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Effect of acute and chronic lipopolysaccharide (LPS) administration on reticuloendothelial system (RES) phagocytic activity in vivo
Bums (1992) 18, (2), 107-108
Printed in Great Britain
107
Effect of acute and chronic lipopolysaccharide (LPS) administration on reticuloendothelial system (RES) phagocytic activity in vivo M. Trop, E. J. SchifYrin and E. A. Carter Departments of Pediatrics, Combined Hospital, Boston, USA
Program in Pediatric Gastroenterology
The e&t of injedion or chronic infusion of lipopolysaccharide (LPS) into unanaesfhetized rats on the distribution of p’Tc”‘]SC has been defermined. At a dose of 2.5 mg/kg, LPS injection calrsed a marked alteration in the distribution of the radiolabelled material, with more uptake being achieved in the lung while less was taken up by the spleen. Kidney and liver uptake were also changed, Chronic infusion of LPS at a similar dose (3 mg/kg in 24 h) caused a marked increase in the uptake of the radioactive material by the lung only. These data are cmistent with a working hypothesis that the alterafions in RES phagocytic activity of the lung observed in rats suhecfed to burn trauma could be related in part to LPS, either coming as a bolus, or being continumsly presented.
Introduction Septicaemia is the major cause of morbidity and mortality in burned patients, including multiple organ system failure, in which the lung is the most common organ involved. The mechanism(s) whereby the acute respiratory distress syndrome (ARDS) develop, and hence the proper therapeutic strategies to use to prevent the outcome, are not clearly established. Lipopolysaccharide (LPS) from the cell wall of Gram-negative microorganisms is the main endotoxin that contributes to the local inflammation and systemic toxicity of Gram-negative infections. The aim of the present study was to determine if acute or chronic LPS administration would reproduce the changes in [99Tc”]SC uptake in vivo we have previously observed in burned rats (Trop et al., 1989, 1990).
Methods and materials Female CD rats (175-200 g, Charles River Breeding Farms, Wilmington, MA, USA) were used throughout this study. The animals were fasted 18 h prior to anaesthesia by ether at which time an indwelling tail catheter was placed. Upon recovery the animals were injected intravenously with saline or LPS at a dose of 2.5mg/kg (Salmonella typhosa 0901, Boivin extract, DIFCO, Detroit, MI, USA). One group of animals received Alzet pumps (Alzet Corp., CA, USA) in the peritoneal cavity, containing either saline or the LPS solution. The dose of LPS was 3 mg/kg/d. The Alzet osmotic infusion pump is a rigid case made of a semipermeable polymer containing hyperosmotic fluid. The 0 1992 Butterworth-Heinemann 0305-4179/92/020107-02
Ltd
and Nutrition,
Massachusetts
General
case also contains a flexible reservoir which holds the experimental solution which is displaced from the pump at a constant rate as free water moves through the semipermeable case into the hyperosmotic fluid. The pumps were placed in animals that were anaesthetized with ether, then a small incision (approximately 3 cm) was made in the lower abdomen, through both the skin and then the muscle wall. The LPS was dissolved in saline (20 ml added to 100 mg) prior to use. Using the needle provided with the Alzet pump, the LPS was introduced into the pump (approximately 2 ml). The filled pump was kept in a vertical position until it was placed within the peritoneum of the rat, with the pumping end placed towards the head of the animal. The muscle layer was sealed with sutures and the skin layer with autoclips. The control animals were handled the same way and received pumps containing saline only. Radiolabelled technetium-99m sulphur colloid ([99Tc”‘]SC) material (prepared daily in Massachusetts General Radiopharmacy) was injected into the rats 5 min prior to sacrifice by cervical dislocation, at which time the liver, spleen, kidneys and lung were removed, washed, weighed and then counted in an LKB gammacounter. All experimental manipulations were completed within 10 h after the preparation of the radiolabelled colloid.
Results The single dose of LPS given intravenously to nonanaesthetized rats at two doses resulted in significant alterations of [99Tcm]SC uptake (TableI, Figure I). As can be seen in Table1 there was a marked reduction in uptake of [99Tc”lSC by the spleen and kidney. Chronic administration of LPS had no effect on liver, kidney or spleen uptake of the [99Tcm]SC (Table I). By contrast, both doses of LPS enhanced the uptake of the material by the lung (Figttre I).
Discussion The major cause of morbidity and mortality in bum patients is the development of septicaemia (Rocco et al., 1989). One of the reasons for the development of this septicaemia could be the inability of the RES to clear microorganisms from the blood efficiently. We have been carrying out extensive examinations using a bum rat model in which we have
Bums (1992) Vol. IS/No.
10s
Table 1. Effect of acute and chronic LPS treatment [99T~m]SCby liver, spleen and kidney in vivot Acute LPS Organ
Control
Liver Spleen Kidney
i6.6+2.1 4.9 f 0.7 l.O&O.l
2
on uptake of
Chronic L PS
Treated 13.4*0.6 2.6hO.4’ 0.6ztO.l’
Control 14.7zt2.2 5.3kO.7 0.9+0.1
Treated 11.6f2.7 3.9*2.2 0.8hOo.2
Numbers are mean f s.d., % dose/g tissue. ‘Uptake of the [ggTcm]SC was determined in animals treated with LPS acutely or chronically for 4 days. ‘Indicates statistical significance, PiO.01. There were five or six animals in each group.
Acute
LPS
Chronic
LPS
Figure 1. Effect of acute and chronic LPS treatment on the uptake of [99Tc”‘]SCby rat lung in vivo. There were five or six animals in each group of rats who received either 500 pg of LPS intravenously or LPS infusion for 4 days. ‘Indicates differences that were statistically significant at P< 0.01. 0, Control; ?? , LPS.
shown alterations in technetium-labelled sulphur colloid uptake in vivo (Trop et al., 1989, 1990, 1991). We initially examined the effect of skin removal on the bum-induced labelled sulphur colloid distribution in vivo (Trop et al.,1990). The results showed that rapid removal of the skin or implantation of the skin into the back of a normal animal failed to prevent the redistribution of labelled sulphur colloid observed in the burned animals. Subsequently we observed that skin killed by freeze injury also resulted in a similar alteration in RES function (Trop et al., 1991). In the present study we have examined the effect of administering LPS acutely or chronically into non-anaesthetized animals. In this way we have attempted to duplicate the situation associated with bum injury in which a non-anaesthetized bum victim would suffer extensive tissue necrosis and damage. Subsequently, the wounds become colonized. Dobke et al. (1989) have demonstrated that LPS is present in the blood of burned patients, the amount being directly proportional to the size of the bum. Thus, in the burned patient, LPS, either from the wound bed and/or of translocational origin would be present in the blood at levels much higher than normal. This material could enter the lung via the systemic circulation and hence produce damage. In addition, it could pass to the small intestine where it would induce production of platelet aggregating factor (PAF) via tumour necrosis factor (TNF). PAF has been shown to induce dramatic intestinal necrosis in rats (Sun and Hsueh, 1988). Once the intestine has been damaged more bacteria and endotoxoin could enter the bloodstream setting up a vicious cycle. Finally, it could also pass to the liver, where it would be detoxified but also stimulate the generation of other cytokines, which would also enter the blood and reach the lung, as well as other tissues. Our present results demonstrate that LPS released into the blood, either acutely or chronically, subsequently produce changes in RES function. Our data shows that the effect on the lung can be produced by either acute or chronic LPS treatment, while the changes in the other organs, such as the spleen and kidney, appear limited to the acute bolus LPS model, which produces ‘shock. Continuous LPS infusion, using the technique described here, does not appear to produce shock (work undertaken in this laboratory). These
studies were done on non-anaesthetized rats because it has been demonstrated that LPS is cleared differently when it is injected into either anaesthetized or non-anaesthetized rats. Thus it was observed that radiolabelled LPS has a biological half-life of approximately 3 days, and is excreted in the urine and faeces when it is injected into non-anaesthetized rats, but it is closer to 3 or 4 months when it is injected into anaesthetized animals (Kleine et al., 1985). In conclusion, the present data suggest that LPS entering the bloodstream of non-anaesthetized rats can produce marked alterations in RES function as measured by [99Tc”]SC. Thus, the release of this material from an infected burned wound and/or translocated from other sources, such as the gut, may well be responsible, in part, for the alterations in phagocytic activity with respect to [99Tc”‘]SC uptake observed in vivo following scald bum injury to rats.
References Dobke M. K., Simoni J., Ninnemann J. L. et al. (1989) Endotoxemia after bum injury: effect of early excision on circulating endotoxin levels. J. Bum Cure Rehabil. 10, 107. Kleine B., Frendenberg M. A. and Galanos C. (1985) Excretion of radioactivity in faeces and urine of rats injected with 3H,‘4Clipopolysaccharide. Br. J Exp. Pufhol. 66,303. Rocco J., Goldfarb I. W. and Slater H. (1989) Septic deaths in bum patients: a lack of progress. Proc. Am. Bum Assoc. 21, 107. Sun X. and Hsueh W. (1988) Bowel necrosis induced by tumor necrosis factor in rats is mediated by platelet-activating factor. 1, C/in. Invest. 81, 1328. Trop M., Schiffrin E. J., Jung W. K. et al. (1989) Effect of acute bum trauma on reticuloendothelial phagocytic activity in rats. J. Bum Care Rehabil. 10,388. Trop M., Schiffrin E. J. and Carter E. A. (1990) Role of the skin in the burn-induced reduction of reticuloendothelial phagocytic activity in rats. Bums 16, 57. Trop M., Schiffrin E. J., Jung W. K. et al. (1991) Effect of freeze burn trauma on reticuloendothelial system (RES) phagocytic activity in the rat. 1. Bum Cure R&&l. 12, 97.
Paper accepted 4 October
1991.
Correspondence should be aa%essed to: Dr E. A. Carter, Combined Program in Pediatric Gastroenterology and Nutrition, Massachussetts General Hospital, Bartlett Extension 4, Boston, MA 02114, USA.
Printed in Great Britain
107
Effect of acute and chronic lipopolysaccharide (LPS) administration on reticuloendothelial system (RES) phagocytic activity in vivo M. Trop, E. J. SchifYrin and E. A. Carter Departments of Pediatrics, Combined Hospital, Boston, USA
Program in Pediatric Gastroenterology
The e&t of injedion or chronic infusion of lipopolysaccharide (LPS) into unanaesfhetized rats on the distribution of p’Tc”‘]SC has been defermined. At a dose of 2.5 mg/kg, LPS injection calrsed a marked alteration in the distribution of the radiolabelled material, with more uptake being achieved in the lung while less was taken up by the spleen. Kidney and liver uptake were also changed, Chronic infusion of LPS at a similar dose (3 mg/kg in 24 h) caused a marked increase in the uptake of the radioactive material by the lung only. These data are cmistent with a working hypothesis that the alterafions in RES phagocytic activity of the lung observed in rats suhecfed to burn trauma could be related in part to LPS, either coming as a bolus, or being continumsly presented.
Introduction Septicaemia is the major cause of morbidity and mortality in burned patients, including multiple organ system failure, in which the lung is the most common organ involved. The mechanism(s) whereby the acute respiratory distress syndrome (ARDS) develop, and hence the proper therapeutic strategies to use to prevent the outcome, are not clearly established. Lipopolysaccharide (LPS) from the cell wall of Gram-negative microorganisms is the main endotoxin that contributes to the local inflammation and systemic toxicity of Gram-negative infections. The aim of the present study was to determine if acute or chronic LPS administration would reproduce the changes in [99Tc”]SC uptake in vivo we have previously observed in burned rats (Trop et al., 1989, 1990).
Methods and materials Female CD rats (175-200 g, Charles River Breeding Farms, Wilmington, MA, USA) were used throughout this study. The animals were fasted 18 h prior to anaesthesia by ether at which time an indwelling tail catheter was placed. Upon recovery the animals were injected intravenously with saline or LPS at a dose of 2.5mg/kg (Salmonella typhosa 0901, Boivin extract, DIFCO, Detroit, MI, USA). One group of animals received Alzet pumps (Alzet Corp., CA, USA) in the peritoneal cavity, containing either saline or the LPS solution. The dose of LPS was 3 mg/kg/d. The Alzet osmotic infusion pump is a rigid case made of a semipermeable polymer containing hyperosmotic fluid. The 0 1992 Butterworth-Heinemann 0305-4179/92/020107-02
Ltd
and Nutrition,
Massachusetts
General
case also contains a flexible reservoir which holds the experimental solution which is displaced from the pump at a constant rate as free water moves through the semipermeable case into the hyperosmotic fluid. The pumps were placed in animals that were anaesthetized with ether, then a small incision (approximately 3 cm) was made in the lower abdomen, through both the skin and then the muscle wall. The LPS was dissolved in saline (20 ml added to 100 mg) prior to use. Using the needle provided with the Alzet pump, the LPS was introduced into the pump (approximately 2 ml). The filled pump was kept in a vertical position until it was placed within the peritoneum of the rat, with the pumping end placed towards the head of the animal. The muscle layer was sealed with sutures and the skin layer with autoclips. The control animals were handled the same way and received pumps containing saline only. Radiolabelled technetium-99m sulphur colloid ([99Tc”‘]SC) material (prepared daily in Massachusetts General Radiopharmacy) was injected into the rats 5 min prior to sacrifice by cervical dislocation, at which time the liver, spleen, kidneys and lung were removed, washed, weighed and then counted in an LKB gammacounter. All experimental manipulations were completed within 10 h after the preparation of the radiolabelled colloid.
Results The single dose of LPS given intravenously to nonanaesthetized rats at two doses resulted in significant alterations of [99Tcm]SC uptake (TableI, Figure I). As can be seen in Table1 there was a marked reduction in uptake of [99Tc”lSC by the spleen and kidney. Chronic administration of LPS had no effect on liver, kidney or spleen uptake of the [99Tcm]SC (Table I). By contrast, both doses of LPS enhanced the uptake of the material by the lung (Figttre I).
Discussion The major cause of morbidity and mortality in bum patients is the development of septicaemia (Rocco et al., 1989). One of the reasons for the development of this septicaemia could be the inability of the RES to clear microorganisms from the blood efficiently. We have been carrying out extensive examinations using a bum rat model in which we have
Bums (1992) Vol. IS/No.
10s
Table 1. Effect of acute and chronic LPS treatment [99T~m]SCby liver, spleen and kidney in vivot Acute LPS Organ
Control
Liver Spleen Kidney
i6.6+2.1 4.9 f 0.7 l.O&O.l
2
on uptake of
Chronic L PS
Treated 13.4*0.6 2.6hO.4’ 0.6ztO.l’
Control 14.7zt2.2 5.3kO.7 0.9+0.1
Treated 11.6f2.7 3.9*2.2 0.8hOo.2
Numbers are mean f s.d., % dose/g tissue. ‘Uptake of the [ggTcm]SC was determined in animals treated with LPS acutely or chronically for 4 days. ‘Indicates statistical significance, PiO.01. There were five or six animals in each group.
Acute
LPS
Chronic
LPS
Figure 1. Effect of acute and chronic LPS treatment on the uptake of [99Tc”‘]SCby rat lung in vivo. There were five or six animals in each group of rats who received either 500 pg of LPS intravenously or LPS infusion for 4 days. ‘Indicates differences that were statistically significant at P< 0.01. 0, Control; ?? , LPS.
shown alterations in technetium-labelled sulphur colloid uptake in vivo (Trop et al., 1989, 1990, 1991). We initially examined the effect of skin removal on the bum-induced labelled sulphur colloid distribution in vivo (Trop et al.,1990). The results showed that rapid removal of the skin or implantation of the skin into the back of a normal animal failed to prevent the redistribution of labelled sulphur colloid observed in the burned animals. Subsequently we observed that skin killed by freeze injury also resulted in a similar alteration in RES function (Trop et al., 1991). In the present study we have examined the effect of administering LPS acutely or chronically into non-anaesthetized animals. In this way we have attempted to duplicate the situation associated with bum injury in which a non-anaesthetized bum victim would suffer extensive tissue necrosis and damage. Subsequently, the wounds become colonized. Dobke et al. (1989) have demonstrated that LPS is present in the blood of burned patients, the amount being directly proportional to the size of the bum. Thus, in the burned patient, LPS, either from the wound bed and/or of translocational origin would be present in the blood at levels much higher than normal. This material could enter the lung via the systemic circulation and hence produce damage. In addition, it could pass to the small intestine where it would induce production of platelet aggregating factor (PAF) via tumour necrosis factor (TNF). PAF has been shown to induce dramatic intestinal necrosis in rats (Sun and Hsueh, 1988). Once the intestine has been damaged more bacteria and endotoxoin could enter the bloodstream setting up a vicious cycle. Finally, it could also pass to the liver, where it would be detoxified but also stimulate the generation of other cytokines, which would also enter the blood and reach the lung, as well as other tissues. Our present results demonstrate that LPS released into the blood, either acutely or chronically, subsequently produce changes in RES function. Our data shows that the effect on the lung can be produced by either acute or chronic LPS treatment, while the changes in the other organs, such as the spleen and kidney, appear limited to the acute bolus LPS model, which produces ‘shock. Continuous LPS infusion, using the technique described here, does not appear to produce shock (work undertaken in this laboratory). These
studies were done on non-anaesthetized rats because it has been demonstrated that LPS is cleared differently when it is injected into either anaesthetized or non-anaesthetized rats. Thus it was observed that radiolabelled LPS has a biological half-life of approximately 3 days, and is excreted in the urine and faeces when it is injected into non-anaesthetized rats, but it is closer to 3 or 4 months when it is injected into anaesthetized animals (Kleine et al., 1985). In conclusion, the present data suggest that LPS entering the bloodstream of non-anaesthetized rats can produce marked alterations in RES function as measured by [99Tc”]SC. Thus, the release of this material from an infected burned wound and/or translocated from other sources, such as the gut, may well be responsible, in part, for the alterations in phagocytic activity with respect to [99Tc”‘]SC uptake observed in vivo following scald bum injury to rats.
References Dobke M. K., Simoni J., Ninnemann J. L. et al. (1989) Endotoxemia after bum injury: effect of early excision on circulating endotoxin levels. J. Bum Cure Rehabil. 10, 107. Kleine B., Frendenberg M. A. and Galanos C. (1985) Excretion of radioactivity in faeces and urine of rats injected with 3H,‘4Clipopolysaccharide. Br. J Exp. Pufhol. 66,303. Rocco J., Goldfarb I. W. and Slater H. (1989) Septic deaths in bum patients: a lack of progress. Proc. Am. Bum Assoc. 21, 107. Sun X. and Hsueh W. (1988) Bowel necrosis induced by tumor necrosis factor in rats is mediated by platelet-activating factor. 1, C/in. Invest. 81, 1328. Trop M., Schiffrin E. J., Jung W. K. et al. (1989) Effect of acute bum trauma on reticuloendothelial phagocytic activity in rats. J. Bum Care Rehabil. 10,388. Trop M., Schiffrin E. J. and Carter E. A. (1990) Role of the skin in the burn-induced reduction of reticuloendothelial phagocytic activity in rats. Bums 16, 57. Trop M., Schiffrin E. J., Jung W. K. et al. (1991) Effect of freeze burn trauma on reticuloendothelial system (RES) phagocytic activity in the rat. 1. Bum Cure R&&l. 12, 97.
Paper accepted 4 October
1991.
Correspondence should be aa%essed to: Dr E. A. Carter, Combined Program in Pediatric Gastroenterology and Nutrition, Massachussetts General Hospital, Bartlett Extension 4, Boston, MA 02114, USA.