JOURNAL
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RESEARCH
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(1991)
Immune Suppression after Acute Ethanol Ingestion and Thermal Injury’ MASATO KAWAKAMI, *Department tDepartment
M.D.,* BOYD R. SWITZER, PH.D.,? SANDRA R. HERZOG, B.A., P.A.-C.,* AND ANTHONY A. MEYER, M.D., PH.D.**’
of Surgery and North Carolina Jaycee Burn Center, University of North Carolina School of Medicine, and of Nutrition at University of North Carolina, School of Public Health, Chapel Hill, North Carolina 27599 Submitted for publication June 13, 1990 alcohol ingestion [4]. Despite the frequent association of acute alcohol ingestion and injury, the effects of acute alcohol ingestion on the pathophysiology of trauma patients has not been widely investigated. Several reports suggested that burned patients with ethanol (EtOH) exposure have a greater mortality than those without EtOH exposure [4-6]. Howland and Hingson suggested that the high mortality rate might be due to increased susceptibility to infection induced by EtOH [4]. Like burn injury, acute EtOH ingestion can induce immune suppression by itself. Chronic ingestion of EtOH prior to injury does significantly increase the immune suppression [7]. However, many accident victims do not have a history of chronic, daily alcohol consumption. In such patients a single exposure to EtOH prior to injury may produce greater immune dysfunction than either EtOH alone or injury alone. The purpose of this study was to determine the combined effects of acute EtOH ingestion and bum injury on immune function. We measured immune function after burn injury in rats with a single EtOH ingestion prior to injury. The data analysis examined the effects of both burn injury and acute EtOH ingestion on these parameters of immune function.
Acute alcohol ingestion is commonly associated with burn injury. Both alcohol ingestion and burn injury produce immune suppression, but the combination of these factors on immune function has not been investigated. To study this combined effect, immune function was measured in rats with a 30% burn injury following a single ingestion of 2.4 g/kg of ethanol (EtOH) and compared to that of animals with burn injury only, animals with EtOH only, and animals with neither alcohol nor burn injury. Four days after ethanol and/or burn, animals receiving both ethanol and burn injury had significant suppression of in uivo chemotaxis and lymphocyte responsiveness to lipopolysaccharide (LPS) compared to animals receiving either burn injury alone or EtOH alone (P < 0.05). There was no difference in responsiveness to concanavalin A (Con A). Serum corticosterone was significantly elevated by burn injury but not EtOH ingestion. EtOH treatment prior to injury caused a further increase in corticosterone level that was significantly associated with a decrease in immune function. These results indicate that a single EtOH exposure prior to burn injury produces greater immune suppression than does burn injury alone. This further decrease in immune function may contribute to increased susceptibility to infection and increased mortality in burn patients with acute EtOH ingestion. G IS~I Academic
Press,
Inc.
MATERIALS
Animals
INTRODUCTION
’ Some of this data was presented at the Ninth Annual Meeting of the Surgical Infection Society, April 13-14, 1989, Denver, CO. 2 To whom reprint requests should he addressed at Department of Surgery, University of North Carolina, Campus Box 7210, 164 Burnett-Womack, Chapel Hill, NC 27599-7210. $1.50 1991 by of reproduction
Copyright 0 All
rights
210 Academic Press. Inc. in any
form
reserved.
METHODS
and Materials
Male Sprague-Dawley rats (Charles River Laboratories, Raleigh, N.C.) weighing 300-350 g were used for all experiments. All animals were given food and water ad libitum throughout the experiments. The protocols for procedures, anesthesia, and euthanasia were approved by the Committee on Animal Research, University of North Carolina at Chapel Hill. Tissue culture media and supplements were purchased from the Tissue Culture Facility (Lineberger Cancer Research Center, University of North Carolina, Chapel Hill, NC). All chemicals were purchased from Sigma Chemicals (St. Louis, MO) except where noted otherwise.
Injury is frequently associated with acute alcohol ingestion. Nearly 50% of trauma victims transferred to hospitals were reported to have been drinking alcohol [l]. Alcohol ingestion is also a major risk factor in fire victims [2, 31. A review of several series reported that half of the people who died in fires had evidence of acute
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All animals were randomly divided into four groups: 11 animals underwent burn injury at 4 hr after EtOH ingestion (EtOH+Burn), 11 animals received burn injury alone (Burn), 11 animals ingested EtOH alone (EtOH), and 11 animals served as control (Control). An acute alcohol model was produced by gavage feeding of 2.4 g/kg of body weight of EtOH in 20% solution in distilled water. A 30% total body surface area (TBSA) fullthickness burn injury was produced, as described previously [8]. Briefly, animals were anesthetized by methoxyAurane (Pitman-Moore, Washington Crossing, NJ); and then their backs were shaved and exposed to steam at 100°C for 30 sec. Resuscitation after burn injury was performed by injecting 20 ml of lactated Ringer’s intraperitoneally and 3 mg/kg of morphine sulfate was given subcutaneously for analgesia. All measures of immune function were made 4 days after acute EtOH ingestion and/or burn injury. Blood EtOH Concentration To determine the levels of blood EtOH achieved in this model, six rats were given 2.4 g/kg of EtOH by gavage feeding, and blood EtOH level was measured. Blood samples were taken from the amputated tail tip before EtOH ingestion and at 1, 2, 3, 4, 6, 8, and 24 hr after ingestion. Blood EtOH concentration was measured using an enzymatic method (Sigma Diagnostics No. 332UV, Sigma Chemicals). Briefly, a whole blood sample was mixed with 6.25% (w/v) trichloroacetic acid to prepare a protein-free supernatant, and vortexed. After the mixture was centrifuged, the supernatant was stored at -70°C. On the next day, 100 ~1 of the stored supernatant was mixed with 2.9 ml of 0.5 mole/liter glycine buffer containing 0.6 mmole/ml nicotinamide adenine dinucleotide and 50 U/ml alcohol dehydrogenase. After the mixture was allowed to stand at room temperature for 10 min, the absorbance of the mixture was measured at 340 nm using a Beckman DU-6 uv-visibile spectrophotometer (Beckman Instruments, Inc., Fullerton, CA). EtOH concentration of the sample was calculated from the absorbance using a standard curve. In Viva Ctwmotaxis Chemotaxis was assessed by the amount of myeloperoxidase (MPO) from polymorphonuclear neutrophils in the skin following injection of N-formylmethionylleucylphenylalanine (FMLP). After animals were anesthetized with methoxyflurane vapor, the hair on their ventral surface was clipped and 0.2 ml of 10 PM FMLP was injected intradermally in three sites for in uiuo chemotaxis measurement. Four hours after FMLP inoculation, all rats were reanesthetized and celiotomy was performed. The animals were euthanized by exsanguination from the abdominal aorta after the splenic artery and
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vein were clamped. Then, the spleen was removed for a mitogenic assay of splenic lymphocytes. The skin at the sites of FMLP injection was biopsied using a 6-mm disposable biopsy punch (Acuderm, Inc., Ft. Lauderdale, FL) for the MPO assay. The skin samples were stored at -70°C and a MPO assay was done on the next day. The technique for a calorimetric assay measuring MPO extracted from skin has been previously described [8]. Mitogenesis
Assay
The procedures of the preparation of spleen cell suspension and a mitogenesis assay were described previously [7,9]. In brief, spleen cells were teased out aseptically into 10 ml of culture medium, the red blood cells were lysed, and the cell suspension was incubated in a glass petri dish at 37°C in a humidified atmosphere of 5% CO, in air for 2 hr in order to separate the nonadherent cells. Nonspecific esterase staining (Sigma Chemicals) demonstrated that the nonadherent cell suspension contained less than 0.01% monocytes. Cell viability was consistently 98% or better, as assessed by trypan blue exclusion. The nonadherent splenocytes were cultured with 2.5 rg/ml of Con A and LPS. At 24 hr prior to termination of mitogenesis, 0.05 ml of media containing 1 &i of tritiated thymidine (sp act 6.7 Ci/mmole; ICN Radiochemicals, Irvine, CA) was added to each well. Wells were harvested on a glass-fiber filter using a semiautomatic cell harvester (Skatron, Inc., Sterling, VA), and the amount of tritiated thymidine uptake was determined. The average counts per minute for each culture condition in five replicate wells was calculated. Serum Corticosterone
Level
Serum samples were taken from animals at the time of sacrifice and stored at -7O’C. Serum corticosterone concentration was measured using a ‘%I-corticosterone radioimmunoassay kit (Cambridge Medical Technology, Billerica, MA). Briefly, 100 ~1 of serum sample was mixed with 100 ~1 of ‘251-corticosterone and corticosterone antiserum in a glass tube, vortexed, and incubated for 16-24 hr at 4°C. Then, 100 ~1 of goat anti-rabbit y-globulin was added to the tube. It was vortexed, incubated for 1 hr at room temperature, and centrifuged at 1500-1600g for 15 min at 2 to 8°C. After decanting the supernatant from the tube, the radioactivity of the sediment was counted and hormone concentration was determined from log-log& analysis of the data. Statistical
Analysis
The effects of acute EtOH ingestion and burn injury were analyzed by two-way analysis of variance (ANOVA), and the comparison between the mean of each group was computed by Scheffe’s multiple comparison test. The correlation between serum corticosterone level
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VwW
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1
2
3
4
6
TIME AFTER EtOH INGESTION
8
24
(hours)
FIG. 1. The changes in blood EtOH level after ingestion of EtOH at 2.4 g/kg. The data are expressed as means and standard deviation. The value at 0 hr represents the baseline measured prior to EtOH ingestion.
and each immunologic parameter was analyzed by single regression analysis. P values of less than 0.05 were accepted as statistically significant. RESULTS
Blood EtOH Concentration The blood EtOH levels of six animals receiving 2.4 g/kg of body weight of EtOH reached a peak approximately 1 hr after EtOH ingestion, with an average maximum EtOH level of 108.7 + 19.6 mg/dl (mean and standard deviation). The mean and standard deviation for each time point are shown in Fig. 1. EtOH was still present in the blood 6 hr after EtOH ingestion, but was not detectable 8 hr after ingestion.
EtOH
FIG. 2.
MPO activity of each group. MPO activity is represented by absorbance (mean and standard error). Statistical results are as follows: by ANOVA, EtOH P = 0.044; Bum, P -z 0.001; Interaction, P = 0.792; * by multiple comparison test, P < 0.05 vs Control.
activity, compared to the Control group and the EtOH group (P < 0.05). The animals which received EtOH only (EtOH) had slightly decreased MPO activity, but the difference from the Control group was not statistically significant using Scheffe’s test.
Mitogenesis Assay Responsiveness of nonadherent spleen cells to Con A (T-lymphocyte mitogen) or LPS (B-lymphocyte mito-
q q
In Viva Chemotaxis In uiuo chemotaxis of skin biopsies was measured by MPO activity 4 hr following FMLP inoculation. The MPO activity in three skin samples obtained from each animal was averaged and considered as MPO activity of each rat. Both a single EtOH ingestion (P = 0.044) and burn injury (P < 0.001) produced significant suppression in chemotaxis by ANOVA, as shown in Fig. 2. In comparison between each group using Scheffe’s test, the animals which received both EtOH and burn injury (EtOH + Burn) had the greatest decrease in MPO activity compared to the Control group (P < 0.05). The animals which received burn injury alone (Burn) also had decreased MPO activity (P K 0.05), but not as severe as the animals which received both treatments (EtOH + Burn). Both the Burn group and the EtOH + Burn group showed a statistically significant decrease in MPO
0
BURN EtOH+BURN
NO MKOQEN
2.5 pg/ml
FIG. 3. Lymphocyte response to Con A. The value is represented by the mean and standard error. Statistical results are as follows: by ANOVA, EtOH, P = 0.975; Bum, P = 0.019; Interaction, P = 0.135; * by multiple comparison test, P < 0.05 vs Control in the same batch.
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BURN
(CPM) W0W - 0
q 1
q
CONTROL EtOH BURN EtOH+BURN
l
0
0
NO MITOGEN
2.5 pg/ml
FIG. 4. Lymphocyte response to LPS. The value is represented by the mean and standard error. Statistical results are as follows: by ANOVA, EtOH, P = 0.999; Burn, P < 0.001; Interaction, P = 0.005;by multiple comparison test, *P < 0.05 vs Control in the same batch, **P < 0.05 vs the others in the same batch.
gen) or without mitogens was evaluated by tritiated thymidine uptake of proliferating cells. Under all three culture conditions, consistent results were obtained, as shown in Figs. 3 and 4. The animals receiving both EtOH ingestion and burn injury (EtOH + Burn) showed the least response to both Con A and LPS, and even the mean thymidine uptake of the unstimulated cell in this group was less than those of the other groups. The decrease in the response to LPS was more remarkable than that to Con A, and the combined insult of burn injury and EtOH ingestion was statistically significant compared to the other three groups (P < 0.05). Although the thymidine uptake of the animals receiving EtOH ingestion only (EtOH) was increased under each culture condition, only the increased uptake of unstimulated cells from the EtOH group was statistically significant compared to that of the Control group (P
CONTROL
BURN
EtOH
ElOH+BURN
FIG. 5. The value is presented by the mean and standard error. The numbers of each group are Control = 8, EtOH = 6, Burn = 8, and EtOH + Burn = 8. Statistical results by ANOVA are as follows: EtOH, P = 2.699; Burn, P = 0.030; Interaction, P = 0.193.
= 0.030). The animals receiving both EtOH ingestion and burn injury (EtOH + Burn) had the greatest increase in corticosterone level compared to the Control group, but the difference was not significant because of the large standard error. Linear regression analysis of the effect of serum corticosterone level on the immunologic parameters, MPO activity, lymphocyte responsiveness to Con A and LPS, and unstimulated lymphocyte proliferation, found that there was a statistically significant negative correlation between serum corticosterone level and immunologic function. The value of R2 (variance) in each regression analysis was small. The relationship between serum corticosterone levels and immune function is summarized in Table 1. Figure 6 is a graphic representation of the relationship between serum corticosterone and MPO activity and is similar to the other measures of immune function. High serum corticosterone level (more than 12 pg/dl) was seen only in the EtOH + Burn group, and these animals
< 0.05).
The thymidine uptake of the animals receiving burn injury only (Burn) was decreased or similar in each culture condition compared to that of the Control group. There were no statistically significant differences between the Burn and Control groups. Serum Corticosterone
Level
We measured serum corticosterone concentrations in serum from the animals of each group. Mean corticosterone levels are shown in Fig. 5. An influence of EtOH ingestion on serum corticosterone level 4 days prior to sampling could not be detected (P = 2.699). Burn injury significantly increased serum corticosterone level (P
TABLE
1
Results of Regression Analysis between Serum Corticosterone Levels and Immunologic Parameters Parameters MPO Thymidine uptakes with no mitogen Thymidine uptakes with Con A Thymidine uptakes with LPS
Coefficient of slope
R2
-0.011
0.245
0.006
-421
0.130
0.050
-7421
0.171
0.023
-2718
0.225
0.008
Probability
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OA
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A
EtOH+BURN
F 2 H 0.100.05 -
0.00
J I
r
I
I
4
6
6
10
Serum Corticosterone
\
12
14
16
16
Level (pggldl)
FIG. 6. Scattergram of MPO activity against serum corticosterone level. The variance (I?) is 0.245 and the probability of no correlation is 0.006. The regression line in the chart is Y = -0.011X + 0.182.
showed the most impaired immune function. However, some animals in the EtOH+Burn group showed impaired immune function, even though their corticosterone level was within the normal range. DISCUSSION
Alcohol has been recognized as a predisposing factor to burn injuries [2, 3, lo]. In addition to its role in causing accidents, alcohol exposure prior to injury appears to increase the mortality of injured patients. Some reports have shown a higher mortality in patients whose burn injuries are associated with alcohol than in patients whose injuries are not associated with alcohol [4,5]. Crikerlair et al. pointed out that preexisting disease, including alcoholism, played a prominent role in determining the outcome of patients [5]. Rittenbury et al. documented that chronic alcohol abuse associated with liver disease significantly increased mortality from burn injury [6]. Immune suppression by a single, acute ingestion of EtOH has also been reported [9, 111. Burn injury has been found to impair many components of the immune system [12-181. Immune suppression by major burn injury can contribute to development of fatal infectious complications. Previous studies in this laboratory have noted that chronic EtOH exposure prior to burn injury produced greater immune suppression than either the burn injury or EtOH exposure alone. It is therefore quite possible that a single, acute EtOH exposure could produce further immune dysfunction than does either alcohol ingestion or burn injury alone. The experiments reported here supports this hypothesis. In this study, neutrophil chemotaxis was further suppressed by a single dose of EtOH prior to burn injury. Neutrophils work by bacterial phagocytosis and intracellular killing, which are important in nonspecific host
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defense. Malfunction of neutrophils can lead to increased bacterial infection. Alexander and Wixson reported evidence that neutrophil dysfunction was a major determinant in the development of life-threatening sepsis after thermal injury [19]. Responsiveness of lymphocytes to mitogens is commonly used for the evaluation of cellular immunity. Significant correlation between mortality after a septic challenge and suppression of mitogenic response in burned animals has been reported [20]. Con A was used as the T-cell mitogen in these experiments because preliminary studies found that it produced a higher, more consistent response than phytohemagglutinin. LPS was used as the B-lymphocyte mitogen in this study because LPS can stimulate B-lymphocytes to proliferate specifically, while pokeweed mitogen stimulates both T- and B-lymphocytes. The present study showed that a 30% burn injury produced suppression in the response to Con A and LPS. In other studies, burn injury produced a significantly decreased response to Con A [13,21-231. Generally T-lymphocyte function is more influenced by burn injury and alcohol than B-lymphocyte function [ll, 121. The more pronounced effect on B-lymphocyte function in the present study may be caused by an alteration of absolute and relative numbers of lymphocyte subpopulations in the spleen induced by burn injury, EtOH, and their combination. However, according to several reports, burn injury seems to influence the T-lymphocyte percentage in the spleen, but not the B-lymphocyte percentage [24-271. With respect to alcohol, Lundy et al. reported that the proportion of T-lymphocytes was decreased and that of B-lymphocytes was unchanged in his study of peripheral blood of human alcoholics [28]. The subjects used in this study, however, had a history of long-term and excessive drinking, and the effect of acute EtOH ingestion is unknown. Reports of lymphocyte subpopulation in the spleen after acute EtOH ingestion are not available at this time. The effect of the combination of burn injury and acute EtOH ingestion on the lymphocyte subpopulation in the spleen is also unknown, but further study is planned to investigate it. Several circulating humoral substrates are recognized as factors which mediate the immune suppression after burn injury and EtOH ingestion [12,29]. Corticosteroids are known as strong immune-suppressive agents. The increase of circulating corticosteroids under severe stress like burn injury is well described. It is suggested by Jerrells et al. that corticosteroids can at least partially mediate the immune suppression induced by EtOH [29]. A 30% burn injury significantly increased the corticosterone level in this study. Furthermore, the acute EtOH ingestion prior to burn injury produced a greater increase in serum corticosterone level than burn injury alone. Although a statistical analysis failed to confirm the significant effect of acute EtOH ingestion on cortico-
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sterone level, this may be due to the small sample size of each group. All the animals with high corticosterone levels of more than 12 pg/dl were apparently associated with suppression in the immunologic parameters studied. Interestingly, some animals in the Burn and EtOH+Burn group had impaired immune function, although they had relatively normal corticosterone levels. This finding may indicate that the increase in corticosterone level contributed to the immune suppression, but it was not the sole factor. Biologic systems of this complexity are unlikely to identify a single factor, such as corticosterone, responsible for highly variable biologic response. The present study demonstrates that the combination of acute EtOH ingestion and burn injury produces greater suppression of in uiuo chemotaxis and lymphocyte mitogenic response than either burn injury or acute EtOH ingestion do independently. The clinical implications are that the further immune suppression from a single exposure prior to injury may contribute to increased susceptibility to infection and increased mortality in trauma patients. Although the mechanisms are not clarified in this study, this suppression of several immune mechanisms could cause increased susceptibility to infection and increased mortality in burn patients with a single alcohol exposure at the time of injury.
10. 11. 12. 13.
14.
15. 16.
17.
18. 19. 20.
21. 22.
REFERENCES 1.
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5. 6.
Thal, E. R., Bost, R. O., and Anderson, R. J. Effects of alcohol and other drugs on traumatized patients. Arch. Surg. 120: 708, 1985. Levine, M. S., and Radford, E. P. Fire victims: Medical outcome and demographic characteristics. Am. J. Public Health 67: 1077, 1977. MacArthur, J. D., and Moore, F. D. Epidemiology of burns. JAMA 231: 259, 1975. Howland, J., and Hingson, R. Alcohol as a risk factor for injuries or death due to fires and burns: Review of the literature. Public Health Rep. 102: 475, 1987. Crikerlair, G. F., Symonds, F. C., Ollstein, R. N., and Kirsner, A. I. Burn causation: Its many sides. J. Trauma 8: 572, 1968. Rittenbury, M. S., Schmidt, F. H., Maddox, R. W., Beazley, W. I., Ham, W. T., and Haynes, B. W., Jr. Factors significantly affecting mortality in the burned patient. J. Trauma 5: 587, 1965. Kawakami, M., Meyer, A. A., Johnson, M. C., deserres, S., and Peterson, H. D. Chronic ethanol exposure prior to injury produces greater immune dysfunction after thermal injury in rats. J. Trauma 30: 27, 1990. Manktelow, A., and Meyer, A. A. Lack of correlation between decreased chemotaxis and susceptibility to infection in burned rats. J. Trauma 26: 143, 1986. Kawakami, M., Meyer, A. A., Johnson, M. C., and Rezvani, A. H.
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Immunologic consequences of acute ethanol ingestion in rats. J. Surg. Res. 47: 412, 1989.13. Schmidt, W., and DeLint, J. Causes of death of alcoholics. Q. J. Stud. Alcohol 33: 171,1972. MacGregor, R. R. Alcohol and immune defense. JAMA 256: 1474,1986. Winkelstein, A. What are the immunological alterations induced by burn injury? J. Trauma 24(Suppl): 72, 1984. Munster, A. A., Winchurch, R. A., Birmingham, W. J., and Keeling, P. Longitudinal assay of lymphocyte responsiveness in patients with major burns. Ann. Surg. 192: 772, 1980. Warden, G. D., Mason, A. D., Jr., and Pruitt, B. A., Jr. Evaluation of leukocyte chemotaxis in vitro in thermally injured patients. J. Clin. Invest. 54: 1001, 1974. Alexander, J. W., and Moncrief, J. A. Alterations of the immune response following thermal injury. Arch. Surg. 93: 75, 1966. Arturson, G., Hogman, C. F., Johansson, S. G. O., and Killander, J. Changes in immunoglobulin levels in severely burned patients. Lancet 1: 546, 1969. Miller, C. A., and Baker, C. C. Changes in lymphocyte activity after thermal injury. The role of suppressor cells. J. Clin. Znuest. 63: 202,1979. Fikrig, S. M., Karl, S. C., and Suntharalingam, K. Neutrophil chemotaxis in patients with burns. Ann. Surg. 186: 746, 1977. Alexander, J. W., and Wixson, D. Neutrophil dysfunction and sepsis in burn injury. Surg. Gynecol. Obstet. 130: 431, 1970. Moss, N. M., Gough, D. B., Jordan, A. L., Grbic, J. T., Wood, J. J., Rodrick, M. L., and Mannick, J. A. Temporal correlation of impaired immune response after thermal injury with susceptibility to infection in a murine model. Surgery 104: 882, 1988. Baker, C. C., Miller, C. L., and Trunkey, D. D. Predicting fatal sepsis in burn patients. J. Trauma 19: 641, 1979. Eurenius, K., and Mortensen, R. F. The phytohemagglutinin (PHA) response in the thermally injured rat. Znt. Arch. Albrgy 40: 707, 1971. Mahler, D., and Batchelor, J. R. Phytohaemagglutinin transformation of lymphocytes in burned patients. Transplantation 12: 409,1971. Deitch, E. A., Xu, D., and Qi, L. Different lymphocyte compartments respond differently to mitogenic stimulation after thermal injury. Ann. Surg. 211: 72, 1989. Hansbrough, J. F., and Gadd, M. A. Temporal analysis of murine lymphocyte subpopulations by monoclonal antibodies and dualcolor flow cytometry after burn and nonburn injury. Surgery
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27.
Burleson, D. G., Mason, A. D., Jr., and Pruitt, B. A., Jr. Lymphoid subpopulation changes after thermal injury and thermal injury with infection in an experimental model. Ann. Surg. 207: 208,1988. Organ, B. C., Antonacci, A. C., Chiao, J., Chiao, J., Kumar, A., deRiestha1. H. F.. Yuan. L.. Black. D.. and Calvano. S. E. Changes in lymphocyte number and’ phenotype in seven lymphoid compartments after thermal injury. Ann. Surg. 210: 78,
1989. 28.
29.
Lundy, J., Raaf, J. H., Deakins, S., Wanebo, H. J., Jacobs, D. A., Lee, T.-d., Jacobowitz, D., Spear, C., and Oettgen, H. F. The acute and chronic effects of alcohol on the human immune system. Surg. Gynecol. Obstet. 141: 212, 1975. Jerrells, T. R., Peritt, D., Marietta, C., andEckardt, M. J. Mechanisms of suppression of cellular immunity induced by ethanol. Alcohol. Clin. Exp. Res. 13: 490, 1989.
OF SURGICAL
RESEARCH
61.210-215
(1991)
Immune Suppression after Acute Ethanol Ingestion and Thermal Injury’ MASATO KAWAKAMI, *Department tDepartment
M.D.,* BOYD R. SWITZER, PH.D.,? SANDRA R. HERZOG, B.A., P.A.-C.,* AND ANTHONY A. MEYER, M.D., PH.D.**’
of Surgery and North Carolina Jaycee Burn Center, University of North Carolina School of Medicine, and of Nutrition at University of North Carolina, School of Public Health, Chapel Hill, North Carolina 27599 Submitted for publication June 13, 1990 alcohol ingestion [4]. Despite the frequent association of acute alcohol ingestion and injury, the effects of acute alcohol ingestion on the pathophysiology of trauma patients has not been widely investigated. Several reports suggested that burned patients with ethanol (EtOH) exposure have a greater mortality than those without EtOH exposure [4-6]. Howland and Hingson suggested that the high mortality rate might be due to increased susceptibility to infection induced by EtOH [4]. Like burn injury, acute EtOH ingestion can induce immune suppression by itself. Chronic ingestion of EtOH prior to injury does significantly increase the immune suppression [7]. However, many accident victims do not have a history of chronic, daily alcohol consumption. In such patients a single exposure to EtOH prior to injury may produce greater immune dysfunction than either EtOH alone or injury alone. The purpose of this study was to determine the combined effects of acute EtOH ingestion and bum injury on immune function. We measured immune function after burn injury in rats with a single EtOH ingestion prior to injury. The data analysis examined the effects of both burn injury and acute EtOH ingestion on these parameters of immune function.
Acute alcohol ingestion is commonly associated with burn injury. Both alcohol ingestion and burn injury produce immune suppression, but the combination of these factors on immune function has not been investigated. To study this combined effect, immune function was measured in rats with a 30% burn injury following a single ingestion of 2.4 g/kg of ethanol (EtOH) and compared to that of animals with burn injury only, animals with EtOH only, and animals with neither alcohol nor burn injury. Four days after ethanol and/or burn, animals receiving both ethanol and burn injury had significant suppression of in uivo chemotaxis and lymphocyte responsiveness to lipopolysaccharide (LPS) compared to animals receiving either burn injury alone or EtOH alone (P < 0.05). There was no difference in responsiveness to concanavalin A (Con A). Serum corticosterone was significantly elevated by burn injury but not EtOH ingestion. EtOH treatment prior to injury caused a further increase in corticosterone level that was significantly associated with a decrease in immune function. These results indicate that a single EtOH exposure prior to burn injury produces greater immune suppression than does burn injury alone. This further decrease in immune function may contribute to increased susceptibility to infection and increased mortality in burn patients with acute EtOH ingestion. G IS~I Academic
Press,
Inc.
MATERIALS
Animals
INTRODUCTION
’ Some of this data was presented at the Ninth Annual Meeting of the Surgical Infection Society, April 13-14, 1989, Denver, CO. 2 To whom reprint requests should he addressed at Department of Surgery, University of North Carolina, Campus Box 7210, 164 Burnett-Womack, Chapel Hill, NC 27599-7210. $1.50 1991 by of reproduction
Copyright 0 All
rights
210 Academic Press. Inc. in any
form
reserved.
METHODS
and Materials
Male Sprague-Dawley rats (Charles River Laboratories, Raleigh, N.C.) weighing 300-350 g were used for all experiments. All animals were given food and water ad libitum throughout the experiments. The protocols for procedures, anesthesia, and euthanasia were approved by the Committee on Animal Research, University of North Carolina at Chapel Hill. Tissue culture media and supplements were purchased from the Tissue Culture Facility (Lineberger Cancer Research Center, University of North Carolina, Chapel Hill, NC). All chemicals were purchased from Sigma Chemicals (St. Louis, MO) except where noted otherwise.
Injury is frequently associated with acute alcohol ingestion. Nearly 50% of trauma victims transferred to hospitals were reported to have been drinking alcohol [l]. Alcohol ingestion is also a major risk factor in fire victims [2, 31. A review of several series reported that half of the people who died in fires had evidence of acute
0022.4804/91
AND
KAWAKAMI
Experimental
ET AL.:
IMMUNE
SUPPRESSION
Design
All animals were randomly divided into four groups: 11 animals underwent burn injury at 4 hr after EtOH ingestion (EtOH+Burn), 11 animals received burn injury alone (Burn), 11 animals ingested EtOH alone (EtOH), and 11 animals served as control (Control). An acute alcohol model was produced by gavage feeding of 2.4 g/kg of body weight of EtOH in 20% solution in distilled water. A 30% total body surface area (TBSA) fullthickness burn injury was produced, as described previously [8]. Briefly, animals were anesthetized by methoxyAurane (Pitman-Moore, Washington Crossing, NJ); and then their backs were shaved and exposed to steam at 100°C for 30 sec. Resuscitation after burn injury was performed by injecting 20 ml of lactated Ringer’s intraperitoneally and 3 mg/kg of morphine sulfate was given subcutaneously for analgesia. All measures of immune function were made 4 days after acute EtOH ingestion and/or burn injury. Blood EtOH Concentration To determine the levels of blood EtOH achieved in this model, six rats were given 2.4 g/kg of EtOH by gavage feeding, and blood EtOH level was measured. Blood samples were taken from the amputated tail tip before EtOH ingestion and at 1, 2, 3, 4, 6, 8, and 24 hr after ingestion. Blood EtOH concentration was measured using an enzymatic method (Sigma Diagnostics No. 332UV, Sigma Chemicals). Briefly, a whole blood sample was mixed with 6.25% (w/v) trichloroacetic acid to prepare a protein-free supernatant, and vortexed. After the mixture was centrifuged, the supernatant was stored at -70°C. On the next day, 100 ~1 of the stored supernatant was mixed with 2.9 ml of 0.5 mole/liter glycine buffer containing 0.6 mmole/ml nicotinamide adenine dinucleotide and 50 U/ml alcohol dehydrogenase. After the mixture was allowed to stand at room temperature for 10 min, the absorbance of the mixture was measured at 340 nm using a Beckman DU-6 uv-visibile spectrophotometer (Beckman Instruments, Inc., Fullerton, CA). EtOH concentration of the sample was calculated from the absorbance using a standard curve. In Viva Ctwmotaxis Chemotaxis was assessed by the amount of myeloperoxidase (MPO) from polymorphonuclear neutrophils in the skin following injection of N-formylmethionylleucylphenylalanine (FMLP). After animals were anesthetized with methoxyflurane vapor, the hair on their ventral surface was clipped and 0.2 ml of 10 PM FMLP was injected intradermally in three sites for in uiuo chemotaxis measurement. Four hours after FMLP inoculation, all rats were reanesthetized and celiotomy was performed. The animals were euthanized by exsanguination from the abdominal aorta after the splenic artery and
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vein were clamped. Then, the spleen was removed for a mitogenic assay of splenic lymphocytes. The skin at the sites of FMLP injection was biopsied using a 6-mm disposable biopsy punch (Acuderm, Inc., Ft. Lauderdale, FL) for the MPO assay. The skin samples were stored at -70°C and a MPO assay was done on the next day. The technique for a calorimetric assay measuring MPO extracted from skin has been previously described [8]. Mitogenesis
Assay
The procedures of the preparation of spleen cell suspension and a mitogenesis assay were described previously [7,9]. In brief, spleen cells were teased out aseptically into 10 ml of culture medium, the red blood cells were lysed, and the cell suspension was incubated in a glass petri dish at 37°C in a humidified atmosphere of 5% CO, in air for 2 hr in order to separate the nonadherent cells. Nonspecific esterase staining (Sigma Chemicals) demonstrated that the nonadherent cell suspension contained less than 0.01% monocytes. Cell viability was consistently 98% or better, as assessed by trypan blue exclusion. The nonadherent splenocytes were cultured with 2.5 rg/ml of Con A and LPS. At 24 hr prior to termination of mitogenesis, 0.05 ml of media containing 1 &i of tritiated thymidine (sp act 6.7 Ci/mmole; ICN Radiochemicals, Irvine, CA) was added to each well. Wells were harvested on a glass-fiber filter using a semiautomatic cell harvester (Skatron, Inc., Sterling, VA), and the amount of tritiated thymidine uptake was determined. The average counts per minute for each culture condition in five replicate wells was calculated. Serum Corticosterone
Level
Serum samples were taken from animals at the time of sacrifice and stored at -7O’C. Serum corticosterone concentration was measured using a ‘%I-corticosterone radioimmunoassay kit (Cambridge Medical Technology, Billerica, MA). Briefly, 100 ~1 of serum sample was mixed with 100 ~1 of ‘251-corticosterone and corticosterone antiserum in a glass tube, vortexed, and incubated for 16-24 hr at 4°C. Then, 100 ~1 of goat anti-rabbit y-globulin was added to the tube. It was vortexed, incubated for 1 hr at room temperature, and centrifuged at 1500-1600g for 15 min at 2 to 8°C. After decanting the supernatant from the tube, the radioactivity of the sediment was counted and hormone concentration was determined from log-log& analysis of the data. Statistical
Analysis
The effects of acute EtOH ingestion and burn injury were analyzed by two-way analysis of variance (ANOVA), and the comparison between the mean of each group was computed by Scheffe’s multiple comparison test. The correlation between serum corticosterone level
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VwW
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1
2
3
4
6
TIME AFTER EtOH INGESTION
8
24
(hours)
FIG. 1. The changes in blood EtOH level after ingestion of EtOH at 2.4 g/kg. The data are expressed as means and standard deviation. The value at 0 hr represents the baseline measured prior to EtOH ingestion.
and each immunologic parameter was analyzed by single regression analysis. P values of less than 0.05 were accepted as statistically significant. RESULTS
Blood EtOH Concentration The blood EtOH levels of six animals receiving 2.4 g/kg of body weight of EtOH reached a peak approximately 1 hr after EtOH ingestion, with an average maximum EtOH level of 108.7 + 19.6 mg/dl (mean and standard deviation). The mean and standard deviation for each time point are shown in Fig. 1. EtOH was still present in the blood 6 hr after EtOH ingestion, but was not detectable 8 hr after ingestion.
EtOH
FIG. 2.
MPO activity of each group. MPO activity is represented by absorbance (mean and standard error). Statistical results are as follows: by ANOVA, EtOH P = 0.044; Bum, P -z 0.001; Interaction, P = 0.792; * by multiple comparison test, P < 0.05 vs Control.
activity, compared to the Control group and the EtOH group (P < 0.05). The animals which received EtOH only (EtOH) had slightly decreased MPO activity, but the difference from the Control group was not statistically significant using Scheffe’s test.
Mitogenesis Assay Responsiveness of nonadherent spleen cells to Con A (T-lymphocyte mitogen) or LPS (B-lymphocyte mito-
q q
In Viva Chemotaxis In uiuo chemotaxis of skin biopsies was measured by MPO activity 4 hr following FMLP inoculation. The MPO activity in three skin samples obtained from each animal was averaged and considered as MPO activity of each rat. Both a single EtOH ingestion (P = 0.044) and burn injury (P < 0.001) produced significant suppression in chemotaxis by ANOVA, as shown in Fig. 2. In comparison between each group using Scheffe’s test, the animals which received both EtOH and burn injury (EtOH + Burn) had the greatest decrease in MPO activity compared to the Control group (P < 0.05). The animals which received burn injury alone (Burn) also had decreased MPO activity (P K 0.05), but not as severe as the animals which received both treatments (EtOH + Burn). Both the Burn group and the EtOH + Burn group showed a statistically significant decrease in MPO
0
BURN EtOH+BURN
NO MKOQEN
2.5 pg/ml
FIG. 3. Lymphocyte response to Con A. The value is represented by the mean and standard error. Statistical results are as follows: by ANOVA, EtOH, P = 0.975; Bum, P = 0.019; Interaction, P = 0.135; * by multiple comparison test, P < 0.05 vs Control in the same batch.
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BURN
(CPM) W0W - 0
q 1
q
CONTROL EtOH BURN EtOH+BURN
l
0
0
NO MITOGEN
2.5 pg/ml
FIG. 4. Lymphocyte response to LPS. The value is represented by the mean and standard error. Statistical results are as follows: by ANOVA, EtOH, P = 0.999; Burn, P < 0.001; Interaction, P = 0.005;by multiple comparison test, *P < 0.05 vs Control in the same batch, **P < 0.05 vs the others in the same batch.
gen) or without mitogens was evaluated by tritiated thymidine uptake of proliferating cells. Under all three culture conditions, consistent results were obtained, as shown in Figs. 3 and 4. The animals receiving both EtOH ingestion and burn injury (EtOH + Burn) showed the least response to both Con A and LPS, and even the mean thymidine uptake of the unstimulated cell in this group was less than those of the other groups. The decrease in the response to LPS was more remarkable than that to Con A, and the combined insult of burn injury and EtOH ingestion was statistically significant compared to the other three groups (P < 0.05). Although the thymidine uptake of the animals receiving EtOH ingestion only (EtOH) was increased under each culture condition, only the increased uptake of unstimulated cells from the EtOH group was statistically significant compared to that of the Control group (P
CONTROL
BURN
EtOH
ElOH+BURN
FIG. 5. The value is presented by the mean and standard error. The numbers of each group are Control = 8, EtOH = 6, Burn = 8, and EtOH + Burn = 8. Statistical results by ANOVA are as follows: EtOH, P = 2.699; Burn, P = 0.030; Interaction, P = 0.193.
= 0.030). The animals receiving both EtOH ingestion and burn injury (EtOH + Burn) had the greatest increase in corticosterone level compared to the Control group, but the difference was not significant because of the large standard error. Linear regression analysis of the effect of serum corticosterone level on the immunologic parameters, MPO activity, lymphocyte responsiveness to Con A and LPS, and unstimulated lymphocyte proliferation, found that there was a statistically significant negative correlation between serum corticosterone level and immunologic function. The value of R2 (variance) in each regression analysis was small. The relationship between serum corticosterone levels and immune function is summarized in Table 1. Figure 6 is a graphic representation of the relationship between serum corticosterone and MPO activity and is similar to the other measures of immune function. High serum corticosterone level (more than 12 pg/dl) was seen only in the EtOH + Burn group, and these animals
< 0.05).
The thymidine uptake of the animals receiving burn injury only (Burn) was decreased or similar in each culture condition compared to that of the Control group. There were no statistically significant differences between the Burn and Control groups. Serum Corticosterone
Level
We measured serum corticosterone concentrations in serum from the animals of each group. Mean corticosterone levels are shown in Fig. 5. An influence of EtOH ingestion on serum corticosterone level 4 days prior to sampling could not be detected (P = 2.699). Burn injury significantly increased serum corticosterone level (P
TABLE
1
Results of Regression Analysis between Serum Corticosterone Levels and Immunologic Parameters Parameters MPO Thymidine uptakes with no mitogen Thymidine uptakes with Con A Thymidine uptakes with LPS
Coefficient of slope
R2
-0.011
0.245
0.006
-421
0.130
0.050
-7421
0.171
0.023
-2718
0.225
0.008
Probability
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0.00
J I
r
I
I
4
6
6
10
Serum Corticosterone
\
12
14
16
16
Level (pggldl)
FIG. 6. Scattergram of MPO activity against serum corticosterone level. The variance (I?) is 0.245 and the probability of no correlation is 0.006. The regression line in the chart is Y = -0.011X + 0.182.
showed the most impaired immune function. However, some animals in the EtOH+Burn group showed impaired immune function, even though their corticosterone level was within the normal range. DISCUSSION
Alcohol has been recognized as a predisposing factor to burn injuries [2, 3, lo]. In addition to its role in causing accidents, alcohol exposure prior to injury appears to increase the mortality of injured patients. Some reports have shown a higher mortality in patients whose burn injuries are associated with alcohol than in patients whose injuries are not associated with alcohol [4,5]. Crikerlair et al. pointed out that preexisting disease, including alcoholism, played a prominent role in determining the outcome of patients [5]. Rittenbury et al. documented that chronic alcohol abuse associated with liver disease significantly increased mortality from burn injury [6]. Immune suppression by a single, acute ingestion of EtOH has also been reported [9, 111. Burn injury has been found to impair many components of the immune system [12-181. Immune suppression by major burn injury can contribute to development of fatal infectious complications. Previous studies in this laboratory have noted that chronic EtOH exposure prior to burn injury produced greater immune suppression than either the burn injury or EtOH exposure alone. It is therefore quite possible that a single, acute EtOH exposure could produce further immune dysfunction than does either alcohol ingestion or burn injury alone. The experiments reported here supports this hypothesis. In this study, neutrophil chemotaxis was further suppressed by a single dose of EtOH prior to burn injury. Neutrophils work by bacterial phagocytosis and intracellular killing, which are important in nonspecific host
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defense. Malfunction of neutrophils can lead to increased bacterial infection. Alexander and Wixson reported evidence that neutrophil dysfunction was a major determinant in the development of life-threatening sepsis after thermal injury [19]. Responsiveness of lymphocytes to mitogens is commonly used for the evaluation of cellular immunity. Significant correlation between mortality after a septic challenge and suppression of mitogenic response in burned animals has been reported [20]. Con A was used as the T-cell mitogen in these experiments because preliminary studies found that it produced a higher, more consistent response than phytohemagglutinin. LPS was used as the B-lymphocyte mitogen in this study because LPS can stimulate B-lymphocytes to proliferate specifically, while pokeweed mitogen stimulates both T- and B-lymphocytes. The present study showed that a 30% burn injury produced suppression in the response to Con A and LPS. In other studies, burn injury produced a significantly decreased response to Con A [13,21-231. Generally T-lymphocyte function is more influenced by burn injury and alcohol than B-lymphocyte function [ll, 121. The more pronounced effect on B-lymphocyte function in the present study may be caused by an alteration of absolute and relative numbers of lymphocyte subpopulations in the spleen induced by burn injury, EtOH, and their combination. However, according to several reports, burn injury seems to influence the T-lymphocyte percentage in the spleen, but not the B-lymphocyte percentage [24-271. With respect to alcohol, Lundy et al. reported that the proportion of T-lymphocytes was decreased and that of B-lymphocytes was unchanged in his study of peripheral blood of human alcoholics [28]. The subjects used in this study, however, had a history of long-term and excessive drinking, and the effect of acute EtOH ingestion is unknown. Reports of lymphocyte subpopulation in the spleen after acute EtOH ingestion are not available at this time. The effect of the combination of burn injury and acute EtOH ingestion on the lymphocyte subpopulation in the spleen is also unknown, but further study is planned to investigate it. Several circulating humoral substrates are recognized as factors which mediate the immune suppression after burn injury and EtOH ingestion [12,29]. Corticosteroids are known as strong immune-suppressive agents. The increase of circulating corticosteroids under severe stress like burn injury is well described. It is suggested by Jerrells et al. that corticosteroids can at least partially mediate the immune suppression induced by EtOH [29]. A 30% burn injury significantly increased the corticosterone level in this study. Furthermore, the acute EtOH ingestion prior to burn injury produced a greater increase in serum corticosterone level than burn injury alone. Although a statistical analysis failed to confirm the significant effect of acute EtOH ingestion on cortico-
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sterone level, this may be due to the small sample size of each group. All the animals with high corticosterone levels of more than 12 pg/dl were apparently associated with suppression in the immunologic parameters studied. Interestingly, some animals in the Burn and EtOH+Burn group had impaired immune function, although they had relatively normal corticosterone levels. This finding may indicate that the increase in corticosterone level contributed to the immune suppression, but it was not the sole factor. Biologic systems of this complexity are unlikely to identify a single factor, such as corticosterone, responsible for highly variable biologic response. The present study demonstrates that the combination of acute EtOH ingestion and burn injury produces greater suppression of in uiuo chemotaxis and lymphocyte mitogenic response than either burn injury or acute EtOH ingestion do independently. The clinical implications are that the further immune suppression from a single exposure prior to injury may contribute to increased susceptibility to infection and increased mortality in trauma patients. Although the mechanisms are not clarified in this study, this suppression of several immune mechanisms could cause increased susceptibility to infection and increased mortality in burn patients with a single alcohol exposure at the time of injury.
10. 11. 12. 13.
14.
15. 16.
17.
18. 19. 20.
21. 22.
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