of the clinical effects of trypsin seems to lend support to this hypothesis. In 1933 Tillett' showed that hemolytic streptococci were capable of lysing fibrin clots. Later Christensens and Raplane demonstrated that these streptococcal preparations contained a kinase that was capable of converting the inactive plasminogen of certain species into the active proteolytic enzyme, plasmin. This enzyme, in addition to its ability to attack fibrin, also digested casein and gelatinlo and certain amino acid esters." Since a streptokinase (SK) preparation was readily available to us in the form of Varidase*, we thought it would be of interest to investigate the effect of this preparation on experimental inflammation. Our first consideration was the selection of a method of inducing inflam- mation. Examination of the literature revealed a perplexing number of *Trademark of the American Cyanamid Co. for a mixture of streptococcal enzymes consisting of streptokinase, streptodornase, other proteins, and phosphate buffer. Lederle Laboratories Division, Pearl River, N. Y. 89
Transcript
THE EFFECT OF STREPTOCOCCAL ENZYMES AND OTHER AGENTS ON EXPERIMENTAL INFLAMMATION
By Samuel Gordon and Frank B. Ablondi Biochemical Research Section, Research Division, American Cyanamid Co., Pearl River, N . Y.
The role of proteolytic enzymes in the inflammatory process has been considered by many investigators. Menkin's studies' on the cellular response in inflammation have shown that tissue breakdown products pro- duce inflammatory reactions. Menkin has suggested that some of the characteristics of inflammation-for example, emigration of leukocytes from blood vessels, their accumulation a t a site of injury, and increased capillary permeability-are due to a substance that is polypeptide in nature. He also showed2 that prolonged incubation of serum or of crystalline serum albumin with trypsin yielded products that increased capillary permeability and caused emigration of leukocytes. Duthie and Chains have demonstrated that substances with similar inflammation-inducing properties could be obtained by digesting different proteins with proteolytic enzymes. These substances were obtained from serum albumin, egg albumin, casein, fibrin, serum globulin, gelatin, and edestin. These authors concluded that the active principle was a polypeptide, and made attempts to purify it. Cullum- bine and Rydon4 also investigated protein digests and came to the same conclusion. By careful analysis of the polypeptides, Spectors was able to establish that, in the case of a peptic digest of fibrin, a number of peptides of average chain length of from 8 to 14 amino acid residues could cause leukocyte emigration and increased capillary permeability.
The evidence presented above suggested that polypeptide or other protein breakdown materials were responsible, a t least in part, for the events occur- ring during the inflammatory process. If such were the case, it seemed conceivable that modification of the proteolytic status of the organism might influence the course of inflammation. Indeed, the work of Innerfields in the study of the clinical effects of trypsin seems to lend support to this hypothesis.
In 1933 Tillett' showed that hemolytic streptococci were capable of lysing fibrin clots. Later Christensens and Raplane demonstrated that these streptococcal preparations contained a kinase that was capable of converting the inactive plasminogen of certain species into the active proteolytic enzyme, plasmin. This enzyme, in addition to its ability to attack fibrin, also digested casein and gelatinlo and certain amino acid esters." Since a streptokinase (SK) preparation was readily available to us in the form of Varidase*, we thought it would be of interest to investigate the effect of this preparation on experimental inflammation.
Our first consideration was the selection of a method of inducing inflam- mation. Examination of the literature revealed a perplexing number of
*Trademark of the American Cyanamid Co. for a mixture of streptococcal enzymes consisting of streptokinase, streptodornase, other proteins, and phosphate buffer. Lederle Laboratories Division, Pearl River, N. Y.
89
90 Annals New York Academy of Sciences
methods that have been advocated as a means of evaluating the anti-inflam- matory effect of various substances. These methods range from administra- tion of such simple irritants as egg white, dextran, or yeast extract, into the rat12-14 to formation of granulomas by means of cotton pellets'6 or croton oil in an air pouch.16 Since it had been shown that SK is capable of acti- vating the plasminogen of the rabbit" but not that of the rat,'* the rabbit was selected as the laboratory animal of choice.
Preliminary experiments involving subcutaneous administration in the rabbit of irritants such as formaldehyde, croton oil, mustard oil, or diphtheria toxin proved this approach unsuitable in our laboratory for precise experi- mental study. Encouraging results were obtained in the study of the inflammation produced by the instillation of mustard oil into the rabbit eye.19 The following experimental procedure was adopted:
Twelve male New Zealand albino rabbits, approximately 2 kg. in weight, were used in each test. The animals were kept a t least a day in the labora- tory to check their condition and allow them to adjust to laboratory environ- ment. In random order 6 animals were given the material to be tested, and 6 controls received saline (solutions administered intravenously in 1 ml. doses). Approximately 15 min. after the administration of the test sub- stance or saline, 0.3 ml. of a 15 per cent mustard oil solution (redistilled artificial mustard oil* in mineral oil) was instilled under the lid of one eye. Approximately 2 hours later the extent of inflammation was noted by scoring from 1 to 4 depending upon the degree of inflammation. A score of 4 (FIGURE la) was characterized by marked edema of the conjunctiva, causing the palpebral margins to bulge. This score was given to an eye in which there was involvement of both the upper and lower lids and of the nictitating membrane. There was marked redness and secretion of fluid, and the eyelids were almost completely closed. A score of 1 (FIGURE lb) consisted of greatly reduced edema. However, some indication of irritation of the conjunctiva was still present. The eyelids were open almost to normal position. Scores between 1% and 3% represented intermediate conditions. The animals were graded by several observers and the results were evaluated by the rank method of Wilcoxon.20 The total of the individual scores was used to rank the animals. The sum of the ranks of the treated rabbits was calculated, and the probability that the group treatments differed was determined from the tables of Wilcoxon.21
TABLE 1 shows data of an experiment in which 20,000 units of SK in the form of Varidase were administered intravenously. It will be noted that the animals that received SK displayed lower ranks than the controls. In this experiment the value of the rank sum for the SK-treated animals did not exceed 23, thus representing a probability of 1 in 100 or less that the results could have arisen by chance.
Inasmuch as we were interested primarily in the effect of different strepto- coccal preparations on experimental inflammation, we undertook to test these preparations. Various concentrations of SK in the form of Varidase were tested. Each dose was made up in 1 ml. of 0.9 per cent saline and was
* Amend Drug 8 Chemical Co., Inc., New York, N. Y.
Gordon & Ablondi: Streptococcal Enzymes 91
FIGURES la and lb. (a) Example of score “4” inflammation; note marked edema of (b) Example of score “1 ” inflammation; the conjunctiva exposed by separating the lids.
note reduced edema.
92 Annals New York Academy of Sciences TABLE 1
EFFECT OF 20,000 UNITS SK (VARIDASE) ON EXPERIMENTAL EDEMA
50
20*
P
Rabbit No.
21 0.01
27.5 22 0.01
6 2 4 1 5 7 3
10 8
11 9
12 P
5'
1
Treat- ment
~
S SK S SK S SK S SK S SK S SK
3__
35 26.5
41
Scores at 2 hours by 3 observers
1 ~
1% 1 4 1% 2% 1 4 1 4 1% 4 1 -
2 __
1% 1 4 2 2 1 4 1 4 1% 4 1 -
3
; 4 1 w 2 55 1 4 1 3% 1 4 1 -
Totals
-__
4f5 3
12 5 7 3
12 3
11 w 4
12 3 -
-
Rank
__
6 2.5
11 7 8 2.5
11 2.5 9 5
11 2.5 -
Sum P 2'1 0.0s 24 0.02 23 0.01
From Wilcoxonal
The sum of the ranks of the SK-treated animals is 22.0 (P < 0.01).
TABLE 2 EFFECT OF VARIOUS DOSES OF VAIUDASE ON EXPERIMENTAL EDEMA
Dose units SK X loa
1 Sum of ranks of treated animals P
>0.05
>0.05 - ..,
* More than one experiment was performed and P calculated on the basis of combined results.
administered intravenously. TABLE 2 shows the data obtained. From the column. of probabilities it is evident that a significant difference exists between Varidase-treated animals and controls, even a t concentrations as low as 10,OOO units of SK. It should be emphasized that the determination of the minimum effective dose by this method has its limitations.
Gordon & Ablondi : Streptococcal Enzymes 93
1 min. 15 min.
15 7.5 7 5 9 16 - 26
TABLE 3 EFFECT OF PURIFIED SD (FREE OF SK) AND PURIFIED SK (FREE OF SD) ON
EXPERIMENTAL EDEMA
30 min. 60 min. _ _ ~ _ -
13 17 6 . 5 25 33 - - -
l p Sum of ranks of treated animals Preparation
Purified SD
1 :Y 1 0.01 Purified SK
Since Varidase is a mixture of streptococcal proteins consisting in part of SK and streptodornase (SD), the effect of a purified SD preparation was studied. This preparation displayed negligible SK activity and contained approximately the same concentration of SD as is present in 100,OOO units of Varidase. It will be noted that there was no significant difference between the controls and the SD-treated animals. Furthermore, partially purified preparations of SK, obtained by chromatography and displaying negligible SD activity, were capable of pre- venting the inflammatory changes produced by mustard oil. The data indicate that SD is not the factor responsible for inhibiting the edema.
In order to ascertain whether a correlation exists between the antiedema effect of an SK preparation and its ability to induce a demonstrable fibrino- lytic response, the presence of a fibrinolytic system was determined. This was accomplished by withdrawing a small sample of blood into a capillary tube and noting the lysis time after coagulation. TABLE 4 shows the fibrino- lytic response obtained with various doses of SK (as Varidase). These results are the averages of several determinations and indicate that a demon- strable fibrinolytic response is observed at a level of 500 units of SK. All
TABLE 3 shows the results of this study.
TABLE 4 FIBRINOLYTIC RESPONSE (AVERAGE LYSIS TIME IN MINUTES) OBSERVED WITH
VARIOUS DOSES OF VARIDASE I
- -
Dose units SK X lo8
loo0 100 10 0.5
Time interval between SK administration and withdrawal of blood sample
(-) No lysis occurred during the 6 hours of observation.
94 Annals New York Academy of Sciences TABLE 5
10.000 UNITS SK (VARIDASE) AVERAGE L ~ s r s TIME OF BLOOD SAMPLE REMOVED AFTER ADMINISTRATION OF
Time interval between SK administration and withdrawal of blood samples
* 5 Animals in each group. (-) No lysis occurred during the 8 hours of observation.
effective antiedema concentrations of SK produced a demonstrable fibrino- lytic response in the rabbit.
If fibrin deposition is involved in the edema of this type of inflammation, the prevention of fibrin formation by a suitable agent should result in sup- pression of edema. Accordingly, 10,000 units of heparin, a dose sufficient to render the blood incoagulable during the experimental period, was tested. It was found to be ineffective in reducing the edema, thus suggesting that fibrin deposition is not a major factor in the induction of edema in this type of inflammation. Whether circulating proteolytic activity is a necessary prerequisite for the antiedema effect of streptococcal preparations cannot be answered a t this time. The results with heparin in which prevention of fibrin deposition did not alter the course of the inflammation points out the complexity of this problem. In addition, the intravenous administration of sodium salicylate a t a level of 300 mg./kg. was found to inhibit the edema effectively without evoking a detectable fibrinolytic response. Salicylates have been reported previously to inhibit this type of experimental edema.lg* 23
Varidase preparations have been shown to contain a hyaluronidase.22 Since streptococcal hyaluronidase free of SK is unavailable, testicular hyaluronidase was studied. A dose of fifteen hundred USP units of testicular hyaluronidase* was administered intravenously and found to be inactive.
It was of interest to determine the effect of cortisone on this experimental syndrome. Rabbits were given daily doses of 20 mg. of cortisone acetate and 40,OOO units of penicillin intramuscularly for a period of 7 days. The animals were then challenged with mustard oil and scored 2 hours later as described previously. No significant difference was found between the cortisone-treated and the control animals. No detectable fibrinolytic activ- ity was observed in blood samples taken from the animals.
It is of further interest to note that in other experiments with cortisone we have found that treated rabbits displayed a diminished fibrinolytic response to SK as compared to untreated animals. TABLE 5 shows a comparison between the fibrinolytic responses of treated and control animals after the
* Cudahy Laboratories, Omaha, Neb.
Gordon & Ablondi : Streptococcal Enzymes 95 TABLE 6
EFFECT OF VARIOUS AGENTS ON MUSTARD-OIL EDEMA OF THE RABBIT EYE
* Determined in a blood sample removed approximately 5 minutes after administration of each agent.
intravenous administration of 10,000 units SK. The treated animals received 20 mg. cortisone acetate and 40,000 units of penicillin daily for 6 days.
The diminished response to SK in the cortisone-treated animals may possibly be explained on the basis of reports in the literature. For example, Moll24 recently reported that administration of cortisone to rabbits caused an increase in the serum trypsin inhibitors. In addition, Kwaan26 found an inhibition of clot lysis in man after administration of corticotrophin.
In TABLE 6 are tabulated the results obtained with the various agents studied.
Our experiments may be summarized as follows: inflammation was induced by instillation of mustard oil into the eye of the’ rabbit. The intravenous administration of certain streptococcal preparations prior to the induction of inflammation was shown to be effective in decreasing the severity of the inflammation so produced. The activity of these preparations cannot be explained by the presence of SD, which, in purified form and free of SK, was found to be inactive. Furthermore, a partially purified preparation of SK, free of SD, was active in decreasing the edema. Sodium salicylate also served as an effective antiedema agent without producing fibrinolysis. Although detectable fibrinolytic activity was present after the administration of every effective level of SK, the need for an active circulating fibrinolytic system in order to effect a reduction of this type of experimental edema has not been established. Pretreat men t with test icular hyaluronidase, heparin, or cortisone acetate was ineffective.
A cknowledgment
The authors gratefully acknowledge the capable participation of W. P. Cekleniak and P. K. Siiteri in these experiments. They are also indebted to E. C. De Renzo for his advice and for help in the preparation of the manuscript.
96 Annals New York Academy of Sciences
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13. ADAMKIEWICZ, V. W., W. B. RICE & J. D. MCCOLL.
14. COHEN, H., M. GRAFF & W. KLEINBERG. 15. MEIER, R., W. SCHULER & P. DESAULLES. 16. SELYE, H. 1953. 17. JOHNSON, A. J. & W. S. TILLETT. 18. CLIFFTON, E. E. & D. A. CANNAMELA. 19. HIBSCHHORN, L. & M. G. MULINOS. 20. WILCOXON, F. 1945. Biometrics Bull. 1: 80. 21. WILCOXON, F. 1947. Biornetrics. S: 119. 22. MEYER, K. Trans. N. Y. Acad. Sci. 14(4): 164. 23. JANUSCHKE, H. 1913. Wien. klin. Wochschr. 26: 869. 24. MOLL, F. C. 1956. J. Exptl. Med. 103: 363. 25. KWAAN, H. C. 1956. Lancet. 270: 136.
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1952.
Discussion of the Paper
P. S. NORMAN (Johns IIopkins Medical School, Baltimore, M d . ) : You have made the point that cortisone reduces the fibrinolytic response to strepto- kinase, but does not change the inflammation produced by your test. Does pretreatment with cortisone change the response to streptokinase when you try to inhibit the edema?
S. GORDON: Unfortunately, we have not tried this. L. S. KRYLE (Department of Therapeutics, N e w York University College of
Medicine, New York, N . Y . ) : Did you have occasion to use chymotrypsin or trypsin in similar experiments?
S. GORDON: Yes, we have. With trypsin we have been unable to give sufficiently large doses intravenously. At the dose levels that we have used, approximately 5 mg. of trypsin per rabbit intravenously, we have not achieved the reversal of the edema as established by our criterion. However, at much larger doses, for example, 100 mg. of trypsin intramuscularly, we do see an effect on the edema. This might be explained by the fact that trypsin is readily inactivated, making it necessary to have high levels.
