VIRAL IMMUNOLOGYVolume 8, Number 2, 1995Mary Ann Liebert, Inc.Pp. 75-79

Serum Levels of Tumor Necrosis Factor-a, Interleukin-1,and Interleukin-6 in Hemorrhagic Fever with Renal

Syndrome

TERESA KRAKAUER,1 JAMES W. LEDUC,1'3 and HENRY KRAKAUER2

ABSTRACT

Hemorrhagic fever with renal syndrome is an acute viral disease caused by hantavirus. Onthe basis of clinical observation, the illness is divided into five sequential stages: febrile, hy-potensive, oliguric, diuretic, and convalescent. Because interleukin-1 (IL-1), tumor necrosisfactor-a (TNF-a), and interleukin-6 (IL-6) are mediators responsible for fever, septic shock,and acute phase protein induction, we examined, using ELISA, the presence of these threecytokines in 276 sera collected during the Korean Conflict from 110 patients. Detectable lev-els (>20 pg/ml) of TNF-a, IL-lß, and DL-6 occurred in 14, 14, and 33% of these samples,respectively. There was a significant correlation between serum levels of IL-1/Î and TNF-a(r = 0.66, p <0.001), DL-1/3 and IL-6 (r = 0.59, p <0.001), and IL-6 and TNF-a (r = 0.71,p <0.001). The pathophysiologic processes of HFRS do not have clear or consistent corre-lations with alterations in the levels of the cytokines studied.

INTRODUCTION

Hemorrhagic fever with renal syndrome (HFRS), an infectious disease caused by hantavirus (1, 2), was

initially recognized in Korea during the Korean Conflict, when >3000 cases occurred in United Nationstroops (3). Recently, a new strain of hantavirus was identified as the etiologic agent of hantavirus pulmonarysyndrome, a severe respiratory illness that occurred in southwestern United States in May of 1993 (4). Thecharacteristics of HFRS are high fever, severe headache, hemorrhage, renal failure, and, in about 7% ofcases, death (3, 5). In contrast, the newly described hantavirus pulmonary syndrome is typically associatedwith rapid progressive pulmonary edema and has a much higher case fatality rate (76%) (4). Because ofthe known association of proinflammatory cytokines with certain pathophysiologic processes such as dis-seminated intravascular coagulation that have been reported in HFRS (6), we decided to examine the pres-ence of three key immune mediators: tumor necrosis factor-a (TNF-a), interleukin-1/3 (IL-lß), and inter-leukin-6 (IL-6) in serum of HFRS patients. We measured the presence of these cytokines retrospectively in

'Applied Research Division, United States Army Medical Research Institute of Infectious Diseases, Fort Derrick,Frederick, Maryland 21702.

2Uniform Services University of Health Sciences, Bethesda, Maryland 20814.3Present address: Dr. James W. LeDuc, Division of Communicable Diseases, World Health Organization, Geneva,

Switzerland.

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KRAKAUER ET AL.

a large collection of acute and convalescent sera from serologically confirmed Korean HFRS patients todefine possible associations between these proinflammatory cytokines with different stages of HFRS.

MATERIALS AND METHODS

Serum collection. Sera were obtained from June 1952 to July 1953 from 110 patients with a clinical di-agnosis of HFRS. These samples were collected by the Hemorrhagic Fever Commission and labeled witheach patient's name and number, date of collection, volume, and a "DD" number, representing the "day ofdisease." The information on the label is the only patient information that we were able to locate, and we

used these dates to calculate days after onset of disease. The sera had been lyophilized in glass ampules,and packed in cardboard boxes and kept at

—

30°C. Sera were rehydrated with sterile, distilled water ac-

cording to the volume indicated on each label, mixed thoroughly, then transferred to plastic tubes and storedat

—

70°C until tested. Different aliquots of these sera had been previously examined and shown by enzymeimmunoassays to contain specific IgM and IgG anti-Hantaan virus antibodies. At least one serum samplefrom each patient was shown to contain neutralizing antibodies specific for Hantaan virus (7).

Cytokine detection. Serum levels of IL-Iß were assayed by ELISA kits (Cistron, Pine Brook), accord-ing to instructions from the manufacturer. TNF-a and IL-6 levels were each measured by sandwich ELISAwith cytokine-specific antibodies, as described previously (8, 9). A standard preparation of recombinant cy-tokine (20 to 1000 pg/ml) was included in each plate for calibration. The detection limit of each assay was20 pg/ml of cytokine. For all ELISA, the intra- and interassay coefficients of variation of replicates wereless than 10%. Control sera from 29 healthy volunteers were collected and stored frozen until use. Circulatinglevels of the three cytokines from this control group were measured and used as a comparison.

Statistical analysis. All values were expressed as mean ± SD. Statistical analyses were carried out byusing the Stata software package (State Corporation, College Station, TX). Correlation analysis was per-formed by Pearson's correlation test.

RESULTS

A total of 276 serum samples from 110 patients were studied, and 14, 14, and 33% of samples had de-tectable levels (>20 pg/ml) of TNF-a, IL-Iß, and IL-6, respectively (Table 1). The proportion of samplesin HFRS with detectable IL-lß was higher than in normal controls (14 ± 1 vs 0%). The percentage of sam-

ples with detectable TNF-a in HFRS (14 ± 1%) was not significantly different from that of normal con-

Table 1. Serum TNF-a, IL-1/3, and IL-6 in HFRS and Normal Subjects

Percentsamples with

measureable cytokines 10%

Percentiles"

50% 90%

HFRSIL- IßTNF-aIL-6

Healthy volunteersIL-1/3TNF-aIL-6

14 ± 114 ± 133 ±3

024 ±869 ±9

000

000

000

00

214

63 pg/ml101 pg/ml804 pg/ml

0 pg/ml733 pg/ml750 pg/ml

"Percent of samples with less than the indicated value.

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HEMORRHAGIC FEVER WITH RENAL SYNDROME

trois (24 ± 8%). In contrast, percentage of HFRS samples with detectable levels of IL-6 (33 ± 3%) was

significantly lower than that of normal controls (69 ± 9%).Because we were interested in following the levels of these cytokines as the disease progressed through

its defined clinical stages, 98 samples from 32 patients with multiple (^3) time points were chosen for de-tailed statistical analysis. However, findings obtained for this subset were generally similar to those in theentire set.

The day of disease of HFRS is divided into five phases according to the known clinical stages of epi-demic hemorrhagic fever (3, 5). They are defined as follows: phase 1 (febrile, 0 to 5 days), phase 2 (hy-potensive, 6 to 9 days), phase 3 (oliguric, 10 to 15 days), phase 4 (diuretic, 16 to 21 days), and phase 5(convalescent, 22 to 54 days).

Figure 1 represents serum levels of TNF-a, IL-Iß, and IL-6 at these various phases. Within this groupof 98 samples, 16 ± 4% of samples showed detectable levels of TNF-a; 18 ± 4 and 39 ± 5% had mea-sureable levels of IL-1/3 and IL-6, respectively. IL-Iß tended to appear in the convalescent phase of thedisease. For example, convalescent serum constituted 44% of the total samples, yet 72% of the sampleswith detectable levels of IL-Iß were found in the convalescent phase. The proportions of sera with IL-6were more evenly distributed among the different phases of HFRS. The mean level of IL-1/3 was highestin the convalescent phase (505 ± 1228 pg/ml, mean ± standard deviation). Mean IL-6 levels were highestduring the hypotensive and convalescent phases (549 ± 1416 and 491 ± 1003 pg/ml, respectively).

A high correlation between serum levels of IL- Iß and TNF-a was found (r = 0.66, p < 0.001). Significantcorrelation also exists between concentrations of IL-6 and IL-1/3 (r = 0.59, p < 0.001) and high levels ofTNF-a (r = 0.71, p < 0.001).

DISCUSSION

Cytokines, produced by the host in response to infection, are thought to be responsible for the patho-physiological manifestations of many disease states. Because the symptoms of HFRS include fever and hy-potension and the principal pathogenetic process affects the vascular endothelium (3, 5), we hypothesizedthat the proinflammatory cytokines, IL-1, TNF-a, and IL-6, might be involved with HFRS. All three cy-tokines have overlapping multiple biological activities and act synergistically with each other (10). IL-1 isan endogenous pyrogen and an activator of lymphocytes (11). TNF-a is also an important mediator of sep-sis (12). Both IL-1 and TNF-a are known to upregulate the expression of adhesion molecules on endothe-lial cells, resulting in enhanced leukocyte adhesion (13). In addition, both stimulate prostaglandins and pro-coagulant activity in endothelial cells (14). IL-6 is a B cell stimulatory factor and an inducer of acute phaseproteins (15).

This study demonstrates that 14% of sera from HFRS patients had measurable levels of IL-1/3. In con-trast, IL-Iß was undetectable in normal subjects. There was no significant difference in the percentages ofsamples with measureable TNF-a in HFRS and normal controls. The detection of TNF-a in various dis-eases has been inconsistent and its correlation with outcome is also highly variable (16-18).

We also found that IL-6 was detected less frequently in patients with HFRS than in normal controls. Thepercentage of measureable IL-6 in the convalescent phase of HFRS was closer to that seen in normal vol-unteers. The lower percentage of measureable IL-6 in HFRS might be indicative of immune inadequacy,since IL-6 is a differentiation factor for B cells and promotes immunoglobulin production.

We also found strong correlation among these three proinflammatory cytokines in HFRS. Overall, we

cannot attribute the pathophysiologic processes of HFRS to the cytokines that we evaluated.

ACKNOWLEDGMENTS

We thank Marilyn Buckley for technical assistance, Katheryn Kenyon for editorial assistance, and DavidElia for assistance in the preparation of graphs.

77

KRAKAUER ET AL.

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FIG. 1. Serum TNF-a, IL-1/3, and IL-6 from HFRS patients over the five clinical phases. Detection limit for eachcytokine is 20 pg/ml. Phase 1, febrile phase, 0-5 days. Phase 2, hypotensive phase, 6-9 days. Phase 3, oliguric phase,10-13 days. Phase 4, diuretic phase, 14-21 days. Phase 5, convalescent phase, 22-54 days.

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HEMORRHAGIC FEVER WITH RENAL SYNDROME

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Address reprint requests to:Dr. Teresa Krakauer

Applied Research DivisionBldg 1425, USAMRIID, Ft. Detrick

Frederick, MD 21702-5011

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