내독소 내성(Endotoxin Tolerance)에 관한 실험적 연구

Abstract

[한글]

An Experimental Study on Endotoxin Tolerance in Dogs

Eui Ho Hwang, M.D.

Department of Medical Science The Graduate School, Yonsei University

(Directed by Professors: Kyu Chul Whang, M.D. and Sei Ok Yoon, M.D.)

Endotoxin is a lido-polysaccharide protein complex derived from gram-negative

bacteria, which can be introduced into the blood stream of the human body in

various circumstances and onuses so-called "Sedotoxin shock. " Septicemia caused by

gram-negative bacteria has been recognized at least since the report of Jacob

(1909) and Felty and Keefer (1924). However, septic shock was first described as a

clinical entity by Waisbren (1951), and in subsequent years many experimental

studies were conducted using endotoxin in dogs.

Endotoxin, given in appropriate doses to the dog, produces hypotension, rice in

portal pressure with simultaneous constriction of hepatic veins, great increase in

the weight of the liver, and mesenteric ischemia (Sabiston, 1972). Recently, it has

been generally considered that any irreversibility of shock is a result from the

insult of endotoxin (Smiddy and Fine, 1957; Fine et al., 1958; Schweinburg and

Fine, 1960; Ravin et al., 1960; Sieged et al., 1967).

Beeson (1947) suggested possible tolerance to the pyrogenic effect of endotoxin,

and Atkins and Wood (1955) observed that tolerant animals cleared endotoxin, which

was administered intravenously, more rapidly from the blood stream, and numerous

experimental studies have been made on endotoxin shock (Dubos and Schaedler, 1956;

Schweinburg et al., 1956; Smith et al., 1957; Carey et al., 1958; Freedman, 1960;

Greisman et al., 1963; Rutenburg et al., 1967; Filkins and Di Luzio, 1978; Haugen,

1972; Trejo and Di Luzio, 1971).

Endotoxin tolerance is the state of enhanced resistance of animals against the

toxic effects of bacterial endotoxins, and the tolerance state is usually induced

by the prior administration of multiple sublethal doses of endotoxin (Beeson, 1947

; Carey et al., 1958; Freedman, 1960; Greisman et al., 1963 ; Tsagaris et al.,

1969; Trejo and Di Luzio, 1972),

Although the mechanism of endotoxin tolerance is still in controversy, it is

generally thought that the endotoxin tolerance state is intimately related to

phagocytic activity of the reticuloendothelial system (Good and Thomas, 1952 ;

Schweinburg and Fine, 1955; Schweinburg, et al., 1955; Zweifach et al., 1957 ; Fine

et al., 1958; Carey et al., 1958; Greisman et al., 1963; Arredondo and Kampschmidt,

1963 ; Filkins and Di Luzio, 1968), and, furthermore, related to the phagocytic

activity of macrophages of the liver and spleen (Collins and Wood, 1959; Rutenburg

et al., 1960, Cohn and Wiener, 1963; Cline et al., 1968; Filkins, 1971 ; Fine,

1972).

Tsagaris et al. (1969) demonstrated in the hemodynamic study of endotoxin shock

that tolerant animals were less susceptible than the nontolerant to hemodynamic

changes such as blood pressure and cardiac output, and blood pressure and cardiac

output were restored more rapidly in the tolerant animals than in the nontolerant.

In mice pretreated with various agents, such as BCG and diethylstilbesterol, to

activate the reticuloendothelial function, increased susceptibility to endotoxin

challenge was observed, and it was suggested that endotoxin tolerance is not

related to the activation of reticuloendothelial function (Suter et al., 1958;

Benacerraf et al., 1959; Stuart and Cooper, 1962). Moreover, tolerance to

pyrogenicity of endotoxin was observed in the presence of normal phagocytic

activity of the reticuloendothelial system, and activation of the

reticuloendothelial system was not necessarily required in the development of

endotoxin tolerance (Greisman et al., 1963).

It is well known that liver and spleen consist of major reticuloendothelial

tissue but it is not yet clear which organ participates more actively to detoxify

endotoxin in the blood stream. It has been also Suggested that distribution of

endotoxin in the body is different between the tolerant and the nontolerant

animals. (Smith et al., 1957; Carey et al., 1958 ; Rutenburg et al., 1965; Alper et

at., 1967).

In order to provide additional information on endotoxin tolerance, a comparative

study was conducted on the hemodynamic changes, antibody formation, endotoxin

uptake, and histopathologic alterations in the reticuloendothelial system of

endotoxin tolerant and nontolerant dogs.

Materials and Methods

A. Materials

1. Animals.

a. Dogs: Mongrel dogs, weighing 12 to 18 kg were used.

b. Rabbits: Female albino rabbits, weighing 2.5 to 3.0 kg were used for

preparation of rabbit anti-endotoxin anti-serum

2. E. coli strain. E. coli 0111 : B4 NIH (USA), obtained from NIH, Korea and

subcultures in trypticase soy agar media (BBL, USA).

3. Endotoxins.

a. Crude endotoxin: prepared by the method of Rubenstein et al. (1962) using

aforementioned E. coli strain (Fig. 1).

b. Purified endotoxin: Bacto lipo-polysaccharide E. coli 0111 : B4 (Difco Lab.

Detroit, Michigan, U.S.A.).

4. Fluorescein-tagged antibody

IgG fraction, FITC conjugated groat anti-rabbit IgG. (Research Product Division,

Miles Lab. Inc., U.S.A.).

B. Methods

1. Preparation of rabbit anti-endotoxin anti-serum.

An albino rabbit was inimunized over an 8-month period with 7 courses of

intravenous endotoxin injections into a marginal ear vein as follows: day 1, 0.25

ml: day 3, 0.5 ml; day 5, 0.75 ml; day 7, 1.0 ml; day 9, 1.25 ml; day 11, 1.5. On

day 14 after the last course, total blood was harvested from a razor-nicked ear

vein. After 2 hours at room temperature, the clot was rimmed and the blood was

refrigerated at 4℃ for 18 hours. Serum with free cells was decanted and

centrifuged at 2,000 rpm for 30 minutes at 4℃. The cell-free serum was filtered

into a sterile rubber stoppered bottle and stored at 4℃.

2. Preparation of tolerant dogs.

Endotoxin tolerant dogs were prepared according to the schedule shown in Table 2

over a 9 to 11 week period.

3. Demonstration of antibody formation in dogs.

By agar gel double diffusion method (Ouchterlony, 1958), precipitating antibody

to endotoxin was demonstrated in the serum of endotoxin tolerant animals.

4. Hemodynamic observations.

Experimental animals were divided into 4 groups and the hemodynamic changes were

observed.

Grovp Ⅰ -Tolerant dogs with purified endotoxin

Group Ⅱ -Tolerant dogs with crude endotonin

Group Ⅲ -Non-tolerant dogs with purified endotoxin

Group Ⅳ -Non-tolerant dogs with crude endotoxin

Experimental animals were anesthetized with sodium pentothal (30 mg/kg), and the

femoral artery was catheterized with a polyethylene tube which was connected to a

kymograph to record the blood pressure. At the same time electrocardiographic

findings were recorded. Purified endotoxin, 3 mg/kg, was injected intravenously in

Group Ⅰ and Group H animals, and crude endotoxin, 27.6 mg/kg, was injected

intravenously in Group Ⅱ and Group 1 animals. Again, blood pressure and

electrocardiograph were recorded with observation of animals from 2 to 5 hours.

5. Localization of endotoxin.

Tissues were obtained from the animals after hemodynamic study for localization

of endotoxin. The tissue was sectioned by cryostat 3 to 5 μ thick, and stained

with fluorescent dye by the method of Rubenstein et al. (1962).

6. Histo-pathologic observation.

With H·E stain, tissues were examined under microscope to determine whether

histologic changes, if any, and proliferation and activation of macrophages were

present.

Results

A. Detection of antibodies

1. Rabbit anti-endotcxin anti-serum.

Rabbit anti-endotoxin anti·serum was caused to react with purified endotoxin in

0.85 percent agar plate, and a single identical precipitation line was observed in

the area of rabbit anti-endotoxin anti-serum. This precipitation line was not

observed when the nonimmunized rabbit strum was used (Fig. 3). After the antibody

of rabbit anti-endotoxin anti-serum was reacted with crude endotoxin, the above

mentioned precipitation line was not observed (Fig. 4).

2. Endotoxin-tolerant serum of dogs.

Serum obtained from endotoxin tolerant dogs was made to react with endotoxin to

verify whether the serum of endotoxin tolerant dogs also produced any antibody to

endotoxin and the same precipitation line as seen in the case of rabbit

anti-endotoxin antiserum was observed. This precipitation was observed in 48 hours

when crude endotoxin was used and in 4 days when purified endotoxin used.

B. Hemodynamic changes

1. Changes of blood pressure.

When endotoxin tolerant dogs were challenged with a shock dose of endotoxin by

the intravenous route, blood pressure fell rapidly up to 15-20% of initial

pressure. About 5 minutes after endotoxin challenge, blood pressure recovered

gradually over a 2 to 5 hour period, the recovery pattern being shown in Fig. 6, 7,

8, 9. Endotoxin tolerant and non-tolerant dogs showed a similar recovery pattern of

blood pressure. However, tolerant dogs skewed very little secondary fall of blood

pressure(Fig. 6 & 7) and non-tolerant dogs did not recover from the first fall of

blood pressure (Fig. 8) or skewed a profound secondary fall of blood pressure and

this finding was core remarkable in the dogs when crude endotoxin was used (Fig.

10).

2. Electrocardiographic change.

Tolerant dogs showed no change in pulse rate or ECG tracing, but non-tolerant

dogs showed Persistent depression of S-T segments in the ECG tracing, which

suggested myocardial ischemia (Fig. 11).

C. Localization of endotoxion

A large amount of specific fluorescent material wart observed in the sinusoid,

central vein and interlobular space of liver, splenic sinusoid, and alveolar cell

of the lung. Especially, in the liver, cytoplasm of macrophages contained a large

amount of fluorescent material, and this was not observed in spleen and lung. There

was no difference in amount of fluorescent material between spleen and liver

tissue.

D. Histopathologic observation

By H-E stain, macrophages proliferated prominently in the liver of endotoxin

tolerant dogs to form granuloma which contained particulated materials which might

be hemosiderin, and this finding suggested activation of macrophages. In the liver

of the non-tolerant dogs proliferation of macrophages was not observed nor in the

spleen and Lung of the tolerant and the non-tolerant dogs.

Tissue of non·tolerant dogs challenged with a single lethal dose of endotoxin

showed no remarkable change by H-E stain.

Conclusion and Summary

1. The agar gel double diffusion method showed the presence of a specific

antibody to purified endotoxin by apperance of a single identical precipitation

line which is identical with one in rabbit anti-endotoxin anti-serum.

2. Fluorescent antibody technique demonstrated a significant amount of specific

fluorescent material in the sinusoid, central vein and interlobular space of the

liver, the sinusoid of spleen, and in the alveolar cell of lung in both tolerant

and nontolerant dogs. There observed no apparent difference in the amount of

fluorescent material of liver and spleen between the tolerant and nontolerant dogs.

3. Recovery from endotoxin shock in the hemodynamic study was far better in

tolerant dogs than in nontolerant ones, and myocardial ischemia was demonstrated by

ECG reoording in the nontolerant after endotoxin challenge.

4. Histopathologic examination of liver tissue fron tolerant dogs revealed a

feature of macrophage activation, but such a finding was not observed in the liver

from nontolerant dogs and in the spleen and lung from the tolerant and nontolerant

dogs.

[영문]

Endotoxin is a lido-polysaccharide protein complex derived from gram-negative bacteria, which can be introduced into the blood stream of the human body in various circumstances and onuses so-called "Sedotoxin shock. " Septicemia caused by gram-negative bacteria has been recognized at least since the report of Jacob

(1909) and Felty and Keefer (1924). However, septic shock was first described as a clinical entity by Waisbren (1951), and in subsequent years many experimental studies were conducted using endotoxin in dogs.

Endotoxin, given in appropriate doses to the dog, produces hypotension, rice in portal pressure with simultaneous constriction of hepatic veins, great increase in the weight of the liver, and mesenteric ischemia (Sabiston, 1972). Recently, it has been generally considered that any irreversibility of shock is a result from the insult of endotoxin (Smiddy and Fine, 1957; Fine et al., 1958; Schweinburg and Fine, 1960; Ravin et al., 1960; Sieged et al., 1967).

Beeson (1947) suggested possible tolerance to the pyrogenic effect of endotoxin, and Atkins and Wood (1955) observed that tolerant animals cleared endotoxin, which was administered intravenously, more rapidly from the blood stream, and numerous

experimental studies have been made on endotoxin shock (Dubos and Schaedler, 1956; Schweinburg et al., 1956; Smith et al., 1957; Carey et al., 1958; Freedman, 1960; Greisman et al., 1963; Rutenburg et al., 1967; Filkins and Di Luzio, 1978; Haugen,

1972; Trejo and Di Luzio, 1971).

Endotoxin tolerance is the state of enhanced resistance of animals against the toxic effects of bacterial endotoxins, and the tolerance state is usually induced by the prior administration of multiple sublethal doses of endotoxin (Beeson, 1947 ; Carey et al., 1958; Freedman, 1960; Greisman et al., 1963 ; Tsagaris et al., 1969; Trejo and Di Luzio, 1972),

Although the mechanism of endotoxin tolerance is still in controversy, it is generally thought that the endotoxin tolerance state is intimately related to phagocytic activity of the reticuloendothelial system (Good and Thomas, 1952 ; Schweinburg and Fine, 1955; Schweinburg, et al., 1955; Zweifach et al., 1957 ; Fine et al., 1958; Carey et al., 1958; Greisman et al., 1963; Arredondo and Kampschmidt, 1963 ; Filkins and Di Luzio, 1968), and, furthermore, related to the phagocytic activity of macrophages of the liver and spleen (Collins and Wood, 1959; Rutenburg et al., 1960, Cohn and Wiener, 1963; Cline et al., 1968; Filkins, 1971 ; Fine, 1972).

Tsagaris et al. (1969) demonstrated in the hemodynamic study of endotoxin shock that tolerant animals were less susceptible than the nontolerant to hemodynamic changes such as blood pressure and cardiac output, and blood pressure and cardiac output were restored more rapidly in the tolerant animals than in the nontolerant.

In mice pretreated with various agents, such as BCG and diethylstilbesterol, to activate the reticuloendothelial function, increased susceptibility to endotoxin challenge was observed, and it was suggested that endotoxin tolerance is not

related to the activation of reticuloendothelial function (Suter et al., 1958; Benacerraf et al., 1959; Stuart and Cooper, 1962). Moreover, tolerance to pyrogenicity of endotoxin was observed in the presence of normal phagocytic activity of the reticuloendothelial system, and activation of the

reticuloendothelial system was not necessarily required in the development of endotoxin tolerance (Greisman et al., 1963).

It is well known that liver and spleen consist of major reticuloendothelial tissue but it is not yet clear which organ participates more actively to detoxify endotoxin in the blood stream. It has been also Suggested that distribution of

endotoxin in the body is different between the tolerant and the nontolerant animals. (Smith et al., 1957; Carey et al., 1958 ; Rutenburg et al., 1965; Alper et at., 1967).

In order to provide additional information on endotoxin tolerance, a comparative study was conducted on the hemodynamic changes, antibody formation, endotoxin uptake, and histopathologic alterations in the reticuloendothelial system of

endotoxin tolerant and nontolerant dogs.

Materials and Methods

A. Materials

1. Animals.

a. Dogs: Mongrel dogs, weighing 12 to 18 kg were used.

b. Rabbits: Female albino rabbits, weighing 2.5 to 3.0 kg were used for preparation of rabbit anti-endotoxin anti-serum

2. E. coli strain. E. coli 0111 : B4 NIH (USA), obtained from NIH, Korea and subcultures in trypticase soy agar media (BBL, USA).

3. Endotoxins.

a. Crude endotoxin: prepared by the method of Rubenstein et al. (1962) using aforementioned E. coli strain (Fig. 1).

b. Purified endotoxin: Bacto lipo-polysaccharide E. coli 0111 : B4 (Difco Lab. Detroit, Michigan, U.S.A.).

4. Fluorescein-tagged antibody

IgG fraction, FITC conjugated groat anti-rabbit IgG. (Research Product Division, Miles Lab. Inc., U.S.A.).

B. Methods

1. Preparation of rabbit anti-endotoxin anti-serum.

An albino rabbit was inimunized over an 8-month period with 7 courses of intravenous endotoxin injections into a marginal ear vein as follows: day 1, 0.25 ml: day 3, 0.5 ml; day 5, 0.75 ml; day 7, 1.0 ml; day 9, 1.25 ml; day 11, 1.5. On day 14 after the last course, total blood was harvested from a razor-nicked ear

vein. After 2 hours at room temperature, the clot was rimmed and the blood was refrigerated at 4℃ for 18 hours. Serum with free cells was decanted and centrifuged at 2,000 rpm for 30 minutes at 4℃. The cell-free serum was filtered into a sterile rubber stoppered bottle and stored at 4℃.

2. Preparation of tolerant dogs.

Endotoxin tolerant dogs were prepared according to the schedule shown in Table 2 over a 9 to 11 week period.

3. Demonstration of antibody formation in dogs.

By agar gel double diffusion method (Ouchterlony, 1958), precipitating antibody to endotoxin was demonstrated in the serum of endotoxin tolerant animals.

4. Hemodynamic observations.

Experimental animals were divided into 4 groups and the hemodynamic changes were observed.

Grovp Ⅰ -Tolerant dogs with purified endotoxin

Group Ⅱ -Tolerant dogs with crude endotonin

Group Ⅲ -Non-tolerant dogs with purified endotoxin

Group Ⅳ -Non-tolerant dogs with crude endotoxin

Experimental animals were anesthetized with sodium pentothal (30 mg/kg), and the femoral artery was catheterized with a polyethylene tube which was connected to a kymograph to record the blood pressure. At the same time electrocardiographic

findings were recorded. Purified endotoxin, 3 mg/kg, was injected intravenously in Group Ⅰ and Group H animals, and crude endotoxin, 27.6 mg/kg, was injected intravenously in Group Ⅱ and Group 1 animals. Again, blood pressure and electrocardiograph were recorded with observation of animals from 2 to 5 hours.

5. Localization of endotoxin.

Tissues were obtained from the animals after hemodynamic study for localization of endotoxin. The tissue was sectioned by cryostat 3 to 5 μ thick, and stained with fluorescent dye by the method of Rubenstein et al. (1962).

6. Histo-pathologic observation.

With H·E stain, tissues were examined under microscope to determine whether histologic changes, if any, and proliferation and activation of macrophages were present.

Results

A. Detection of antibodies

1. Rabbit anti-endotcxin anti-serum.

Rabbit anti-endotoxin anti·serum was caused to react with purified endotoxin in 0.85 percent agar plate, and a single identical precipitation line was observed in the area of rabbit anti-endotoxin anti-serum. This precipitation line was not

observed when the nonimmunized rabbit strum was used (Fig. 3). After the antibody of rabbit anti-endotoxin anti-serum was reacted with crude endotoxin, the above mentioned precipitation line was not observed (Fig. 4).

2. Endotoxin-tolerant serum of dogs.

Serum obtained from endotoxin tolerant dogs was made to react with endotoxin to verify whether the serum of endotoxin tolerant dogs also produced any antibody to endotoxin and the same precipitation line as seen in the case of rabbit anti-endotoxin antiserum was observed. This precipitation was observed in 48 hours when crude endotoxin was used and in 4 days when purified endotoxin used.

B. Hemodynamic changes

1. Changes of blood pressure.

When endotoxin tolerant dogs were challenged with a shock dose of endotoxin by the intravenous route, blood pressure fell rapidly up to 15-20% of initial pressure. About 5 minutes after endotoxin challenge, blood pressure recovered gradually over a 2 to 5 hour period, the recovery pattern being shown in Fig. 6, 7,

8, 9. Endotoxin tolerant and non-tolerant dogs showed a similar recovery pattern of blood pressure. However, tolerant dogs skewed very little secondary fall of blood pressure(Fig. 6 & 7) and non-tolerant dogs did not recover from the first fall of blood pressure (Fig. 8) or skewed a profound secondary fall of blood pressure and this finding was core remarkable in the dogs when crude endotoxin was used (Fig. 10).

2. Electrocardiographic change.

Tolerant dogs showed no change in pulse rate or ECG tracing, but non-tolerant dogs showed Persistent depression of S-T segments in the ECG tracing, which suggested myocardial ischemia (Fig. 11).

C. Localization of endotoxion

A large amount of specific fluorescent material wart observed in the sinusoid, central vein and interlobular space of liver, splenic sinusoid, and alveolar cell of the lung. Especially, in the liver, cytoplasm of macrophages contained a large amount of fluorescent material, and this was not observed in spleen and lung. There was no difference in amount of fluorescent material between spleen and liver tissue.

D. Histopathologic observation

By H-E stain, macrophages proliferated prominently in the liver of endotoxin tolerant dogs to form granuloma which contained particulated materials which might be hemosiderin, and this finding suggested activation of macrophages. In the liver

of the non-tolerant dogs proliferation of macrophages was not observed nor in the spleen and Lung of the tolerant and the non-tolerant dogs.

Tissue of non·tolerant dogs challenged with a single lethal dose of endotoxin showed no remarkable change by H-E stain.

Conclusion and Summary

1. The agar gel double diffusion method showed the presence of a specific antibody to purified endotoxin by apperance of a single identical precipitation line which is identical with one in rabbit anti-endotoxin anti-serum.

2. Fluorescent antibody technique demonstrated a significant amount of specific fluorescent material in the sinusoid, central vein and interlobular space of the liver, the sinusoid of spleen, and in the alveolar cell of lung in both tolerant and nontolerant dogs. There observed no apparent difference in the amount of

fluorescent material of liver and spleen between the tolerant and nontolerant dogs.

3. Recovery from endotoxin shock in the hemodynamic study was far better in tolerant dogs than in nontolerant ones, and myocardial ischemia was demonstrated by ECG reoording in the nontolerant after endotoxin challenge.

4. Histopathologic examination of liver tissue fron tolerant dogs revealed a feature of macrophage activation, but such a finding was not observed in the liver from nontolerant dogs and in the spleen and lung from the tolerant and nontolerant dogs.