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Tissue oxidation after endotoxin
301
Bioelecfrochemisfry and Bioenergetics, 18 (1987) 301-306 A section of J. Electroanal. Chem., and constituting Vol. 232 (1987) Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands
1016 -
TISSUE
J.M. STARK,
OXIDATION
SK. JACKSON,
AFTER
BRIDGET
ENDOTOXIN*
WOODHEAD
and H.C. RYLEY
Department of Medical Microbiology, University of Wales Cotiege of Medicine, Heath Park, Cardiff CF4 4XN, Wales (Great Britain)
SUMMARY Macrophages from the spleens of mice sensitized with BCG produce oxygen radicals in vitro after stimulation with endotoxin as demonstrated by electron spin resonance spectroscopy. The TBA method for measuring lipid peroxidation products showed that, surprisingly, spleen homogenates from sensitized mice injected with endotoxin showed a reduced amount of TBA-reactive material after incubation in vitro. This result may be explained if endotoxin induces increases in tissue antioxidants. Splenic macrophages may play a critical role in this process since splenectomy of sensitized animals was seen to reduce the sensitivity to endotoxin.
INTRODUCTION
Improvement in the treatment of otherwise frequently fatal endotoxic shock depends on a better understanding of its pathogenesis. There is a large species-variation in sensitivity to endotoxins: mice are relatively resistant to endotoxin but may be sensitized by pre-injection with BCG [l]. The mechanisms underlying this sensitizing process remain unclear, but activated macrophages and their products may take part. Recent evidence suggests that endotoxin stimulates macrophages to produce a variety of active compounds including free radicals which may be damaging to the host’s tissues. The present investigation was undertaken in an attempt to show that free radicals are produced from activated macrophages in response to endotoxin challenge in vitro and that such free radicals could initiate damaging lipid peroxidation reactions. The idea that macrophages are important in the events of endotoxic shock was demonstrated by removing the spleen (a major source of macrophages) from sensitized animals.
l Presented at the Bioelectrochemical Biological Systems”, Obemai, 22-24
0302-4598/87/$03.50
Society Meeting October 1986.
0 1987 Elsevier Sequoia
S.A.
on
“Formation
and Reactions
of Peroxides
in
302 MATERIALS
AND METHODS
Sensitization to endotoxin Sensitization to endotoxin was induced by giving intravenously l/6 vial percutaneous BCG (Glaxo) (an attenuated strain of Mycobacterium tuberculosis) warmed, vasodilated adult male TO mice 14-18 days before endotoxin.
of to
Endotoxin Lipopolysaccharide B from Escherichia coli 026:B6 (Difco) was given in 10 pg amounts intraperitoneally in 0.4 cm3 phosphate buffered saline (PBS). Splenectomies in BCG-treated animals Spleens, enlarged 12 days after BCG were removed under ether anaesthesia from 15 animals. Fourteen animals survived the operation. A further 15 mice given BCG were sham-operated after the same time. The animals were given 2 pg endotoxin i.p. two days later and their survival monitored for 48 hours. Free radical production Spleens were removed from freshly killed mice 14-18 days after injection with BCG and carefully minced in pyrogen-free balanced salts solution. Tissue debris was removed. The cells were then washed and resuspended in serum-free medium and incubated in a quartz-aqueous flat electron spin resonance (e.s.r.) cell for several hours at 37’C to allow the macrophages to adhere to the glass walls. The tube was then washed through twice and a solution of 100 mM 5,5-dimethyl-l-pyrroline-N-oxide (DMPO) (Sigma Chemical Co. Ltd., Poole, England) spin trap containing lo-100 pg endotoxin was added and the tube incubated again before running e.s.r. spectra. e.s.r. spectra were run in the Chemistry Department, University College, Cardiff, on a Varian El09 ESR spectrometer. Lipid peroxidation Thiobarbituric acid (TBA) reactive substances ing products of lipid peroxidation were measured for 90 minutes at 37’ C by the TBA assay [2].
which are important and damageither directly or after incubation
Fatty acid analysis g.1.c. analysis of methylated fatty acids was performed extraction of the lipid materials [3].
after chloroform/methanol
RESULTS
Effect of splenectomy Splenectomy of BCG-primed mice gave rise to a significantly prolonged survival time (p < 0.02 by Student’s t test) after challenge with endotoxin when compared to sham-operated controls. The log mean time (hours) to death of the sham-operated animals (no survivors) was 1.13 _t 0.36 whereas that of the splenectomized animals was 1.48 f 0.36 with 3 survivors.
303
II
Fig. 1. e.s.r. spectrum of the hydroxyl BCG-primed macrophages.
0
'/2
3
6
P-=i radical
24
Fig. 2. Determination of lipid peroxidation at various times after endotoxin treatment for 90 minutes (B). Statistical comparisons time.
adduct
of DMPO
produced
from endotoxin-stimulated
48
in spleens from normal and BCG-treated mice before (0) and in vim. Spleens were unincubated (A) or incubated at 37 o C are made by the t test against the same preparation at zero
Fig. 3. Determination of lipid peroxidation in tissues of BCG-primed mice 6 hours after injection with endotoxin (t = 6) to untreated controls (t = 0). All tissues were incubated for 90 minutes in vitro at 37 ’ C prior to TBA/MDA assay (n = 4 for all groups).
Electron spin resonance A typical e.s.r. spectrum of the spin-trapped radicals produced from the endotoxin-stimulated splenic macrophages is shown in Fig. 1: The spectrum is characteristic of the hydroxyl radical adduct of DMPO (aN = aH = 1.48 mT).
Lipid peroxidation products The TBA reactive substances of the spleens of normal sensitized to endotoxin by BCG did not differ significantly
Fig. 4. GLC-polyunsaturated with and without injection
fatty acid (PUFA) of endotoxin.
analysis
animals and of animals except at 6 hours after
of spleens from normal
and BCG-primed
mice
305
endotoxin when the BCG treated animals showed a significant reduction in the amount of TBA reactive substances in the tissues (Fig. 2A). When the tissues, taken at intervals after endotoxin (O-48 h) were incubated for 90 minutes, the TBA reactive substances increased in all samples due to oxidation in vitro (Fig. 2B). However at 6 h in the unsensitized animals the product was significantly less than at zero time. When other tissues were similarly incubated (Fig. 3) all except blood and illeum showed this reduction in TBA reactive substances at 6 h when compared with the product of tissues taken before endotoxin (Fig. 3, t = 0). Fatty acid content of spleen Arachidonic acid (C20:4 w6) and C22:6.w3 were significantly increased in the BCG spleens and C18:1.6c (petroselenic acid) and C18:2c.w6 (linoleic acid) were significantly reduced. Endotoxin did not significantly alter these in either normal or primed animals (Fig. 4). DISCUSSION
The fact that splenectomy reduces susceptibility to endotoxin in BCG treated animals focuses attention on the metabolic changes in the spleen in the endotoxic crisis. This work has therefore examined free radical production and TBA reactive substances in the spleens of normal and endotoxin sensitized animals. The e.s.r. studies reveal a strong signal characteristic of the hydroxyl radical adduct after the introduction of endotoxin into splenic cell suspensions suggesting that this macrophage product may be produced in life by the cells of the BCG modified spleen. The sensitization process changes the fatty acid profile of the organ with a notable increase of the pharmacologically important arachidonic acid. However, the giving of endotoxin did not result in any significant change in the profiles. The TBA reactive substances in unincubated tissues reflects the tissue oxidation in life and did not show any increase after endotoxin in the examined spleens. However, when these tissues were allowed to oxidize in vitro a major difference was found in the tissues taken from the animals at 3 h and 6 h after endotoxin. These surprisingly showed a reduction in TBA reaction product. This appears to be a widespread feature of tissues at this interval after endotoxin as all tissues tested except blood and ileum behaved in the same way. This result may imply that tissue has been oxidized and removed from its in uiuo site. Yoshikawa et al. [4] have found TBA reactive material in the bloodstream after endotoxin. An alternative explanation is that these tissues are more resistant to oxidation because their antioxidant content has been increased during the endotoxic crisis. Antioxidants acting intracellularly, extracellularly or within the lipid membranes should now be examined for altered activity during endotoxic shock. ACKNOWLEDGEMENT
Our thanks
are due to Dr. J.C. Evans for use of the e.s.r. facility.
306 REFERENCES 1 2 3 4
E. Suter and E.M. Kirsanow, Immunology, 4 (1961) 354. A.D. Heys and T.L. Dormandy, CIin Sci., 60 (1981) 295. J. Folch, I. AscoIi, M. Lees, J.A. Meath and F.N. LeBaron, J. Biol. Chem., 191 (1951) 833. T. Yoshikawa, M. Murakami, Y. Furukawa, H. Kato, S. Takemura and M. Kondo, Thromb. Haemost., 49 (1983) 214.
Bioelecfrochemisfry and Bioenergetics, 18 (1987) 301-306 A section of J. Electroanal. Chem., and constituting Vol. 232 (1987) Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands
1016 -
TISSUE
J.M. STARK,
OXIDATION
SK. JACKSON,
AFTER
BRIDGET
ENDOTOXIN*
WOODHEAD
and H.C. RYLEY
Department of Medical Microbiology, University of Wales Cotiege of Medicine, Heath Park, Cardiff CF4 4XN, Wales (Great Britain)
SUMMARY Macrophages from the spleens of mice sensitized with BCG produce oxygen radicals in vitro after stimulation with endotoxin as demonstrated by electron spin resonance spectroscopy. The TBA method for measuring lipid peroxidation products showed that, surprisingly, spleen homogenates from sensitized mice injected with endotoxin showed a reduced amount of TBA-reactive material after incubation in vitro. This result may be explained if endotoxin induces increases in tissue antioxidants. Splenic macrophages may play a critical role in this process since splenectomy of sensitized animals was seen to reduce the sensitivity to endotoxin.
INTRODUCTION
Improvement in the treatment of otherwise frequently fatal endotoxic shock depends on a better understanding of its pathogenesis. There is a large species-variation in sensitivity to endotoxins: mice are relatively resistant to endotoxin but may be sensitized by pre-injection with BCG [l]. The mechanisms underlying this sensitizing process remain unclear, but activated macrophages and their products may take part. Recent evidence suggests that endotoxin stimulates macrophages to produce a variety of active compounds including free radicals which may be damaging to the host’s tissues. The present investigation was undertaken in an attempt to show that free radicals are produced from activated macrophages in response to endotoxin challenge in vitro and that such free radicals could initiate damaging lipid peroxidation reactions. The idea that macrophages are important in the events of endotoxic shock was demonstrated by removing the spleen (a major source of macrophages) from sensitized animals.
l Presented at the Bioelectrochemical Biological Systems”, Obemai, 22-24
0302-4598/87/$03.50
Society Meeting October 1986.
0 1987 Elsevier Sequoia
S.A.
on
“Formation
and Reactions
of Peroxides
in
302 MATERIALS
AND METHODS
Sensitization to endotoxin Sensitization to endotoxin was induced by giving intravenously l/6 vial percutaneous BCG (Glaxo) (an attenuated strain of Mycobacterium tuberculosis) warmed, vasodilated adult male TO mice 14-18 days before endotoxin.
of to
Endotoxin Lipopolysaccharide B from Escherichia coli 026:B6 (Difco) was given in 10 pg amounts intraperitoneally in 0.4 cm3 phosphate buffered saline (PBS). Splenectomies in BCG-treated animals Spleens, enlarged 12 days after BCG were removed under ether anaesthesia from 15 animals. Fourteen animals survived the operation. A further 15 mice given BCG were sham-operated after the same time. The animals were given 2 pg endotoxin i.p. two days later and their survival monitored for 48 hours. Free radical production Spleens were removed from freshly killed mice 14-18 days after injection with BCG and carefully minced in pyrogen-free balanced salts solution. Tissue debris was removed. The cells were then washed and resuspended in serum-free medium and incubated in a quartz-aqueous flat electron spin resonance (e.s.r.) cell for several hours at 37’C to allow the macrophages to adhere to the glass walls. The tube was then washed through twice and a solution of 100 mM 5,5-dimethyl-l-pyrroline-N-oxide (DMPO) (Sigma Chemical Co. Ltd., Poole, England) spin trap containing lo-100 pg endotoxin was added and the tube incubated again before running e.s.r. spectra. e.s.r. spectra were run in the Chemistry Department, University College, Cardiff, on a Varian El09 ESR spectrometer. Lipid peroxidation Thiobarbituric acid (TBA) reactive substances ing products of lipid peroxidation were measured for 90 minutes at 37’ C by the TBA assay [2].
which are important and damageither directly or after incubation
Fatty acid analysis g.1.c. analysis of methylated fatty acids was performed extraction of the lipid materials [3].
after chloroform/methanol
RESULTS
Effect of splenectomy Splenectomy of BCG-primed mice gave rise to a significantly prolonged survival time (p < 0.02 by Student’s t test) after challenge with endotoxin when compared to sham-operated controls. The log mean time (hours) to death of the sham-operated animals (no survivors) was 1.13 _t 0.36 whereas that of the splenectomized animals was 1.48 f 0.36 with 3 survivors.
303
II
Fig. 1. e.s.r. spectrum of the hydroxyl BCG-primed macrophages.
0
'/2
3
6
P-=i radical
24
Fig. 2. Determination of lipid peroxidation at various times after endotoxin treatment for 90 minutes (B). Statistical comparisons time.
adduct
of DMPO
produced
from endotoxin-stimulated
48
in spleens from normal and BCG-treated mice before (0) and in vim. Spleens were unincubated (A) or incubated at 37 o C are made by the t test against the same preparation at zero
Fig. 3. Determination of lipid peroxidation in tissues of BCG-primed mice 6 hours after injection with endotoxin (t = 6) to untreated controls (t = 0). All tissues were incubated for 90 minutes in vitro at 37 ’ C prior to TBA/MDA assay (n = 4 for all groups).
Electron spin resonance A typical e.s.r. spectrum of the spin-trapped radicals produced from the endotoxin-stimulated splenic macrophages is shown in Fig. 1: The spectrum is characteristic of the hydroxyl radical adduct of DMPO (aN = aH = 1.48 mT).
Lipid peroxidation products The TBA reactive substances of the spleens of normal sensitized to endotoxin by BCG did not differ significantly
Fig. 4. GLC-polyunsaturated with and without injection
fatty acid (PUFA) of endotoxin.
analysis
animals and of animals except at 6 hours after
of spleens from normal
and BCG-primed
mice
305
endotoxin when the BCG treated animals showed a significant reduction in the amount of TBA reactive substances in the tissues (Fig. 2A). When the tissues, taken at intervals after endotoxin (O-48 h) were incubated for 90 minutes, the TBA reactive substances increased in all samples due to oxidation in vitro (Fig. 2B). However at 6 h in the unsensitized animals the product was significantly less than at zero time. When other tissues were similarly incubated (Fig. 3) all except blood and illeum showed this reduction in TBA reactive substances at 6 h when compared with the product of tissues taken before endotoxin (Fig. 3, t = 0). Fatty acid content of spleen Arachidonic acid (C20:4 w6) and C22:6.w3 were significantly increased in the BCG spleens and C18:1.6c (petroselenic acid) and C18:2c.w6 (linoleic acid) were significantly reduced. Endotoxin did not significantly alter these in either normal or primed animals (Fig. 4). DISCUSSION
The fact that splenectomy reduces susceptibility to endotoxin in BCG treated animals focuses attention on the metabolic changes in the spleen in the endotoxic crisis. This work has therefore examined free radical production and TBA reactive substances in the spleens of normal and endotoxin sensitized animals. The e.s.r. studies reveal a strong signal characteristic of the hydroxyl radical adduct after the introduction of endotoxin into splenic cell suspensions suggesting that this macrophage product may be produced in life by the cells of the BCG modified spleen. The sensitization process changes the fatty acid profile of the organ with a notable increase of the pharmacologically important arachidonic acid. However, the giving of endotoxin did not result in any significant change in the profiles. The TBA reactive substances in unincubated tissues reflects the tissue oxidation in life and did not show any increase after endotoxin in the examined spleens. However, when these tissues were allowed to oxidize in vitro a major difference was found in the tissues taken from the animals at 3 h and 6 h after endotoxin. These surprisingly showed a reduction in TBA reaction product. This appears to be a widespread feature of tissues at this interval after endotoxin as all tissues tested except blood and ileum behaved in the same way. This result may imply that tissue has been oxidized and removed from its in uiuo site. Yoshikawa et al. [4] have found TBA reactive material in the bloodstream after endotoxin. An alternative explanation is that these tissues are more resistant to oxidation because their antioxidant content has been increased during the endotoxic crisis. Antioxidants acting intracellularly, extracellularly or within the lipid membranes should now be examined for altered activity during endotoxic shock. ACKNOWLEDGEMENT
Our thanks
are due to Dr. J.C. Evans for use of the e.s.r. facility.
306 REFERENCES 1 2 3 4
E. Suter and E.M. Kirsanow, Immunology, 4 (1961) 354. A.D. Heys and T.L. Dormandy, CIin Sci., 60 (1981) 295. J. Folch, I. AscoIi, M. Lees, J.A. Meath and F.N. LeBaron, J. Biol. Chem., 191 (1951) 833. T. Yoshikawa, M. Murakami, Y. Furukawa, H. Kato, S. Takemura and M. Kondo, Thromb. Haemost., 49 (1983) 214.