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Alterations in intracellular lymphocyte metabolism induced by infection and injury
JOURNAL
OF SURGICAL
53,
RESEARCH
293-297
Alterations YUAN-LIN Shriners
DONG, M.D., Burns
Institute
(1992)
in Intracellular Lymphocyte Metabolism Induced by Infection and Injury’
TINA YAN, B.S., DAVID N. HERNDON,
Galveston
Unit
and Department
of Surgery,
M.D.,
University
Submitted for publication
AND J. PAUL WAYMACK,
of Texas
Medical
Branch,
M.D.,
Galveston,
Texas
SC.D.~ 77550
April 5, 1991
ria 161.To date there have been minimal investigations of the effects of burn injury and sepsis on intracellular leukocyte metabolism. Herein we describe investigations on the effect of burn injury on leukocyte metabolism with particular attention to glucose utilization, amino acid metabolism, and nucleic acid metabolism.
The effects of burn injury and sepsis on intracellular lymphocyte metabolism were evaluated using a rat model. Adult Lewis rats were subjected to a sham burn, a 30% full-thickness burn, or a 30% full-thickness burn which was infected with Pseudomonas aeruginosa. One week later the animals were sacrificed, and the splenic lymphocytes were harvested and cultured for 24 hr with mitogen stimulation. Lymphocytes from the burned-infected rats were found to utilize more glucose and certain amino acids than did lymphocytes obtained from the other two groups. Lymphocytes obtained from the burned-infected group had lower levels of the immunologically important enzyme, adenosine deaminase, than did the lymphocytes obtained from the other two groups. In summary, sepsis appears to alter a number of intracellular lymphocyte metabolic processes. These alterations may be found to be predictive of early sepsis. 0 1992 Academic Press, Inc.
MATERIALS
AND
METHODS
Animals
INTRODUCTION
Infection remains the most frequent cause of death in burn patients who survive the initial 24 hr following their injury [l]. This remains true despite the use of aggressive surgical therapy to obtain coverage of the burn wound and thus eliminate the wound as a site of entry for pathogenic bacteria. Rather, this therapy has merely changed the portal of entry for bacteria from the burn wound to the lung. As a result, pneumonias now most frequently lead to the patient’s ultimate demise [2]. This continued infection diathesis is in part the result of a burn-induced immunosuppression [3,4]. The immunosuppression is broad in nature and appears to have a particularly severe effect on lymphocyte function [5]. Most investigations of the effect of burn injury on immune function have centered on functional assays of leukocytes. These have included blastogenic response, lymphokine production, and cytotoxicity against bacte-
Adult male Lewis rats weighing approximately 250 g were used. The animals were housed in individual stainless steel hanging cages and were allowed food and water ad libitum throughout the experiment. The animals were observed for 1 week prior to entry into the study in order to exclude the presence of any preexisting diseases. The animals were divided into a control group (n = lo), a burned group (n = lo), and a burned-infected group (n = 10). The care of all animals was in accordance with the guidelines set forth by the Animal Welfare Act and other federal statutes and regulations relating to animals and studies involving animals and with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication 86-23). Burn Injury The animals in each of the three groups were anesthetized with an intraperitoneal injection of 45 mg/kg body weight of sodium pentobarbital. The backs were clipped of all hair. The animals were placed in an asbestos coated template which exposed 30% of the total body surface area. The control group (sham burn) was immersed for 10 set in a room temperature water bath. The burn and burn-infected groups were immersed for 10 set in 95°C water bath. The infected group had its burn wounds covered with 1 X lo7 Pseudomonas aeruginosa stain 1244. The animals in each of the three groups were then resuscitated with 10 ml of lactated Ringers intraperitoneally. Lymphocyte Harvesting
I Supported by the Shriners of North America and by NIH Grants T32 GM08256-OlAl, and ROl HL37411-OlAl. 2 To whom correspondence and reprint requests should be addressed at Shriners Burns Institute, 815 Market St., Galveston, TX
One week following burn injury the animals were sacrificed. The peritoneal cavities were entered in an aseptic manner and the spleens removed. The spleens were bivalved and lavaged with M-199 media containing 10%
77550.
293
0022-4so4/92
$4.00
Copyright 0 1992 by Academic Press, Inc. All rights
of reproduction
in any form
reserved.
294
JOURNAL
OF
SURGICAL
RESEARCH:
fetal calf serum, penicillin, and streptomycin. The resulting cell suspensions were further purified by FicollHypaque density gradient centrifugation. Contaminating red blood cells were removed by hypotonic lysis using sterile distilled water. The suspensions were washed three times with complete M-199 media. After the final washing, they were resuspended in sufficient M-199 media to obtain a concentration of 1 X lo7 cells/ml of M-199 media. One-milliliter aliquots of each suspension were placed in flat-bottom polystyrene culture plates. The suspensions underwent mitogen stimulation using concanavalin A (Con A). Con A was utilized since it has previously been demonstrated that rat lymphocytes respond to Con A but not to PHA [7]. In lymphocytes cultured without mitogen stimulation the rate of metabolism was too low to detect amino acid catabolism by our methods. All lymphocyte cultures were therefore cultured with Con A stimulation. The plates were incubated at 37°C in a 5% CO, atmosphere. After culturing for 24 hr the plates were removed and the suspensions harvested. Duplicates were run on all samples. One set of samples were frozen and thawed three times at -70°C to obtain lysis of the cells. A second set underwent centrifugation with the resulting cellular pellet being resuspended in 1 ml of phosphate-buffered saline. This was frozen and thawed three times at -70°C. The resulting lysates were assayed for glucose and amino acid content, nucleotide triphosphate levels (ATP plus GTP), and adenosine deaminase activity. The levels of glucose and amino acids in the lysate from the cells that had been resuspended in phosphate buffered saline were noted to be too low to be measureable by our assays. Thus the data showing the rate of amino acid and glucose metabolism by the lymphocytes are based on their rate of disappearance from the culture media. Since it has been previously demonstrated that lymphocytes can not perform gluconeogenesis, but can only catabolize glucose, the rate of glucose disappearance from the media is equal to the rate of lymphocyte metabolism of glucose. The same is true for amino acids since lymphocytes can only catabolize amino acids to form less complex amino acids or their carbon skelaton [8]. Amino Acid Assay The concentrations of free amino acids were determined following deproteinization using 3 vol of 5% sulfosalicyclic acid solution which contained thienylalanine as an internal standard. The samples were vortexed, centrifuged, and filtered. Amino acid levels were determined using an automated amino acid analyzer (Beckman) which employed a free buffer lithium citrate solution. Glucose Assay The glucose concentrations were determined using an automated Beckman glucose analyzer which measures glucose by oxidation.
VOL.
53, NO.
Adenosine
3, SEPTEMBER
Deaminase
1992
Activity
Adenosine deaminase activity was assayed using a colorimeteric method described by Giusti [9]. Briefly, 0.05 ml aliquots of the thawed lymphocyte lysate were added to 1 ml of buffered adenosine solution. After incubation for 60 min at 37”C, 3 ml of pheno-nitroprusside solution and 3 ml of alkaline hypochlorite were added. After incubation for 30 min at 37°C absorbance at 628 nm was measured. Volume activity (E) was described as follows: E =
Nucleotide
E sample - E sample blank E standard - E reagent blank
Triphosphate
(NTP)
.
Assay
NTP levels (ATP plus GTP) were measured using a coupled enzymatic reaction [lo]. The two reactions were: NTP + 3-phosphoglycerate + NDP + 1,3-diphosphoglycerate [l]; 1,3-diphosphoglycerate + NADH + Glyceraldehyde 3-triphosphate + NAD + phosphate [2]. Reaction 1 was catalyzed by phosphoglycerate kinase and reaction 2 was catalyzed by glyceraldehyde phosphate dehydrogenase. The change in absorbance at 340 nm which occurs when NADH is oxidized to NAD was measured. Statistical
Analysis
All data are expressed as means f standard of the mean. Comparisons between groups were measured using ANOVA. Statistical significance was assumed at a P < 0.05. RESULTS
There were noted to be significant alterations in the rate of glucose metabolism between the three groups. Lymphocytes obtained from the sham-burned animals were found to metabolize 508 +- 44 pg of glucose/l X lo7 cells/24 hr. Lymphocytes obtained from the burned animals metabolized 562 & 32 pg of glucose/l X 107/24 hr, and lymphocytes from the burned-infected rats metabolized 837 + 32 pug/l X lo7 cells/24 hours. These differences were statistically significant (P < 0.0001). The nucleotide triphosphate levels were not significantly altered by either injury or infection. The NTP concentrations in lymphocytes obtained from shamburned, burned, and burned-infected rats were 59.2 +10.9 pg/107 cells, 45.8 f 15.3 pg/107 cells, and 37.8 f 8.0 pg/lO’ cells, respectively (P = 0.44). The intracellular concentrations of adenosine deaminase were 14.10 + 0.75 units/lo7 cells for lymphocytes from the sham-burned animals, 10.56 f 0.25 units/lo7 cells for lymphocytes obtained from the burned rats, and 8.40 f 0.71 units/IO7 cells for lymphocytes obtained from burned-infected rats. These differences were statistically significant (P < 0.0001).
DONG
ET
AL.:
ALTERATIONS
IN
INTRACELLULAR
TABLE Rate
of Amino
Acid
-62 +80 f67 +116 -264 +92 +43 +lO +71 +4 -40 -688 -107 -324 -200 -27
t + + 4 i f f f k + * + k + t k
in nm/lO’
Burn 45 47 42 84 48 20 51 42 30 12 13 72 16 21 34 7
-235 -13 -29 -50 -531 +105 -11 +41 +48 -40 -79 -711 -132 -319 -324 -69f
There was noted to be an increased rate of utilization of the amino acids aspartate (P = 0.0003), threonine (P = 0.0039), serine (P = 0.0042), glutamate (P = 0.0020), glutamine (P = 0.0083), proline (P = 0.0015), glycine (P = 0.00017), alanine (P = 0.00002), valine (P = 0.00006), isoleucine (P = 0.01351), tyrosine (P = 0.04675), lysine (P = 0.00485), and histidine (P = 0.0044) by lymphocytes obtained from the burned and burned-infected animals. These data are shown in Table 1. Negative values indicate the rate of disappearance of the amino acid from the media over time. Positive values indicate a net increase in the concentration of the amino acid in the culture media over time.
295
METABOLISM
1
Disappearance
Control Aspartate Threonine Serine Glutamate Glutamine Proline Glycine Alanine Valine Cystiune Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine
LYMPHOCYTE
cell/24
Hr
P
Infected + i + +k -t f c f + f k + + k
31 38 35 70 24 33 50 43 32 18 10 68 17 22 17 5
-360 -166 -160 -317 -419 -43 -280 -264 -156 -8 -91 -862 -174 -369 -308 -59
+ f + + + + + f + k f f * i f *
47 49 47 91 80 25 32 20 27 16 11 90 20 27 20 11
0.0003 0.0039 0.0042 0.0020 0.0083 0.0015 0.00017 0.00002 0.00006 0.1395 0.01351 0.2499 0.04675 0.27538 0.00485 0.0044
ations in which they differ from helper T lymphocytes. Further studies utilizing cultures of pure T helper and T suppressor lymphocyte populations obtained through the use of the fluorescence-activated cell sorter would appear to be indicated in order to establish this fact. The increased rate of utilization of glucose by lymphocytes obtained from burn-infected rats is consistent with the finding that glucose is one of the preferred substrates for activated leukocytes. Im and Hoopes found that surgical wounds utilize large quantities of glucose [12]. Since the two cell types which predominate in such wounds are fibroblasts and leukocytes, the demonstration of metabolism of large amounts of glucose by the lymphocytes in our study is not surprising. It also would have been anticipated that lymphocytes obtained from DISCUSSION burned infected rats would be metabolically more active Our data indicated a number of significant alterations since they would have a considerable septic foci to deal in the intracellular metabolism of lymphocytes obtained with. However, the lymphocytes obtained from all three from burned and burned-infected rats compared to non- groups in our study were stimulated the Con A mitogen. traumatized, noninfected rats. These included an in- This mitogen is supposed to stimulate all T lymphocytes creased rate of utilization of glucose, an increased rate of to a very high level of activity. It would therefore appear utilization of certain amino acids, and a decreased level possible that the burn injury plus the septic foci may of activity of adenosine deaminase. These alterations upregulate the level of T lymphocyte metabolism in rewere also noted to be greater in those animals which sponse to antigenic stimulation, including that by nonwere infected compared to noninfected burned animals. specific mitogens. Such an upregulation is consistent The fact that burn injury itself was not able to induce with recent theories that one of the causes of mortality alterations as large as with the addition of a septic foci is in septic patients is an overly active immunologic reconsistent with the previous work of Burleson et al. [111. sponse. Such a response may result not only in the deThey noted that burn injury alone failed to alter the struction of bacteria but also of the patient’s own organs numbers of the various T lymphocyte subsets. However, and tissues [13]. when the burn wound was infected with P. aeruginosa, We have also noted an enhanced rate of utilization of the helper/suppressor T lymphocyte ratio of blood de- certain amino acids by lymphocytes obtained from creased significantly due to a decrease in the number of burned and burned-infected rats compared to nonhelper T cells. One possible explanation for our findings burned noninfected rats, even when the lymphocytes might therefore be that the suppressor T lymphocyte were exposed to a large dose of the nonspecific T lympopulation has a number of intracellular metabolic alterphocyte mitogen Con A. The explanation for this eleva-
296
JOURNAL
OF
SURGICAL
RESEARCH:
tion is probably the same as for the increased rate of metabolism of glucose by the lymphocytes. Several of the amino acid metabolic changes we noted bear further comment. Newsholme et al. have previously described the importance of glutamine as an oxidative fuel for lymphocytes [8, 141. Lymphocytes metabolize glutamine to glutamate and then either excrete the glutamate or continue metabolizing it to aspartate, pyruvate, and lactate. Our studies provide further support for the concept that glutamine is a vital energy source for lymphocytes and that trauma increases the rate of glutamine metabolism by the lymphocytes. Our studies also demonstrated a net increase in the concentration of glutamate in the media containing lymphocytes obtained from the sham-burned rats and a net decrease in the concentration of glutamate in the media containing lymphocytes from the burned and burned-infected rats. This would appear to indicate that the lymphocytes from the sham-burned rats were releasing glutamate into the media during the culture process, while those from the burned and burned-infected rats were metabolizing glutamate. Our data would therefore appear to indicate that burn injury and especially burn injury plus sepsis result in a decreased rate of release of glutamate and an increased rate of metabolism of glutamate by the lymphocytes. This would be consistent with an increased metabolic rate since it would indicate that the lymphocytes were metabolizing the glutamine through as many steps as possible prior to releasing the final metabolite in order to maximize the amount of biochemical energy generated from the glutamine. Our data also demonstrated that burn injury and sepsis significantly increased aspartate metabolism by lymphocytes. This is not surprising since aspartate is metabolized through the same pathway as glutamine [8]. We also noted a marked preference for branch chain amino acids by lymphocytes obtained from the burned and septic rats. This is consistent with the previous demonstration that in septic animals and patients there is an increased rate of metabolism of branch chain amino acids by the entire body [15, 161. Finally, we noted that the lymphocytes obtained from the burned-infected rats had a decreased adenosine deaminase level. This enzyme is important for lymphocyte metabolism and proliferation [ 171. It is necessary for the generation of the high energy nucleotide, adenosine triphosphate, which is essential for normal cellular functions and for cell division. In fact, individuals with a congenital deficiency of this enzyme suffer from an immunodeficiency state which predisposes them to infectious complications [ 181. In both transplant patients and animal models, adenosine deaminase activity has been shown to exhibit a biphasic response to CMV infections. During the early phase of CMV infections, adenosine deaminase activity increases [19]. In those patients who go on to die from
VOL.
53, NO.
3, SEPTEMBER
1992
CMV infections, there is a subsequent decrease in adenosine deaminase activity to subnormal levels [20]. Rats subjected to the burn wound infection protocol we utilized herein have previously been demonstrated to have a 100% mortality rate due to the infectious process [21]. The decreased level of adenosine deaminase activity in the burn-infected rats may therefore be indicative that in lethal bacterial infections, as in lethal viral infections, the activity of this enzyme is at subnormal levels. The impairment in immune function in these patients may therefore be in part a result of inadequate levels of adenosine deaminase to meet the lymphocyte’s metabolic demands for ATP generation. If this is found to be the case, pharmacologic supplementation might offer potential therapeutic benefit. In conclusion, sepsis appears to alter a number of aspects of intracellular lymphocyte metabolism. These include the rate of glucose and amino acid utilization, plus the levels of adenosine deaminase. Such alterations may offer diagnostic and therapeutic possibilities. REFERENCES 1.
Sevitt, S. A review of the complications of burns, their origin importance for illness and death. J. Trauma 19: 358, 1979.
2.
Shirani, K. Z., Pruitt, B. A., Jr., and Mason, A. D., Jr. The influence of inhalation injury and pneumonia on burn mortality. Ann. Surg. 205: 82, 1987. Alexander, J. W., Ogle, C. K., Stinnett, J. D., and Macmillan, B. G. A sequential, prospective analysis of immunologic abnormalities and infection following severe thermal injury. Ann. Surg. 188: 809,1978. Roe, E. A. The burn bactericidal index: A bactericidal index specific for burn patients. Burns Incl. Therm. Inj. 5: 241, 1979.
3.
4.
and
5.
Miller, C. L., and Baker, after thermal injury: The 63: 202, 1979.
6.
Markley, K., Smallman, E. T., and LaJohn, L. A. The thermal trauma in mice on cytotoxicity of lymphocytes Proc. Sot. Exp. Biol. Med. 154: 72, 1977.
7.
Deitch, E. A., Xu, D. Z., and Qi, L. Different partments respond differently to mitogenic thermal injury. Ann. Surg. 211: 72,199O.
8.
Newsholme, E. A., Crabtree, B., and Ardawi, M. S. M. Review article. Glutamine metabolism in lymphocytes: its biochemical, physiological and clinical importance. Q. J. Exp. Physiol. 70: 473,1985. Gusti, G. Adenosine deaminase determination. In H. U. Bergmeyer (Ed.), Methods of Enzymatic Analysis. New York: Academic Press, 1974. Vol. 3, pp. 1092-1099. Adams, H. Adenosine 5’triphosphate determination with phosphoglycerate kinase. In H. U. Bergmeyer (Ed.), Methods of Enzymatic Analysis. New York: Academic Press, 1965. Vol. 10, pp. 539-543. Burleson, D. G., Vaughn, G. K., Mason, A. D., Jr., and Pruitt, B. A., Jr. Flow cytometric measurement of rat lymphocyte subpopulations after burn injury and burn injury with infection. Arch. Surg. 122: 216, 1987. Im, M. J. C., and Hoopes, J. E. Energy metabolism in healing skin wounds. J. Surg. Res. 10: 459, 1970. Tennenberg, S. D., Jacobs, M. P., and Solomkin, J. S. Comple-
9.
10.
11.
12. 13.
C. C. Changes in lymphocyte activity role of suppressor cells. J. C&z. Inuest.
lymphocyte stimulation
effect of (39607). comafter
DONG
ET
AL.:
ALTERATIONS
ment-mediated neutrophil activation in sepsislated adult respiratory distress syndrome. Arch. 1987. 14.
15.
16.
IN
INTRACELLULAR
and trauma-reSurg. 122:
Carson, D. A., and Seegmiller, nase inhibition upon human Invest. 57: 274, 1976.
J. E. Effect of adenosine lymphocyte blastogenesis.
deamiJ. Clin.
297
Giblett, E. R., Anderson, J. E., Cohen, F., Pollara, B., and Meuwissen, H. J. Adenosine-deaminase deficiency in two patients with severely impaired cellular immunity. Lancet 2: 1067, 1972.
19.
Lum, C. T., Najarian, J. S., Sutherland, D. E. R., Seibel, J. R., Balfour, H. H., Jr., Yasmineh, W. G., and Howard, R. J. ADA activity in spleen cells from mice infected with cytomegalovirus. Surg. Forum 30: 276, 1979.
20.
Lum, C. T., Sutherland, D. E. R., Yasmineh, W. G., Fryd, D. S., Howard, R. J., and Najarian, J. S. Adenosine deaminase activity in cytomegalovirus-related graft and patient loss. Transplant. hoc. 11: 83, 1979.
21.
Waymack, J. P., Robb, E., and Alexander, J. W. Effect of transfusion on immune function in a traumatized animal model. II. effect on mortality rate following septic challenge. Arch. Surg.
Snelling, C. F., Woolf, L. I., Groves, A. C., Moore, J. P., and Duff, J. H. Amino acid metabolism in patients with severe burns. Sur-
gery91:474,1982. 17.
METABOLISM
18.
26,
Ardawi, M. S. M., and Newsholme, E. A. The transport of glutamine into rat mesenteric lymphocytes. Biochim. Biophys. Acta 856: 413, 1986. Freund, H. R., James, J. H., and Fischer, J. E. Nitrogen-sparing mechanisms of singly administered branched-chain amino acids in the injured rat. Surgery 90: 237, 1981.
LYMPHOCYTE
122:935,1987.
OF SURGICAL
53,
RESEARCH
293-297
Alterations YUAN-LIN Shriners
DONG, M.D., Burns
Institute
(1992)
in Intracellular Lymphocyte Metabolism Induced by Infection and Injury’
TINA YAN, B.S., DAVID N. HERNDON,
Galveston
Unit
and Department
of Surgery,
M.D.,
University
Submitted for publication
AND J. PAUL WAYMACK,
of Texas
Medical
Branch,
M.D.,
Galveston,
Texas
SC.D.~ 77550
April 5, 1991
ria 161.To date there have been minimal investigations of the effects of burn injury and sepsis on intracellular leukocyte metabolism. Herein we describe investigations on the effect of burn injury on leukocyte metabolism with particular attention to glucose utilization, amino acid metabolism, and nucleic acid metabolism.
The effects of burn injury and sepsis on intracellular lymphocyte metabolism were evaluated using a rat model. Adult Lewis rats were subjected to a sham burn, a 30% full-thickness burn, or a 30% full-thickness burn which was infected with Pseudomonas aeruginosa. One week later the animals were sacrificed, and the splenic lymphocytes were harvested and cultured for 24 hr with mitogen stimulation. Lymphocytes from the burned-infected rats were found to utilize more glucose and certain amino acids than did lymphocytes obtained from the other two groups. Lymphocytes obtained from the burned-infected group had lower levels of the immunologically important enzyme, adenosine deaminase, than did the lymphocytes obtained from the other two groups. In summary, sepsis appears to alter a number of intracellular lymphocyte metabolic processes. These alterations may be found to be predictive of early sepsis. 0 1992 Academic Press, Inc.
MATERIALS
AND
METHODS
Animals
INTRODUCTION
Infection remains the most frequent cause of death in burn patients who survive the initial 24 hr following their injury [l]. This remains true despite the use of aggressive surgical therapy to obtain coverage of the burn wound and thus eliminate the wound as a site of entry for pathogenic bacteria. Rather, this therapy has merely changed the portal of entry for bacteria from the burn wound to the lung. As a result, pneumonias now most frequently lead to the patient’s ultimate demise [2]. This continued infection diathesis is in part the result of a burn-induced immunosuppression [3,4]. The immunosuppression is broad in nature and appears to have a particularly severe effect on lymphocyte function [5]. Most investigations of the effect of burn injury on immune function have centered on functional assays of leukocytes. These have included blastogenic response, lymphokine production, and cytotoxicity against bacte-
Adult male Lewis rats weighing approximately 250 g were used. The animals were housed in individual stainless steel hanging cages and were allowed food and water ad libitum throughout the experiment. The animals were observed for 1 week prior to entry into the study in order to exclude the presence of any preexisting diseases. The animals were divided into a control group (n = lo), a burned group (n = lo), and a burned-infected group (n = 10). The care of all animals was in accordance with the guidelines set forth by the Animal Welfare Act and other federal statutes and regulations relating to animals and studies involving animals and with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication 86-23). Burn Injury The animals in each of the three groups were anesthetized with an intraperitoneal injection of 45 mg/kg body weight of sodium pentobarbital. The backs were clipped of all hair. The animals were placed in an asbestos coated template which exposed 30% of the total body surface area. The control group (sham burn) was immersed for 10 set in a room temperature water bath. The burn and burn-infected groups were immersed for 10 set in 95°C water bath. The infected group had its burn wounds covered with 1 X lo7 Pseudomonas aeruginosa stain 1244. The animals in each of the three groups were then resuscitated with 10 ml of lactated Ringers intraperitoneally. Lymphocyte Harvesting
I Supported by the Shriners of North America and by NIH Grants T32 GM08256-OlAl, and ROl HL37411-OlAl. 2 To whom correspondence and reprint requests should be addressed at Shriners Burns Institute, 815 Market St., Galveston, TX
One week following burn injury the animals were sacrificed. The peritoneal cavities were entered in an aseptic manner and the spleens removed. The spleens were bivalved and lavaged with M-199 media containing 10%
77550.
293
0022-4so4/92
$4.00
Copyright 0 1992 by Academic Press, Inc. All rights
of reproduction
in any form
reserved.
294
JOURNAL
OF
SURGICAL
RESEARCH:
fetal calf serum, penicillin, and streptomycin. The resulting cell suspensions were further purified by FicollHypaque density gradient centrifugation. Contaminating red blood cells were removed by hypotonic lysis using sterile distilled water. The suspensions were washed three times with complete M-199 media. After the final washing, they were resuspended in sufficient M-199 media to obtain a concentration of 1 X lo7 cells/ml of M-199 media. One-milliliter aliquots of each suspension were placed in flat-bottom polystyrene culture plates. The suspensions underwent mitogen stimulation using concanavalin A (Con A). Con A was utilized since it has previously been demonstrated that rat lymphocytes respond to Con A but not to PHA [7]. In lymphocytes cultured without mitogen stimulation the rate of metabolism was too low to detect amino acid catabolism by our methods. All lymphocyte cultures were therefore cultured with Con A stimulation. The plates were incubated at 37°C in a 5% CO, atmosphere. After culturing for 24 hr the plates were removed and the suspensions harvested. Duplicates were run on all samples. One set of samples were frozen and thawed three times at -70°C to obtain lysis of the cells. A second set underwent centrifugation with the resulting cellular pellet being resuspended in 1 ml of phosphate-buffered saline. This was frozen and thawed three times at -70°C. The resulting lysates were assayed for glucose and amino acid content, nucleotide triphosphate levels (ATP plus GTP), and adenosine deaminase activity. The levels of glucose and amino acids in the lysate from the cells that had been resuspended in phosphate buffered saline were noted to be too low to be measureable by our assays. Thus the data showing the rate of amino acid and glucose metabolism by the lymphocytes are based on their rate of disappearance from the culture media. Since it has been previously demonstrated that lymphocytes can not perform gluconeogenesis, but can only catabolize glucose, the rate of glucose disappearance from the media is equal to the rate of lymphocyte metabolism of glucose. The same is true for amino acids since lymphocytes can only catabolize amino acids to form less complex amino acids or their carbon skelaton [8]. Amino Acid Assay The concentrations of free amino acids were determined following deproteinization using 3 vol of 5% sulfosalicyclic acid solution which contained thienylalanine as an internal standard. The samples were vortexed, centrifuged, and filtered. Amino acid levels were determined using an automated amino acid analyzer (Beckman) which employed a free buffer lithium citrate solution. Glucose Assay The glucose concentrations were determined using an automated Beckman glucose analyzer which measures glucose by oxidation.
VOL.
53, NO.
Adenosine
3, SEPTEMBER
Deaminase
1992
Activity
Adenosine deaminase activity was assayed using a colorimeteric method described by Giusti [9]. Briefly, 0.05 ml aliquots of the thawed lymphocyte lysate were added to 1 ml of buffered adenosine solution. After incubation for 60 min at 37”C, 3 ml of pheno-nitroprusside solution and 3 ml of alkaline hypochlorite were added. After incubation for 30 min at 37°C absorbance at 628 nm was measured. Volume activity (E) was described as follows: E =
Nucleotide
E sample - E sample blank E standard - E reagent blank
Triphosphate
(NTP)
.
Assay
NTP levels (ATP plus GTP) were measured using a coupled enzymatic reaction [lo]. The two reactions were: NTP + 3-phosphoglycerate + NDP + 1,3-diphosphoglycerate [l]; 1,3-diphosphoglycerate + NADH + Glyceraldehyde 3-triphosphate + NAD + phosphate [2]. Reaction 1 was catalyzed by phosphoglycerate kinase and reaction 2 was catalyzed by glyceraldehyde phosphate dehydrogenase. The change in absorbance at 340 nm which occurs when NADH is oxidized to NAD was measured. Statistical
Analysis
All data are expressed as means f standard of the mean. Comparisons between groups were measured using ANOVA. Statistical significance was assumed at a P < 0.05. RESULTS
There were noted to be significant alterations in the rate of glucose metabolism between the three groups. Lymphocytes obtained from the sham-burned animals were found to metabolize 508 +- 44 pg of glucose/l X lo7 cells/24 hr. Lymphocytes obtained from the burned animals metabolized 562 & 32 pg of glucose/l X 107/24 hr, and lymphocytes from the burned-infected rats metabolized 837 + 32 pug/l X lo7 cells/24 hours. These differences were statistically significant (P < 0.0001). The nucleotide triphosphate levels were not significantly altered by either injury or infection. The NTP concentrations in lymphocytes obtained from shamburned, burned, and burned-infected rats were 59.2 +10.9 pg/107 cells, 45.8 f 15.3 pg/107 cells, and 37.8 f 8.0 pg/lO’ cells, respectively (P = 0.44). The intracellular concentrations of adenosine deaminase were 14.10 + 0.75 units/lo7 cells for lymphocytes from the sham-burned animals, 10.56 f 0.25 units/lo7 cells for lymphocytes obtained from the burned rats, and 8.40 f 0.71 units/IO7 cells for lymphocytes obtained from burned-infected rats. These differences were statistically significant (P < 0.0001).
DONG
ET
AL.:
ALTERATIONS
IN
INTRACELLULAR
TABLE Rate
of Amino
Acid
-62 +80 f67 +116 -264 +92 +43 +lO +71 +4 -40 -688 -107 -324 -200 -27
t + + 4 i f f f k + * + k + t k
in nm/lO’
Burn 45 47 42 84 48 20 51 42 30 12 13 72 16 21 34 7
-235 -13 -29 -50 -531 +105 -11 +41 +48 -40 -79 -711 -132 -319 -324 -69f
There was noted to be an increased rate of utilization of the amino acids aspartate (P = 0.0003), threonine (P = 0.0039), serine (P = 0.0042), glutamate (P = 0.0020), glutamine (P = 0.0083), proline (P = 0.0015), glycine (P = 0.00017), alanine (P = 0.00002), valine (P = 0.00006), isoleucine (P = 0.01351), tyrosine (P = 0.04675), lysine (P = 0.00485), and histidine (P = 0.0044) by lymphocytes obtained from the burned and burned-infected animals. These data are shown in Table 1. Negative values indicate the rate of disappearance of the amino acid from the media over time. Positive values indicate a net increase in the concentration of the amino acid in the culture media over time.
295
METABOLISM
1
Disappearance
Control Aspartate Threonine Serine Glutamate Glutamine Proline Glycine Alanine Valine Cystiune Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine
LYMPHOCYTE
cell/24
Hr
P
Infected + i + +k -t f c f + f k + + k
31 38 35 70 24 33 50 43 32 18 10 68 17 22 17 5
-360 -166 -160 -317 -419 -43 -280 -264 -156 -8 -91 -862 -174 -369 -308 -59
+ f + + + + + f + k f f * i f *
47 49 47 91 80 25 32 20 27 16 11 90 20 27 20 11
0.0003 0.0039 0.0042 0.0020 0.0083 0.0015 0.00017 0.00002 0.00006 0.1395 0.01351 0.2499 0.04675 0.27538 0.00485 0.0044
ations in which they differ from helper T lymphocytes. Further studies utilizing cultures of pure T helper and T suppressor lymphocyte populations obtained through the use of the fluorescence-activated cell sorter would appear to be indicated in order to establish this fact. The increased rate of utilization of glucose by lymphocytes obtained from burn-infected rats is consistent with the finding that glucose is one of the preferred substrates for activated leukocytes. Im and Hoopes found that surgical wounds utilize large quantities of glucose [12]. Since the two cell types which predominate in such wounds are fibroblasts and leukocytes, the demonstration of metabolism of large amounts of glucose by the lymphocytes in our study is not surprising. It also would have been anticipated that lymphocytes obtained from DISCUSSION burned infected rats would be metabolically more active Our data indicated a number of significant alterations since they would have a considerable septic foci to deal in the intracellular metabolism of lymphocytes obtained with. However, the lymphocytes obtained from all three from burned and burned-infected rats compared to non- groups in our study were stimulated the Con A mitogen. traumatized, noninfected rats. These included an in- This mitogen is supposed to stimulate all T lymphocytes creased rate of utilization of glucose, an increased rate of to a very high level of activity. It would therefore appear utilization of certain amino acids, and a decreased level possible that the burn injury plus the septic foci may of activity of adenosine deaminase. These alterations upregulate the level of T lymphocyte metabolism in rewere also noted to be greater in those animals which sponse to antigenic stimulation, including that by nonwere infected compared to noninfected burned animals. specific mitogens. Such an upregulation is consistent The fact that burn injury itself was not able to induce with recent theories that one of the causes of mortality alterations as large as with the addition of a septic foci is in septic patients is an overly active immunologic reconsistent with the previous work of Burleson et al. [111. sponse. Such a response may result not only in the deThey noted that burn injury alone failed to alter the struction of bacteria but also of the patient’s own organs numbers of the various T lymphocyte subsets. However, and tissues [13]. when the burn wound was infected with P. aeruginosa, We have also noted an enhanced rate of utilization of the helper/suppressor T lymphocyte ratio of blood de- certain amino acids by lymphocytes obtained from creased significantly due to a decrease in the number of burned and burned-infected rats compared to nonhelper T cells. One possible explanation for our findings burned noninfected rats, even when the lymphocytes might therefore be that the suppressor T lymphocyte were exposed to a large dose of the nonspecific T lympopulation has a number of intracellular metabolic alterphocyte mitogen Con A. The explanation for this eleva-
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tion is probably the same as for the increased rate of metabolism of glucose by the lymphocytes. Several of the amino acid metabolic changes we noted bear further comment. Newsholme et al. have previously described the importance of glutamine as an oxidative fuel for lymphocytes [8, 141. Lymphocytes metabolize glutamine to glutamate and then either excrete the glutamate or continue metabolizing it to aspartate, pyruvate, and lactate. Our studies provide further support for the concept that glutamine is a vital energy source for lymphocytes and that trauma increases the rate of glutamine metabolism by the lymphocytes. Our studies also demonstrated a net increase in the concentration of glutamate in the media containing lymphocytes obtained from the sham-burned rats and a net decrease in the concentration of glutamate in the media containing lymphocytes from the burned and burned-infected rats. This would appear to indicate that the lymphocytes from the sham-burned rats were releasing glutamate into the media during the culture process, while those from the burned and burned-infected rats were metabolizing glutamate. Our data would therefore appear to indicate that burn injury and especially burn injury plus sepsis result in a decreased rate of release of glutamate and an increased rate of metabolism of glutamate by the lymphocytes. This would be consistent with an increased metabolic rate since it would indicate that the lymphocytes were metabolizing the glutamine through as many steps as possible prior to releasing the final metabolite in order to maximize the amount of biochemical energy generated from the glutamine. Our data also demonstrated that burn injury and sepsis significantly increased aspartate metabolism by lymphocytes. This is not surprising since aspartate is metabolized through the same pathway as glutamine [8]. We also noted a marked preference for branch chain amino acids by lymphocytes obtained from the burned and septic rats. This is consistent with the previous demonstration that in septic animals and patients there is an increased rate of metabolism of branch chain amino acids by the entire body [15, 161. Finally, we noted that the lymphocytes obtained from the burned-infected rats had a decreased adenosine deaminase level. This enzyme is important for lymphocyte metabolism and proliferation [ 171. It is necessary for the generation of the high energy nucleotide, adenosine triphosphate, which is essential for normal cellular functions and for cell division. In fact, individuals with a congenital deficiency of this enzyme suffer from an immunodeficiency state which predisposes them to infectious complications [ 181. In both transplant patients and animal models, adenosine deaminase activity has been shown to exhibit a biphasic response to CMV infections. During the early phase of CMV infections, adenosine deaminase activity increases [19]. In those patients who go on to die from
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CMV infections, there is a subsequent decrease in adenosine deaminase activity to subnormal levels [20]. Rats subjected to the burn wound infection protocol we utilized herein have previously been demonstrated to have a 100% mortality rate due to the infectious process [21]. The decreased level of adenosine deaminase activity in the burn-infected rats may therefore be indicative that in lethal bacterial infections, as in lethal viral infections, the activity of this enzyme is at subnormal levels. The impairment in immune function in these patients may therefore be in part a result of inadequate levels of adenosine deaminase to meet the lymphocyte’s metabolic demands for ATP generation. If this is found to be the case, pharmacologic supplementation might offer potential therapeutic benefit. In conclusion, sepsis appears to alter a number of aspects of intracellular lymphocyte metabolism. These include the rate of glucose and amino acid utilization, plus the levels of adenosine deaminase. Such alterations may offer diagnostic and therapeutic possibilities. REFERENCES 1.
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