EXPERIMENTAL HEATSTROKE IN BABOON: ANALYSIS OF THE SYSTEMIC INFLAMMATORY RESPONSE : Shock

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EXPERIMENTAL HEATSTROKE IN BABOON: ANALYSIS OF THE SYSTEMIC INFLAMMATORY RESPONSE

Bouchama, Abderrezak*; Mohanna, Falah Al*; El-Sayed, Raafat*; Eldali, Abdelmoneim; Saussereau, Elodie; Chollet-Martin, Sylvie; Ollivier, Véronique; de Prost, Dominique‡,¶; Roberts, George§

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Shock 24(4):p 332-335, October 2005. | DOI: 10.1097/01.shk.0000180620.44435.9c
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Abstract

INTRODUCTION

Classic heatstroke is a lethal condition characterized by a rapid increase in core temperature to more than 40°C and widespread tissue injury in which encephalopathy predominates after exposure to high ambient temperature (1). Prompt recognition of this condition and initiation of cooling dramatically improves survival (1). Nonetheless, despite cooling and normalization of the core temperature, up to one-third of patients who survive the initial deleterious effect of hyperthermia can progress to multiorgan dysfunction syndrome, culminating in death (1-3). Mortality rate among these patients requiring intensive care ranges from 21 to 67% (2, 3). Moreover, an additional 29% mortality at 1 year following discharge from hospital, and attributed to the neurological damage sustained by heatstroke survivors, has been reported (2).

Findings in humans and experimental animals with heatstroke suggest that excessive activation of inflammation may contribute to the multiorgan dysfunction syndrome and death (4-11). Concentrations of circulating pro- and anti-inflammatory cytokines are significantly increased in heatstroke patients and experimental animal models in correlation with severity and outcome (4-12). Therapeutic intervention with steroidal anti-inflammatory drugs or IL-1 receptor antagonists (IL-1ra) protects rats and rabbits against lethal heatstroke, suggesting that immunomodulation of the host responses may be beneficial (9-11). However, studies of the inflammatory response to heatstroke in these small laboratory animals were limited to the inflammatory pathways, namely IL-1β and TNFα (9-11). Moreover, extrapolation of data from small animal models to humans cannot predict reliably the human response because of interspecies difference.

We have recently established an experimental baboon model of classic heatstroke that replicates the cellular injury, organ failure, and death seen in human heatstroke (12). Moreover, we found that IL-6 was dramatically increased and associated with outcome (12). Therefore, using the plasma collected from these animals, we comprehensively analyzed the nature and time course of the inflammatory response to heatstroke with a view to identifying potential targets for therapeutic interventions. The baboon, the closest species to humans, was used so that therapeutic perspectives could be evaluated as a basis for human trials.

MATERIALS AND METHODS

Animals

The present investigation was part of a project aimed at characterizing the hemostatic, inflammatory, and clinical changes in a baboon model of heatstroke with a view to developing novel therapeutic strategies. The first set of results of this work has been published elsewhere (12). Using a protocol reviewed and approved by the Basic Research Committee and the Animal Care and Use Committee of King Faisal Specialist Hospital and Research Center, 12 healthy juvenile baboons (Papio hamadryas, 4-5 kg) handled in accordance with the American Physiological Society guiding principles in the care and use of animals, were fasted overnight with water ad libitum and then immobilized with ketamine chloride (15 mg/kg) on the morning of the investigation as described previously (12). Briefly, the animals were intubated and allowed to respire spontaneously in room air. Venous and arterial catheters were placed aseptically via femoral cutdown for administration of drugs and fluids, continuous monitoring of arterial blood pressure, and blood sampling. General anesthesia was maintained with ketamine infusion and diazepam with a concomitant infusion of dextrose normal saline at 5 mL/kg/h to prevent dehydration and hypoglycemia.

Methods

Induction of moderate and severe heatstroke-

The animals were assigned randomly to a study (moderate or severe heatstroke) or control group. Both study groups were subjected to environmental heat stress in a prewarmed neonatal incubator (Isolette Infant Incubator; Air-shield, Hatboro, PA) maintained at 44°C to 47°C and a relative humidity of 33% to 39% until core temperature reached 42.5°C (moderate heatstroke) or until systolic blood pressure fell to <90 mmHg, which was taken as the onset of severe heatstroke. The latter occurred at a core temperature of 43.3°C ± 0.1°C. Sham-heated baboons serving as a control group were handled in an identical manner including anesthesia, instrumentation, and duration inside the incubator but were not subjected to heat stress.

After heat stress, the study animals were removed from the incubator and allowed to cool passively at an ambient temperature of 26°C to 29°C. None of the animals survived severe heatstroke. All of the animals with moderate heatstroke survived. None had evidence of bleeding, but three of four animals had neurologic changes manifest as lethargy and limb weakness. The neurologic changes subsided at 72 h.

Blood sampling-

Blood samples were collected in EDTA-treated tubes. The samples were collected before heat exposure or baseline, at the end of heat exposure (T + 0), and at 1 (T + 1), 2 (T + 2), 3 (T + 3), 12 (T + 12), and 36 (T + 36) h.

On collection, the blood samples were immediately centrifuged at 4°C for 20 min at 1600 g. Plasma samples were stored at -80°C until assayed.

Exploration of inflammatory and regulatory mediators-

Plasma cytokines, chemokine, and soluble cytokine receptors were assayed using specific enzyme-linked immunoabsorbant assays (Quantikine; R&D Systems, Minneapolis, MN) according to the instructions of the manufacturer. The cytokine profile chosen was as follows: proinflammatory cytokines TNFα and IL-1β (detection limit of 5 and 1 pg/mL, respectively), regulatory and anti-inflammatory cytokines IL-12p40, IL-4, IL-10, and IL-1ra, soluble TNF receptors (sTNFrI and II; detection limit of 15, 5, 4, 22, 3, and 1 pg/mL, respectively), and chemokine IL-8 (detection limit of 10 pg/mL). The intra- and interassay precisions were tested and evidenced coefficients of variability between 2% and 6% depending upon the mediators.

Statistical analysis

Values are expressed as mean ± SEM. Comparison between values at baseline and at the end of heat exposure for each group was performed by Student's t test and the nonparametric Kruskal-Wallis test. Comparison between groups during the observation period was performed using repeated-measures analysis of variance (ANOVA). ANOVA was used to determine significance of difference in means between groups at given times. Spearman's rank test was used to calculate probability levels for correlation. Differences were considered significant at P < 0.05.

RESULTS

Rectal temperature at baseline and during cooling

The baseline core temperature was similar between the three groups (control, moderate, and severe; Table 1).

T1-6
Table 1:
Core temperature in baboons subjected to moderate and severe heatstroke compared with sham-heated controls*

Release of cytokines and cytokine receptors in moderate and severe heatstroke

The baseline values before heat exposure or baseline for TNFα, IL-1β, and IL-4 were below the limit of detection in all 12 animals. The baseline levels for other cytokines were similar between the three groups (control, moderate, and severe, respectively; P > 0.05) were: IL-8, 281 ± 46 pg/mL, 285 ± 70 pg/mL, and 250 ± 30 pg/mL; IL-10, 9 ± 2 pg/mL, 8 ± 1 pg/mL, and 6 ± 1 pg/mL); IL1ra, 445 ± 166 pg/mL, 565 ± 195 pg/mL, and 628 ± 318 pg/mL; sTNFrI, 433 ± 47 pg/mL, 346 ± 27 pg/mL, and 435 ± 30 pg/mL; sTNFrII, 304 ± 42 pg/mL, 341 ± 38 pg/mL, and 314 ± 61 pg/mL; and IL-12p40, 159 ± 19 pg/mL, 223 ± 28 pg/mL, and 232 ± 35 pg/mL. These baseline levels remained unchanged in the control group throughout the experiment (Fig. 1, A and B). TNFα, IL-1β, and IL-4 were not detected in the circulation in any group during the entire study period.

F1-6
FIG. 1:
(A) Inflammatory response patterns in control and heat-stressed study groups at induction of heatstroke. Values represent mean ± SEM in plasma concentrations of IL-10, IL-8, IL-1ra, IL-12p40, and sTNFrI and II at baseline and at the end of heat exposure or onset of heatstroke (T + 0 h). Statistical comparisons were made using Student's t test and Kruskal-Wallis test for skewed variables. An asterisk indicates that the difference between groups was significant (P < 0.05). A double asterisk indicates that the difference between groups was significant (P < 0.01). (B) Time course of the inflammatory response in control and heat-stressed study groups. Values represent mean ± SEM in plasma concentrations of IL-10, IL-8, IL-1ra, IL-12p40, and sTNFrI and II at baseline (B), at the onset of heatstroke (T + 0 h), during passive cooling (T + 1, T + 2, and T + 3 h), and at recovery (T + 12 and T + 36 h). The difference between the three groups was significant by repeated-measures ANOVA. A significant difference in plasma IL-10, IL-8, sTNFrI and II (P < 0.01) was evident, but not for IL12p40 and IL-1ra, when severe heatstroke was compared with moderate heatstroke. A significant difference in plasma IL-10, IL12p40, IL-1ra and sTNFrI and II was evident, but not for IL-8, when moderate heatstroke is compared with control.

At the onset of severe heatstroke (T + 0 h), circulating levels of anti-inflammatory cytokine (IL-10), cytokine inhibitors (IL-1ra and sTNFrI and II), and chemokine (IL-8) were significantly increased and regulatory cytokine IL-12p40 levels were significantly decreased from their baseline levels (Fig. 1A). A similar pattern was observed in animals with moderate heatstroke, except that statistical significance was not reached for sTNFr I and IL-12p40 (Fig. 1A). The intensity of the cytokine responses (IL-10, IL-1ra, and sTNFrII) at induction of heatstroke was significantly higher when severe is compared with moderate heatstroke.

Time course of cytokines during passive cooling and recovery

Figure 1B shows that circulating levels of IL-8, IL-10, IL-1ra, and sTNFrI and II increased markedly in animals with severe heatstroke during the cooling period (T +1, T + 2, and T + 3 h). Plasma IL-10 and sTNFrI levels peaked at T + 1 h, and IL-8, IL-1rA, and sTNFrII peaked at T + 3 h. There was a significant correlation between plasma IL-8 and IL-1ra concentrations (r = 0.734; P < 0.0001), with both curves appearing strictly parallel until T + 2 h (Fig. 1B).

In contrast, animals with moderate heatstroke exhibited a mild to moderate variation of circulating cytokines from their peak observed at onset of heatstroke (T + 0 h; Fig. 1B). As a result, the magnitude of the cytokine response, including, IL-8, IL-10, and sTNFr I and II and except for IL-1ra and IL-12p40, was significantly different in animals with severe compared with moderate heatstroke (P < 0.01). The magnitude of the cytokine levels coincided with the outcome, namely, intense inflammatory response was associated with mortality and lower intensity with survival. In survivors, the inflammatory response remained prolonged during the recovery period as IL-1ra and sTNFrI were still detectable at T + 12, and IL-1ra up to T + 36 h.

DISCUSSION

This study was designed to describe the cascade of cytokines together with their physiological regulators from their early appearance in a nonhuman primate model of moderate and severe heatstroke. The present data confirm our initial observation concerning IL-6 and allows us to go further into the mechanisms of heatstroke-induced inflammation and regulation (12).

This study reveals that heatstroke activates a complex inflammatory response characterized by an early and simultaneous release of anti-inflammatory cytokine (IL-10), soluble cytokine receptors (IL-1ra and sTNFrI and II), and chemokine (IL-8), and a decrease in regulatory cytokine (IL-12p40). During passive cooling, the circulating cytokines fluctuated moderately from their levels attained at induction of moderate heatstroke, but exhibited an uninterrupted rise, culminating with the demise of the animals in severe heatstroke. The findings in the present study support the hypothesis that in human heatstroke, a dysregulated inflammatory response might contribute to tissue injury and death. However, they do not concur with previous work where TNFα and IL-1β were suggested to be important mediators of tissue injury and death in small animal models of heatstroke (9-11).

Previous studies in humans with classic heatstroke have documented an increase in levels of circulating IL-6, IL-10, and sTNFrI and II, associated with outcome, whereas TNFα and IL-1β were detected inconsistently (4-8). In these studies, only few cytokines were examined, each time at differing severity of disease and/or time course (4-8). The present study confirms these findings and extends them by unraveling their dynamics and identifying IL-8, IL-1ra, and IL-12p40, but not IL-4, as part of the network of cytokines at play in classic heatstroke. TNFα and IL-1β were not detected in this study, which concurs with some but differs from other clinical studies (4, 6, 7). It might be that TNFα and IL-1β were not detected because their release is early and transitory and our sampling started only at the very high core temperature of 42.5°C (13-15). This agrees with the observation that patients undergoing whole-body hyperthermia as an adjuvant therapy against cancer show that TNFα and IL-1β are induced at temperatures as low as 41.8°C, and that TNFα was detectable in only a few patients (15).

Another finding in this study is the decreased circulating levels of IL-12p40 in heatstroke. IL-12p40 together with IL-12p70 isoforms compose IL-12, a critical cytokine that promotes the differentiation of T helper (Th) to Th-1 cells, which release proinflammatory cytokines such as interferon-γ or IL-2, in contrast to Th-2 cells, which produce anti-inflammatory cytokines such as IL-10 or IL-4 (13, 16-18). The IL-12p40 isoform behaves as the natural inhibitor of IL-12p70 and thus regulates inflammation (17). Decreased productions of IL-12p40 have been described during severe sepsis, trauma, and thermal injury, and have been suggested to signal a shift in the balance Th2/Th1 in favor of Th2 (13, 16-18). In this study, the decreased circulating levels of IL-12 p40 were associated with increased levels of IL-10 without any detectable level of IL-4, making the identification of the T helper phenotype in heatstroke difficult.

It is not known what causes the exacerbation of the inflammatory response in animals with severe compared with moderate heatstroke. Our data suggest that it is independent of the core temperature once heatstroke is established and cooling is commenced because the changes of circulating cytokines remained steady or continued to rise in moderate and severe heatstroke, respectively, even though the core temperature was subsiding to normal. A possible explanation is heat-induced tissue damage, particularly of the endothelium, which is commonly observed in experimental and human heatstroke (12, 19-21). Injured/activated endothelium can release a variety of mediators, including pro- and anti-inflammatory mediators, and thus may have contributed to the exaggeration of the host responses (12, 20, 21). Alternatively, endotoxin, a major cell wall component of gram-negative bacteria, has been shown to enter the circulation in monkeys subjected to heatstroke and reaches a maximum concentration at a temperature above 43°C (22, 23). Endotoxin is a powerful inducer of the synthesis of proinflammatory cytokines and thus could have fueled the inflammatory response in the animals with severe heatstroke. Indeed, in an in vitro model, DuBose et al. (24) provided evidence that endotoxin was required to provoke cytokine mRNA expression and production by human whole blood cells exposed to heat (24).

In conclusion, this study shows that heatstroke activates complex, systemic, inflammatory, and regulatory responses associated with outcome. Further studies are needed to better define the inflammatory response before successful modulation of cytokines as an adjunct to cooling in the treatment of life-threatening heatstroke may become conceivable.

ACKNOWLEDGMENTS

The authors thank Catalino Santos, Ludivina Apilado, Sahar Salem, Julius Mabborang, Crisologo Caliao, Heidi Davis, and Yvonne Lock; King Faisal Specialist Hospital and Research Center (Riyadh, Saudi Arabia) for their technical assistance.

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Keywords:

Heat stress; hyperthermia; chemokine; cytokine; baboons

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