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Division of Nephrology, Department of Internal Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, Republic of China
Division of Nephrology, Department of Internal Medicine, Cardinal Tien Hospital, Taipei, Taiwan, Republic of China
Division of Nephrology, Department of Internal Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, Republic of China
Division of Nephrology, Department of Internal Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, Republic of China
Division of Nephrology, Department of Internal Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, Republic of China
Division of Nephrology, Department of Internal Medicine, Tri-Service General Hospital, No 325, Section 2, Cheng-Kung Rd., Neihu 114, Taipei, Taiwan, ROC
Division of Nephrology, Department of Internal Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, Republic of China
Increased susceptibility to infections has been shown in patients with classic heat stroke. Although immunologic and inflammatory responses may be important factors, the direct role of circulating neutrophil phagocytosis and lymphocyte adhesion molecule expression has yet to be investigated in exertional heatstroke (ExHS).
Design
Circulating neutrophil phagocytosis and lymphocyte adhesion molecule CD11a and CD11b expression were examined in 17 patients with ExHS and 17 exertional control subjects (ExC).
Results
Patients with ExHS showed significantly increased total leukocyte, neutrophil, and lymphocyte counts, attenuated neutrophil phagocytosis ability, and higher expression of CD11a and CD11b in the acute phase of ExHS, compared with the recovery phase of ExHS and ExC. Although there were no correlations between body temperature and phagocyte function or adhesion molecules, a negative correlation between phagocytosis and CD11a/CD11b was present.
Conclusion
Increased leukocyte count with decreased circulating neutrophil phagocytic capacity and increased expression of lymphocyte adhesion molecules may in part explain the susceptibility to infections in ExHS.
Exertional heat stroke (ExHS) is defined as a condition in which body temperature is elevated to levels that cause damage to the body's tissues and gives rise to a characteristic clinical and pathological syndrome of multiorgan dysfunction.
hepatic failure, and severe neurologic sequelae. The severity of the illness depends on the degree of hyperthermia and its duration—a function of the temperature-duration area above a critical temperature.
Subjects are often healthy young adults with no apparent clinical or biological deficits.
The immune response to exercise has received heightened interest. Several studies have demonstrated that regular moderate physical activity may lead to lowered susceptibility to viral and bacterial infections.
Prolonged physical activity outdoors is a common story elicited from patients with ExHS. Increased suspectibility to infections, endotoxemia, and evidence of endothelial cell activation and/or injury have been reported in classic heat stroke,
However, there have been no studies in the literature about circulating neutrophil phagocytosis capacity in ExHS.
Herein, heat stroke also resembles sepsis in many aspects, and there is growing evidence that endotoxemia and cytokine production may be implicated in its pathogenesis.
Whether lymphocyte adhesion molecules are activated in ExHS and whether this can be attributed to lymphokine and monokine production as well as impaired phagocytosis ability has not been studied. The purpose of this study was to examine circulating neutrophil phagocytosis and lymphocyte adhesion molecules, such as CD11a and CD11b expression, in patients with ExHS.
Subjects and Methods
All procedures and protocols involving human subjects complied with the principles of the Helsinki Declaration and were approved by the Human Ethics Committee of the National Defense Medical Center. Informed consent for participation was obtained from each patient with ExHS upon recovery of consciousness. Criteria for the diagnosis of ExHS included a rectal temperature >40°C and presence of severe neurologic abnormalities (delirium, convulsion, or coma). From December 2000 to November 2002, we encountered 17 recruits with ExHS after heavy physical training including 5000 meters of running, push-ups, and sit-ups. The duration of exertion was from 4 to 10 hours. Their age ranged between 21 and 23 years. All patients received active cooling with cold, wet towels applied to the forehead, hand, neck, axilla, trunk, and extremities immediately on arrival to the hospital. Normal saline (1000 mL in 30 minutes) was administered in an attempt to raise the urine flow rate to at least 1 mL/min. The acute phase was defined as the period during which patients arrived and cooling commenced; the recovery phase was the time during which clinical manifestations and biochemical data returned toward normal. Another 17 age-matched healthy subjects who had gone through the same physical training in the hot environment on the same day without developing ExHS were selected as exertional controls (ExC).
Blood samples were drawn into sterile sodium heparin vacutainers at acute and recovery phases. Blood samples were obtained for biochemical data, including serum creatinine, serum urea nitrogen, aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatine phosphokinase (CPK), sodium (Na+), potassium (K+), and bicarbonate (HCO3−) (AV 5000 chemistry analyzer; Olympus, Tokyo, Japan). In addition, neutrophil phagocytosis and lymphocyte adhesion molecule determinations were performed. Another 3 mL of blood was collected in Vacutainers (BD Biosciences, San Jose, CA) containing sodium EDTA and analyzed on an automated cell analysis system (Gen System; Beckman Coulter, Fullerton, CA) Measurements included quantification of total leukocytes, neutrophils, and lymphocytes.
Neutrophil Phagocytosis Assay
The phagocytosis of Escherichia coli K12 bacteria by human neutrophils was investigated using the commercially available Phagotest from Orpegen-Pharma (Heidelberg, Germany). The test was performed as described previously,
using blood from the subjects and nonopsonized, fluorescein isothiocyanate-labeled E coli. Heparinized whole blood was centrifuged to separate plasma from cells, and the latter were subsequently washed 3 times with phosphate-buffered saline at pH 7.4. After addition of the washed, unfractionated blood cells and the labeled E coli, the reaction mixture was incubated for 10 minutes at 37°C. The ratio of E coli to white blood cells was 25:1. Internalization of E coli by neutrophils was visualized by FACscan (BD Biosciences Immunocytometry Systems, San Jose, CA), and analysis of these measurements performed with the CELL Quest software version 3.0.1. Phagocytosis ability is expressed as mean channel fluorescence (MCF) intensity.
Adhesion Molecules
Adhesion molecules CD11a and CD11b expressed on lymphocytes were analyzed by FACscan with monoclonal antibodies.
Lymphocytes bearing CD11a or CD11b were identified using antihuman leukocyte function associated antigen (LFA)-1 and antiMac-1, respectively (BD Biosciences). CD11a and CD11b of ExHS are expressed as percentage of mean fluorescence intensity compared with those of ExC.
Statistical Analysis
Data are presented as mean ± SD. The differences in biochemical data, leukocyte count with differential, neutrophil phagocytic capacity, and lymphocyte adhesion molecule expression among groups of acute and recovery phases of ExHS and ExC were analyzed by one-way analysis of variance test. Correlation analysis was made by linear regression. A p value less than 0.05 was considered statistically significant.
Results
Clinical and Biochemical Data
In the acute phase, ExHS patients showed higher body temperature, heart rate, respiratory rate, lower Glasgow coma scores; and urinary output; higher serum creatinine, AST, ALT, CPK, and lower plasma HCO3− levels compared with recovery and those in the ExC, as shown in Table 1. All of these abnormal data returned to normal in the recovery phase. There were no differences in hematocrit and serum potassium levels among the 3 groups.
Table 1Clinical and Biochemical Data in 17 Patients with ExHS and 17 ExC
As shown in Table 2, total leukocyte counts increased significantly in the acute phase of ExHS compared with the recovery phase and ExC (19.6 ± 3.6 versus 8.2 ± 1.3 × 109L, P < 0.01; 19.6 ± 3.6 versus 8.5 ± 1.7 × 109/L, P < 0.01, respectively). The heat stress-induced leukocytosis included increases in neutrophils (15.7 ± 2.5 versus 5.7 ± 1.8 × 109/L, P < 0.01; 15.7 ± 2.5 versus 5.2 ± 1.0 × 109/L, P < 0.01, respectively) and lymphocytes (3.3 ± 0.3 versus 2.1 ± 0.2 × 109/L, P < 0.05; 3.3 ± 0.3 versus 2.5 ± 0.3 × 109/L, P < 0.05, respectively).
Table 2Leukocyte, Neutrophil, and Lymphocyte Counts and Lymphocyte Adhesion Molecule (CD11a, CD11b) Expression in 17 Patients with ExHS and 17 ExC
The neutrophils' phagocytosis function in the 17 ExHS and 17 ExC patients is shown in Figure 1. Phagocytic ability as expressed by MCF of fluorescein isothiocyanate E coli is significantly attenuated in the acute phase compared with the recovery phase and ExC (103 ± 8 versus 130 ± 13 MCF, P < 0.05; 103 ± 8 versus 150 ± 12 MCF, P < 0.01, respectively). This impaired phagocytosis capacity never fully normalized during the recovery phase of ExHS.
Figure 1Comparison of phagocytosis ability by circulating neutrophils among acute phase, recovery phase, and healthy control subjects. **, P < 0.01, acute phase versus ExC; *, P < 0.05, recovery phase versus ExC.
Lymphocyte adhesion molecules CD11a and CD11b expression in response to heat-exertion stress was shown in Table 2. Comparison of the lymphocyte adhesion molecule expression revealed that the acute phase of ExHS was associated with significantly higher levels of CD11a and CD11b compared with recovery phase of ExHS recovery and ExC (CD11a 176 ± 27 versus 127 ± 18% fluorescence, P < 0.05; 176 ± 27 versus 100% fluorescence, P < 0.05 and CD11b 278 ± 34 versus 142 ± 21% fluorescence, P < 0.01; 278 ± 34 versus 100% fluorescence, P < 0.01; respectively).
Correlation
There is no correlation between phagocytosis ability and either body temperature or duration of exertion (data not shown). There are significant negative correlations between either phagocytosis ability and CD11a (r = −0.662, P < 0.001) or phagocytosis ability and CD11b (r = -0533, P < 0.001) (Figure 2, Figure 3).
Figure 2Correlation between phagocytosis ability and CD11a in 17 patients with ExHS.
little attention has been paid to the potentially causative roles of changes in circulating neutrophil phagocytic ability and lymphocyte adhesion molecule expression in exertional heat stroke. Our results have demonstrated decreased circulating neutrophil phagocytic capacity and increased expression of lymphocyte adhesion molecule in ExHS patients.
Intense exercise induces a profound leukocytosis that includes an increase in the number of polymorphonuclear cells (PMN). PMN counts are increased during and immediately after exercise of various degrees of intensity and duration, most probably as a consequence of demargination, mediated by altered hemodynamics and catecholamines.
A second delayed neutrophilia occurs several hours after exercise as a result of mobilization from bone marrow in response to elevated cortisol levels or humoral signals.
Our data revealed obvious leukocytosis in the acute phase of ExHS, which resolved during the recovery phase, suggesting the underlying role of catecholamines, similar to previously reported results.
Neutrophils play an important role in the nonspecific killing of infectious agents, especially bacteria. Neutrophil uptake of pathogens by phagocytosis triggers a series of non–oxygen-dependent and oxygen-dependent processes that damages and ultimately destroys the microbe within the confines of the cell.
Neutrophil responses to a single episode of exercise are, in general, intensity-dependent. Although exercise at low to moderate intensity enhances some aspects of PMN function, maximal exercise is generally suppressive as shown in our ExHS patients.
However, there is little change in either chemiluminescence or superoxide production per cell in blood sampled when the core temperature has been increased to 39.5°C.
Our study showed suppressed neutrophil phagocytosis ability in the acute phase of ExHS, which may be attributed to both rigor of exertion and body temperature. This may mediate the higher susceptibility to infections in ExHS. However, data from our patients with a high degree of heat injury after strenuous exercise did not produce a direct correlation between phagocytosis ability and body temperature or duration of exertion. Body temperature and duration of exertion, although acting in concert, are not the only important factors influencing the phagocytosis. Other factors such as stress, catecholamine release, endotoxemia, endothelial dysfunction, activated cytokine/chemokine production, and altered adhesion molecule expression present in ExHS
may also take part in the impaired phagocytosis. The presence of these factors may account for the lack of significant correlation between phagocytosis ability and body temperature or duration of exertion in this study. Nevertheless, there is good correlation between phagocytosis and lymphocyte adhesion molecules, suggesting common intermediary cytokines that activate lymphocyte adhesion molecules and decrease phagocytosis activity.
The integrins are a set of cell surface adhesion molecules that regulate cell-cell and cell-extracellular matrix interactions. As such, they play a very important role in a wide array of biologic processes, including organogenesis, tissue remodeling, thrombosis, and leukocyte migration.
The integrins involved in the leukocyte-endothelial cell adhesion are α4β2 (very late antigen-4, or CD49d/CD29), αLβ2 (lymphocyte functional antigen-1, or CD11a/CD18), αmβ2 (Mac-1, CD11b/CD18), and αxβ2 (CD11c/CD18).
Stimulation of leukocytes by several chemotactic factors can activate conformational changes in the adhesion molecules, exposing their ligand-binding sites, leading to an integrin-mediated firm adhesion process, and extravascular migration of leukocytes.
The adhesion molecules expressed on the cell surface then mediate several lymphocyte functions, such as T cell-mediated killing, T-helper functions, T cell proliferative response, and B cell activation.
It could be the case that activation of lymphocyte adhesion molecules CD11a and CD11b results in secretion of pro-inflammatory molecules, such as Th1 cytokines (interferon γ, interleukin-2), monokines (interleukin-1, tumor necrosis factor α, interleukin-6), and chemokines (monocyte chemoattractant protein-1). Th1 cytokine has been proven to activate monokines and Th2 cytokine production in classic heatstroke models. In addition, it has been reported that monokines can trigger further lymphocyte proliferation.
demonstrated positive correlations between simplified physiology scores and interleukin-6, indicating that cytokine activation is associated with disease severity in ExHS. Thus, augmented lymphocyte adhesion molecule expression in ExHS might lead to cytokine/chemokine production that aggravates tissue injury and attenuates neutrophil function, as seen by the negative correlation between adhesion molecules and phagocytosis ability. The mechanism that lymphocyte adhesion molecule activation attenuates phagocytosis ability is unclear and merits further investigation.
In conclusion, we have demonstrated profound leukocytosis, decreased circulating neutrophil phagocytic function, and increased expression of lymphocyte adhesion molecules CD11a and CD11b in ExHS. Such observations may provide a therapeutic strategy for the future treatment of ExHS. Further study is required to understand mechanistically how ExHS affects immune function.
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