Vox Sang. 35: 420-422 ( I 978)
Inhibition of the Local Shwartzman Reaction by Turpentine, Prostaglandin EZand Carbon Tetrachloride Bong-Sop Shim and In-Kyung Kim Department of Biochemistry, Catholic Medical College, Seoul
Abstract. The conventional local Shwartzman reaction (LSR) did not occur in rabbits whose plasma haptoglobin (Hp) was lowered by treatment with carbon tetrachloride, nor in animals in whom Hp was elevated after treatment with turpentine, or PGE, or endotoxin. The possible mechanisms of such inhibition of the LSR are discussed.
Introduction In a preceeding paper [ 1 I ] we demonstrated that haptoglobin (Hp) can participate in the production of a local Shwartzmanlike phenomenon (LSLP) after complexing with hemoglobin (Hb). To clarify the role of Hp in the conventional local Shwartzman reaction (LSR), we have tested the development of LSR in rabbits which were treated with turpentine, prostaglandin E, (PGE,) or carbon tetrachloride. Turpentine [3,4,6, 71 and PGE, [ O ] raise the plasma Hp level markedly, whereas carbon tetrachloride [3, 4, 61 decreases the level.
Materials and Methods Korean albino rabbits (body weight: 1.5-2.0 kg) were used throughout the experiment. For the production of LSR, various amounts of endotoxin (from 20 to 80 !rg) in less than 0.3 ml
were injected intradernially i n the abdominal region, and 501rg of endotoxin was injected intravenously 24 h later. Endotoxin (lipopolysaccharide. W., Esclrerichici coli 0127:B,, Difco, Detroit, Mich.) was donated by Dr. S. Chrr (Brown University, Providence, K.I.). In endotoxin-treated rabbits, 20 !‘g of endotoxin was injected intradermally daily for 7 days. on the 8th day 40-80 !rg of endotoxin was injected intraderrnally and on the 9th day SO!cg of endotoxin was administered intravenously. I n carbon tetrachloride-treated rabbits, 0.0s nil/ kg of 20% CCI, in olive oil was injected intramuscularly once a day on the 1st and 3rd day, on the 4th day endotoxin was injected intraderrnally in the abdominal region, and on the 5th day SO !cg of endotoxin was injected intravenously in each rabbit. In turpentine-treated rabbits, 1.0 ml of turpentine was injected subcutaneously in the dorsal region of each rabbit. 24 h later when plasma H p was maximally elevated [4,6, 71, endotoxin was administered intradermally in the abdominal region and followed by intravenous injection of SO !‘g of endotoxin 24 h later. In PGE,-treated rabbits, 30 !rglkg of PGE, was injected intravenously once daily for 4 days.
Inhibition of Local Shwartzman Reaction
On the 5th day endotoxin was injected intradermally and on the 6th day intravenously. PGE, solution was prepared as previously described [9]. In indomethacin-treated rabbits, 10 mg/kg of indomethacin was injected intraperitioneally twice with an interval of 12 h on the 1st day. On the 2nd day 30 rnin after indomethacin injection, endotoxin was administered intradermally at the abdominal region, then indomethacin was injected again 1 and 2 h later. On the 3rd day 50 pg of endotoxin was injected intravenously and indomethacin was injected twice 1 and 2 h later. 2% indomethacin solution was prepared with ethanol and diluted with water to 0.1% prior to use.
Results and Discussion None of the rabbits treated with turpentine, PGE, or carbon tetrachloride showed the conventional LSR; nor did the rabbits given repeated injections of endotoxin (table I). We demonstrated in the preceding paper that Hp in the form of Hp-Hb complex participated in the development of LSLP [ l l ] . If Hp participates in LSR as well as in LSLP, then the inhibition of LSR by carbon tetrachloride could be easily understood, since it depletes plasma Hp [3,4,6]. But in rabbits treated with turpentine which elevates plasma Hp [3,4,6,7], LSR was not observed either. This suggests that Hp may in some way inhibit the development of LSR. Turpentine increases plasma Hp by releasing stored Hp in the liver to the circulation [3,9] and it produces severe inflammation at the injected area. Therefore, the inhibitory action of turpentine might not be related to the increased plasma Hp level. PGE, increases plasma Hp by stimulating Hp synthesis in the liver [3,9]; and was also found to inhibit the development of LSR, even when administered intravenously
42 1
Table I. Modification of the conventional local Shwartzman reaction in rabbits Pretreatment (see ‘Methods’)
Control rabbits Endotoxin-treated rabbits CCI,-treated rabbits Turpentine-treated rabbits PGE,-treated rabbits Indomethacin-treated rabbits
Amount of intradermal endotoxin, pglra bbi t
20
40
80
014 016 0111 014 -
4/4l 015 016 0/11 014 416
4/4l 015 016 0111 014 416
The figures indicate the number of positive rabbits among the rabbits tested. All rabbits were provoked with intravenous injection of 50pg of endotoxin 24 h after the intradermal challenge.
to avoid local inflammation. Endotoxin also increases plasma Hp [5]. Therefore, it is possible that Hp may participate in the inhibition of LSR by turpentine, PGE, and endotoxin itself. However, at present we do not know how these agents act. Recently, Shim [lo] and Saeed et al. [8] suggested that Hp may be an endogenous inhibitor of PG synthetase. Macrophages from C3H/HeJ mice, which are genetically resistant to endotoxin, do not produce PG on stimulation with endotoxin [personal commun. from Dr. 1. L. Ryan, Yale University]. However, indomethacin treatment inhibited the development of LSR in only one-third of the rabbits tested (table I). Hence, other possibilities such as the action on the blood coagulation and complement system of the above agents cannot be excluded, since anticoagulants [2] and depletion of complement [ l ] inhibit the development of LSR. Further studies will be necessary to clarify these interrelationships.
Shim/Kim
422
Acknowledgements We thank Dr. John E. Pike (Upjohn Co.) for PGE,, Prof. S. Cha (Brown University) for endotoxin and Prof.11-Seon Y u n and Chae-Koo Lee for criticism on the manuscript. This work was supported by grants from the Asan Foundation for Social Welfare and the Research Fund of the Catholic Medical Center, Seoul.
References 1 Brown, D. L. and Lachmann, P. J.: The behaviour of complement and platelets in lethal endotoxin shock in rabbits. Int. Archs Allergy appl. Immun. 45: 193-205 (1973). 2 Good, R.A. and Thomas, L.: Studies on the generalized Shwartzman reaction. IV. Prevention of the local and generalized Shwartzman reaction with heparin. J. exp. Med. 97: 871888 (1953). 3 Hong, K.-J. and Shim, B.-S.: The effect of prostaglandin E, and turpentine on the protein synthesis in the liver. Korean J. Biochem. 9: 5-13 (1977). 4 Kim, H. C. and Shim, B.-S.: Effect of carbon tetrachloride upon the serum haptoglobin level in experimental inflammation induced with turpentine. J. Cath. med. Coll., Seoul 5: 17-22 (1961). 5 Knyszynski, A. and Burger, M.: Increase of haptoglobin concentration in mouse serum by
endotoxin and by a serum factor. Experientia 27: 838-839 (1971). 6 Mourray, H.: Biosynthise de l'haptoglobine chez le lapin (Foulon, Paris 1966). 7 Murray, R. K. and Connell, G. E.: Elevation of serum haptoglobin in rabbits in response to experimental inflammation. Nature, Lond. 186: 86 (1960). 8 Saeed, S. A.; Gardner, P. J.; Butt, N. M., and Collier, H. 0.J.: Endogenous inhibitor of prostaglandin synthetase. Nature, Lond. 270: 3236 (1977). 9 Shim, B.-S.: Increase in serum haptoglobin stimulated by prostaglandins. Nature, Lond. 259: 326-327 (1976). 10 Shim, B.-S.: A functional inquiry on serum haptoglobin. Haptoglobin may modulate prostaglandin formation. Korean J. Biochem. 8: 7-11 (1976). 11 Shim, B.4. and Kim, I.-K.: Local Shwartzmanlike phenomenon prepared with haptoglobinhemoglobin complex and provoked with prostaglandin. Vox Sang. 35: 414419 (1978).
Received: February 8, 1978 Accepted: March 13, 1978 Prof. Bong-Sop Shim, Department of Biochemistry, Catholic Medical College, Kyongwoon-Dong, Seoul 110 (Korea)
Inhibition of the Local Shwartzman Reaction by Turpentine, Prostaglandin EZand Carbon Tetrachloride Bong-Sop Shim and In-Kyung Kim Department of Biochemistry, Catholic Medical College, Seoul
Abstract. The conventional local Shwartzman reaction (LSR) did not occur in rabbits whose plasma haptoglobin (Hp) was lowered by treatment with carbon tetrachloride, nor in animals in whom Hp was elevated after treatment with turpentine, or PGE, or endotoxin. The possible mechanisms of such inhibition of the LSR are discussed.
Introduction In a preceeding paper [ 1 I ] we demonstrated that haptoglobin (Hp) can participate in the production of a local Shwartzmanlike phenomenon (LSLP) after complexing with hemoglobin (Hb). To clarify the role of Hp in the conventional local Shwartzman reaction (LSR), we have tested the development of LSR in rabbits which were treated with turpentine, prostaglandin E, (PGE,) or carbon tetrachloride. Turpentine [3,4,6, 71 and PGE, [ O ] raise the plasma Hp level markedly, whereas carbon tetrachloride [3, 4, 61 decreases the level.
Materials and Methods Korean albino rabbits (body weight: 1.5-2.0 kg) were used throughout the experiment. For the production of LSR, various amounts of endotoxin (from 20 to 80 !rg) in less than 0.3 ml
were injected intradernially i n the abdominal region, and 501rg of endotoxin was injected intravenously 24 h later. Endotoxin (lipopolysaccharide. W., Esclrerichici coli 0127:B,, Difco, Detroit, Mich.) was donated by Dr. S. Chrr (Brown University, Providence, K.I.). In endotoxin-treated rabbits, 20 !‘g of endotoxin was injected intradermally daily for 7 days. on the 8th day 40-80 !rg of endotoxin was injected intraderrnally and on the 9th day SO!cg of endotoxin was administered intravenously. I n carbon tetrachloride-treated rabbits, 0.0s nil/ kg of 20% CCI, in olive oil was injected intramuscularly once a day on the 1st and 3rd day, on the 4th day endotoxin was injected intraderrnally in the abdominal region, and on the 5th day SO !cg of endotoxin was injected intravenously in each rabbit. In turpentine-treated rabbits, 1.0 ml of turpentine was injected subcutaneously in the dorsal region of each rabbit. 24 h later when plasma H p was maximally elevated [4,6, 71, endotoxin was administered intradermally in the abdominal region and followed by intravenous injection of SO !‘g of endotoxin 24 h later. In PGE,-treated rabbits, 30 !rglkg of PGE, was injected intravenously once daily for 4 days.
Inhibition of Local Shwartzman Reaction
On the 5th day endotoxin was injected intradermally and on the 6th day intravenously. PGE, solution was prepared as previously described [9]. In indomethacin-treated rabbits, 10 mg/kg of indomethacin was injected intraperitioneally twice with an interval of 12 h on the 1st day. On the 2nd day 30 rnin after indomethacin injection, endotoxin was administered intradermally at the abdominal region, then indomethacin was injected again 1 and 2 h later. On the 3rd day 50 pg of endotoxin was injected intravenously and indomethacin was injected twice 1 and 2 h later. 2% indomethacin solution was prepared with ethanol and diluted with water to 0.1% prior to use.
Results and Discussion None of the rabbits treated with turpentine, PGE, or carbon tetrachloride showed the conventional LSR; nor did the rabbits given repeated injections of endotoxin (table I). We demonstrated in the preceding paper that Hp in the form of Hp-Hb complex participated in the development of LSLP [ l l ] . If Hp participates in LSR as well as in LSLP, then the inhibition of LSR by carbon tetrachloride could be easily understood, since it depletes plasma Hp [3,4,6]. But in rabbits treated with turpentine which elevates plasma Hp [3,4,6,7], LSR was not observed either. This suggests that Hp may in some way inhibit the development of LSR. Turpentine increases plasma Hp by releasing stored Hp in the liver to the circulation [3,9] and it produces severe inflammation at the injected area. Therefore, the inhibitory action of turpentine might not be related to the increased plasma Hp level. PGE, increases plasma Hp by stimulating Hp synthesis in the liver [3,9]; and was also found to inhibit the development of LSR, even when administered intravenously
42 1
Table I. Modification of the conventional local Shwartzman reaction in rabbits Pretreatment (see ‘Methods’)
Control rabbits Endotoxin-treated rabbits CCI,-treated rabbits Turpentine-treated rabbits PGE,-treated rabbits Indomethacin-treated rabbits
Amount of intradermal endotoxin, pglra bbi t
20
40
80
014 016 0111 014 -
4/4l 015 016 0/11 014 416
4/4l 015 016 0111 014 416
The figures indicate the number of positive rabbits among the rabbits tested. All rabbits were provoked with intravenous injection of 50pg of endotoxin 24 h after the intradermal challenge.
to avoid local inflammation. Endotoxin also increases plasma Hp [5]. Therefore, it is possible that Hp may participate in the inhibition of LSR by turpentine, PGE, and endotoxin itself. However, at present we do not know how these agents act. Recently, Shim [lo] and Saeed et al. [8] suggested that Hp may be an endogenous inhibitor of PG synthetase. Macrophages from C3H/HeJ mice, which are genetically resistant to endotoxin, do not produce PG on stimulation with endotoxin [personal commun. from Dr. 1. L. Ryan, Yale University]. However, indomethacin treatment inhibited the development of LSR in only one-third of the rabbits tested (table I). Hence, other possibilities such as the action on the blood coagulation and complement system of the above agents cannot be excluded, since anticoagulants [2] and depletion of complement [ l ] inhibit the development of LSR. Further studies will be necessary to clarify these interrelationships.
Shim/Kim
422
Acknowledgements We thank Dr. John E. Pike (Upjohn Co.) for PGE,, Prof. S. Cha (Brown University) for endotoxin and Prof.11-Seon Y u n and Chae-Koo Lee for criticism on the manuscript. This work was supported by grants from the Asan Foundation for Social Welfare and the Research Fund of the Catholic Medical Center, Seoul.
References 1 Brown, D. L. and Lachmann, P. J.: The behaviour of complement and platelets in lethal endotoxin shock in rabbits. Int. Archs Allergy appl. Immun. 45: 193-205 (1973). 2 Good, R.A. and Thomas, L.: Studies on the generalized Shwartzman reaction. IV. Prevention of the local and generalized Shwartzman reaction with heparin. J. exp. Med. 97: 871888 (1953). 3 Hong, K.-J. and Shim, B.-S.: The effect of prostaglandin E, and turpentine on the protein synthesis in the liver. Korean J. Biochem. 9: 5-13 (1977). 4 Kim, H. C. and Shim, B.-S.: Effect of carbon tetrachloride upon the serum haptoglobin level in experimental inflammation induced with turpentine. J. Cath. med. Coll., Seoul 5: 17-22 (1961). 5 Knyszynski, A. and Burger, M.: Increase of haptoglobin concentration in mouse serum by
endotoxin and by a serum factor. Experientia 27: 838-839 (1971). 6 Mourray, H.: Biosynthise de l'haptoglobine chez le lapin (Foulon, Paris 1966). 7 Murray, R. K. and Connell, G. E.: Elevation of serum haptoglobin in rabbits in response to experimental inflammation. Nature, Lond. 186: 86 (1960). 8 Saeed, S. A.; Gardner, P. J.; Butt, N. M., and Collier, H. 0.J.: Endogenous inhibitor of prostaglandin synthetase. Nature, Lond. 270: 3236 (1977). 9 Shim, B.-S.: Increase in serum haptoglobin stimulated by prostaglandins. Nature, Lond. 259: 326-327 (1976). 10 Shim, B.-S.: A functional inquiry on serum haptoglobin. Haptoglobin may modulate prostaglandin formation. Korean J. Biochem. 8: 7-11 (1976). 11 Shim, B.4. and Kim, I.-K.: Local Shwartzmanlike phenomenon prepared with haptoglobinhemoglobin complex and provoked with prostaglandin. Vox Sang. 35: 414419 (1978).
Received: February 8, 1978 Accepted: March 13, 1978 Prof. Bong-Sop Shim, Department of Biochemistry, Catholic Medical College, Kyongwoon-Dong, Seoul 110 (Korea)
