Distribution of peripheral in acute heatstroke

blood leukocytes

ABDERREZAK BOUCHAMA, KHALED AL HUSSEIN, ESSAM AL SHAIL, AND SULTAN AL SEDAIRY

CHAKER

ADRA,

MOHAMED

REZEIG,

Departments of Medicine and Biological and Medical Research, King Faisal Specialist Hospital and Research Centre, Riyadh 11211, Saudi Arabia BOUCEIAMA, ABDERREZAK, KHALED AL HUSSEIN, CHAKER ADRA,MOHAMEDREZEIG, ESSAM AL SHAIL,AND SULTAN AL SEDAIRY. Distribution of peripheral blood leukocytes in acute heatstroke. J. Appl. Physiol. 73(2): 405-409, 1992.-We exam-

ined 11 heatstroke patients (mean rectal temperature 41.4 t 0.3”C) and 40 healthy subjectsto determine the effects of hyperthermia on peripheral blood leukocyte distribution. Precooling sampleswere taken on admission.Whole blood was incubated with conjugated monoclonal antibodies, and erythrocytes were eliminated by FACS lysing solution. Lymphocyte subsetswere detectedby specific mousemonoclonal antibodies: Leu-4/CD3+ (T-cells), Leu-3a/CD4+ (T-helper cells), Leu-2a/CDS+ (T-suppressor-cytotoxic cells), Leu-11/19/CD16+/CD56+ (natural killer cells), and Leu-12/CD19+ (B-cells). Immunofluorescence wasmeasuredwith a flow cytometer. The number of circulating leukocytes and lymphocytes was significantly increased in heatstroke patients. This lymphocytosis was mainly due to an increasein T-suppressor-cytotoxic cellsand natural killer cells. The absolute number of lymphocytes and T-suppressor-cytotoxic cellssignificantly correlated with the degreeof hyperthermia (r = 0.62, P = 0.04; r = 0.751, P = 0.007, respectively). There was a significant decreasein the percentagesof T-, B-, and T-helper cells and increasein T-suppressor-cytotoxic and natural killer cells, giving a marked decreasein the ratio of T-helper to T-suppressor-cytotoxic cells. We conclude that heatstroke is associatedwith leukocytosis and significant alteration in absolute number and percentage of circulating lymphocyte subpopulations. lymphocytes; hyperthermia; flow cytometry

to a high ambient temperature may result in heatstroke, a disorder characterized by hyperthermia and neurological abnormalities such as delirium, convulsions, or coma (7). Heatstroke has also been associated with increased susceptibility to infection (13, 19,20,26). The incidence of pulmonary infection has been reported as 2590% (19, 20), and systemic fungal infection has been described in otherwise healthy patients (19, 26). Furthermore, there is growing evidence that the morbidity and mortality observed in heatstroke are related to endotoxemia (5,12). Consequently, we hypothesized that heatstroke could affect the immune system. The present study was designed to determine the peripheral blood leukocyte distribution in heatstroke and whether leukocyte subsets are numerically altered in heatstroke patients compared with normal healthy controls. EXPOSURE

METHODS

Heatstroke patients. This study was carried out at the Heatstroke Center of King Faisal Hospital, Makkah, during the pilgrimage in July 1990. Adult patients with a diagnosis of heatstroke were enrolled in the study. Criteria for inclusion were a rectal temperature >40.1°C on admission and associated neurological signs such as convulsions, delirium, or coma. Immediate evaluation included recording blood pressure, pulse, and respiration rates. Temperature was recorded continuously on a four-channel readout from thermometers placed on the skin (chest, forearms, thighs) and in the rectum. Neurological status was assessed by the Glasgow coma scale (27), i.e., the sum of the scores for best eye opening and motor and verbal response; a normal person will score 15 and a patient in deep coma will score 3. The patients were cooled according to the method recommended by Weiner et al. (29), which is based on dissipation of heat by evaporation. ControZ subjects. The control group was comprised of 40 healthy blood donors, 37 t 9 yr old. They were sampled in a separate experiment; however, Simultest monoclonal antibody reagents (Becton Dickinson Immunocytometry, San Jose, CA), antibody concentrations, operator, and Simulset software on the FACScan were the same for both control subjects and heatstroke patients. Blood samples. Blood samples for analysis were obtained from 11 consecutive heatstroke patients before the sta .rt of parenteral treatment or cooling. Samples of EDTA whole blood were collected and tested immediately. A complete blood count was done. Plasma electrolytes, creatine kinase activity, and lactate levels were also measured on admission. Immunofluorescence staining. EDTA whole blood from each patient was stained for two-color immunofluorescence analysis. Combinations of fluorescein isothiocyanate-conjugated (FITC) monoclonal antibody and phycoerythrin-conjugated (PE) monoclonal antibody (Simulset reagent, Becton Dickinson Immunocytometry Systems) were used to ch aracterize lymphocyte subsets (Table 1). After 100 ~1 of whole blood had been incubated with 20 ~1 of conjugated monoclonal reagents for 20 min at room temperature, erythrocytes were eliminated by FACS lysing- solution (Becton -Dickinson). Cell pellets were washed in phosphate-buffered saline and then resuspended in 500 ~1 of cold 0.5% paraformaldehyde in

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406

PERIPHERAL

TABLE

BLOOD

LEUKOCYTES

Antibody

CD3/CDl9 CD4/CD8 CD3/CDl6/CD56

Anti-Leu-4/anti-Leu-12 Anti-Leu-3a/anti-Leu-2a Anti-Leu-4/anti-Leu-llc/anti-Leu-19

CD4WCD14

Anti-Leu-HLel/anti-Leu-M3 (1eucoGate)

Distribution

T-lymphocytes/B-lymphocytes Helper-inducer T-cells and monocytes/suppressor-cytotoxic T- and NK cells T-lymphocytes/Fe receptor type III expressed by NK cells and all neutrophils/NK cells Leukocytes/monocytes and macrophages

phosphate-buffered saline and stored at 4°C in the dark until analyzed on a FACScan flow cytometer. Flow cytometry analysis. With the use of forward and side (right angle) light scattering, the subpopulations of leukocytes (lymphocytes, monocytes, and neutrophils) were separated. A three-part differential was calculated with the use of automatic gating. The automatic gating utilized the leukogate reagent (Table 1) to optimize lymphocyte gating. Initially, it identifies lymphocytes on the basis of HLe-1 and Leu-M3 immunofluorescence. Finally, it sets a corresponding light scatter gate that includes >98% of the lymphocytes. Granulocytes, monocytes, erythroid cells, and platelets are excluded. The lymphocyte population was then analyzed for dual fluorescence. The dual FITC and PE were excited at 488 nm and produced maximum emission at 525 (FITC) and 575 nm (PE). This allowed lymphocyte subsets to be discriminated by Simulset software with a Hewlett-Packard 310 computer. Statistical analysis. Data were analyzed on a Tandon AT-compatible microcomputer with the use of a standard statistical package (Statgraphics, STSG, Rockville, MD). Student’s t test was used to compare heatstroke patients with normal control subjects. Linear regression was applied to determine correlation coefficients. Differences were considered significant at P < 0.05, and values are expressed as the means t SE. RESULTS

Table 2 gives the clinical and laboratory data of the heatstroke patients on admission. These data concur with our previous findings (4). Hematocrit and albumin are within the normal ranges, lactate levels are mildly increased, and there is no evidence of overt rhabdomyolysis Leukocytes in heatstrokepatients. The concentration of

Age,yr

HEATSTROKE

1. Monoclonal antibodies used for simultaneous two-color enumeration of leukocytes

Cluster

TABLE

IN ACUTE

2. Clinical characteristics and laboratory data

Sex, M/F Temperature, “C Systolic blood pressure, mmHg Heart rate, beats/min Respiratory rate, breaths/min GCS Lactate, mmol/l Plasma creatinine, pmol/l Creatine kinase, U/l Potassium, mmol/l Albumin, g/l Hematocrit, %

43.2t14.4 10/l 41.4kO.3 120t8 112tl6 3626 9.7tl.6 2.6kO.3 136t32 524tll9 3.8t0.12 44.3t0.6 4lt4

circulating leukocytes was significantly increased in heatstroke patients compared with the normal control subjects (P < 0.001; Table 3). Although there was no significant change in the percentage of lymphocytes (P = 0.09), the lymphocyte count was significantly increased (P < 0.05). There was no significant correlation between leukocyte count and temperature (r = 0.07, P = 0.81). Figure 1 shows that lymphocyte counts significantly correlated with the degree of hyperthermia (r = 0.62, P = 0.04). By contrast, the neutrophil number was not significantly related to the degree of hyperthermia (r = -0.186, P = 0.582). T-, B-, and natural killer (NK) cells in heatstroke patients. Table 4 shows the distribution of lymphocyte sub-

populations expressed in percentage. The percentage of T- and B-cells was significantly decreased compared with the control subjects (P < 0.001 and P < 0.05, respectively), whereas the percentage of NK cells was significantly increased (P < 0.001). Figure 2 shows the same results expressed in absolute number of cells per unit volume. It is interesting to note the striking increase in NK cells compared with the control value. No significant correlation was found between the temperature and the concentration of T- (r = 0.49, P = 0.124), B- (r = 0.547, P = 0.08), or NK cells (r = 0.4968, P = 0.12). T-helper (T,) and T-suppressor-cytotoxic cells (T,,) heatstroke patients. Table 4 gives the percentages

in

of these cells. In the heatstroke patients, the percentage of T,-cells was significantly decreased compared with the normal control subjects (P = 0.001). In contrast, the percentage of T,,,- cells was significantly increased (P < 0.001). When expressed in absolute number per unit volume (Fig. 2), there was a 34% decrease in T,-cells and a 214% increase in T,,c-cells. As a result, the ratio of T,cells to T,,,-cells was markedly reduced. However, when the relationship between the hyperthermia and T,- and T,,-cells is analyzed (Fig. 3), a statistically significant 3. Leukocyte and lymphocyte counts in heatstroke patients and healthy control subjects TABLE

(0.5-2.0) (70-125) (24-295) (3.6-5.2) (40-52) (38-52)

Values are means t SE of 11 heatstroke patients on admission. Values in parentheses are normal range. GCS, Glasgow coma score.

Leukocyte Lymphocyte Lymphocyte, %

Control Subjects

Heatstroke Patients

(n = 40)

(n = 11)

P

13.14t2.56 (2.9-34.8) 3.52kO.92 (0.5-9.4) 27t0.4 (5-57)