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Clinical and Diagnostic Laboratory Immunology, November 2003, p. 1090-1095, Vol. 10, No. 6
1071-412X/03/$08.00+0     DOI: 10.1128/CDLI.10.6.1090-1095.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Characterization of the Cytokine Immune Response in Children Who Have Experienced an Episode of Typical Hemolytic-Uremic Syndrome

Children's Hospital and Department of Pediatric Nephrology of the Technical University of Munich, 80804 Munich,¹ Department of Medical Immunology, Charité, Humboldt-University, 10098 Berlin,² Department of Biochemical Pharmacology, University of Konstanz, 78464 Konstanz, Germany³

Received 19 June 2002/ Returned for modification 18 September 2002/ Accepted 4 August 2003

	ABSTRACT

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The lipopolysaccharide (LPS) of enterohemorrhagic Escherichia coli (EHEC) and Shiga toxin together substantially contribute to the pathophysiology of typical hemolytic-uremic syndrome (HUS). Both factors have been shown to be immune stimulators and could play a key role in the individual innate immune response, characterized by proinflammatory and anti-inflammatory cytokines. By use of a whole blood stimulation model, we therefore compared the LPS- and superantigen-induced cytokine responses in children who had been having recovering from an acute episode of typical HUS for at least 6 months (group 1) with those in controls, who consisted of patients seen in the pediatric neurology outpatient department for routine examination (group 2). Samples were analyzed for cytokine protein levels and the levels of mRNA production. LPS stimulation revealed lower levels of interleukin 10 (IL-10) (P < 0.05) and increased levels of gamma interferon (P < 0.05) and increased ratios of pro- and anti-inflammatory cytokines (P < 0.05 for the IL-1ß/IL-10 ratio; P < 0.05 for the tumor necrosis factor alpha/IL-10 ratio) in group 1. In addition superantigen stimulation showed decreased IL-2 levels in group 1 (P < 0.01). Our results suggest an alteration of the cytokine response characterized by high proinflammatory cytokine levels and low anti-inflammatory cytokine levels as well as low levels of IL-2 production in children who have experienced an episode of typical HUS. We hypothesize that this altered immune response is not a residual effect of the infection but a preexisting characteristic of the patient. This could be one reason why individuals infected with EHEC are potentially predisposed to a systemic disease (HUS).

	INTRODUCTION

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In recent years our understanding of the pathophysiology of hemolytic-uremic syndrome (HUS) has further improved. However, the exact mechanisms leading to typical HUS (D+ HUS, where D+ is gastroenteritis positive) secondary to infection with enterohemorrhagic Escherichia coli (EHEC) are still not completely understood. As HUS is a disease of young children and elderly adults, a substantial role of the immune system has been discussed (6, 12, 13, 19). It is well known that tumor necrosis factor alpha (TNF-) and Shiga toxin (Stx) potentiate the toxicity of each other for endothelial cells (9, 20) and that elevated levels of antibodies to EHEC lipopolysaccharide (LPS) can be found in HUS patients (1, 3, 22). Therefore, a crucial role of the inflammation system has been suggested in HUS. Nevertheless, attempts to prove that patients with an acute episode of HUS have elevated systemic proinflammatory cytokine levels in comparison to those in children with infectious diarrhea have failed (8). Consequently, we and other groups recently added evaluation of anti-inflammatory cytokine profiles to the study design in order to monitor the balance between pro- and anti-inflammatory immune activation (10, 15, 24). We and others observed alterations in anti-inflammatory cytokine levels, even though the results are conflicting: in our D+ HUS patient group, interleukin 10 (IL-10) levels were found to be decreased in patients with EHEC infections in comparison to those in patients with bacterial gastroenteritis of other origins. Consequently, the TNF-/IL-10 ratio for the HUS group was increased when the levels of the two cytokines were compared, reflecting an imbalance toward inflammation. This may result in the expression of an altered immunity in patients with this rare disease. Nevertheless, measurement of systemic cytokine levels is not likely to reflect the whole picture of immune activation, as cytokines mainly act in a paracrine fashion and have short half-lives in plasma (11). Therefore, we raised the question whether the cytokine profiles observed in vivo can be reproduced by ex vivo LPS stimulation of whole blood from patients who had experienced and who were recovering from an acute HUS episode. As the results of studies with animal models suggest compromised immune activity secondary to Stx exposure (17), we decided to study the immune response to superantigen in order to characterize T-cell immunity. Finally, the results were correlated with clinical parameters like the glomerular filtration rate (GFR), blood pressure, protein and erythrocyte levels in urine, and others.

(This study was presented in part at VTEC 2000, Kyoto, Japan, 28 October to 2 November 2000 [S. Westerholt, A.-K. Pieper, B. Klare, P. Emmrich, and R. Oberhoffer, Abstr. VTEC 2000, abstr. 429, 2000; S. Westerholt, A.-K. Pieper, B. Klare, P. Emmrich, and R. Oberhoffer, Abstr. VTEC 2000, abstr. 96, 2000] and at the Jahrestagung der Deutschen Gesellschaft für Kinderheilkunde und Jugendmedizin, Stuttgart, Germany, September 2000 [A.-K. Pieper, S. Westerholt, M. Griebel, P. Emmrich, and R. Oberhoffer, Abstr. Jahr. Dtsch. Gesellschaft Kinderheilkd. Jungendmed., abstr. V121, 2000]. This study contains parts of the M.D. thesis of Anne-Kathrin Pieper.)

	MATERIALS AND METHODS

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Patients. This study was conducted in accordance with the precepts of the Helsinki Declaration and was approved by the Institutional Review Board of the Technical University of Munich, Munich, Germany. All children volunteered to take part in this study. Written informed consent was collected from the patients' parents and from the patients themselves, when appropriate, before entry into the study. This study was approved by the Ethics Committee, Technical University of Munich. All subjects underwent a thorough physical examination to exclude the presence of an acute infection. In addition, blood samples were examined by routine clinical chemistry analyses. We recruited children more than 6 months after an episode of typical HUS (group 1) and controls (group 2), who consisted of patients seen in the pediatric neurology outpatient department for routine examination. Group 2 consisted of patients who had previously had complicated febrile seizures and patients with successfully treated epilepsy. Results were analyzed by linear regression by age and sex. Patients were matched where appropriate. Subjects from group 2 who were using immunoactive drugs, such as adrenocorticotropic hormone, or who were suspected of having a systemic or genetic disease were excluded. Blood was collected from the patients between 9 and 11 a.m. Along with routine blood withdrawal, a 0.5-ml sample that was placed in EDTA (Sarstedt, Nümbrecht, Germany) and a 2-ml sample that was placed in lithium-heparin (Sarstedt) were collected. Clinical data were collected from all patients, including length of illness, blood pressure, use of antihypertensive drugs, protein and erythrocyte levels in urine, and creatinine levels. GFR was estimated by the Schwartz formula. Patients with clinical or laboratory signs of acute inflammatory disease were excluded. Typical HUS was defined according to the present definition of the Centers for Disease Control and Prevention: hemolytic anemia with microangiopathic changes, thrombocytopenia, acute renal injury or failure, and acute symptoms or a history of EHEC-positive gastroenteritis, as proved by fecal culture (n = 31) or a positive result by serological testing (n = 1). All EHEC isolates had to be positive for Stx1 or Stx2, or both. In the case of a negative fecal culture, the titers of antibodies against Stx1 or Stx2, or both, had to be positive; in addition, the titers of antibodies against EHEC LPS had to be positive. None of the patients in group 1 where allowed to be on dialysis or to have a GFR <80 ml/min/1.73 m² at the time of blood sampling.

Blood counts. Complete blood counts were carried out with a Max M counter (Coulter, Krefeld, Germany). All differential white blood cell counts were determined with the same machine.

Incubation of whole blood. To study EHEC LPS-induced cytokine release into whole blood, 800 µl of RPMI 1640 (Bio Whittaker, Boehringer Ingelheim, Ingelheim am Rhein, Germany) supplemented with 2.5 IU of heparin (Liquemin; Hoffmann-La Roche, Grenzach-Whylen, Germany) per ml and 100 IU of penicillin-streptomycin (Gibco BRL, Eggenstein, Germany) per ml was pipetted into a polypropylene reaction tube (Eppendorf, Hamburg, Germany), and 10 µg of endotoxin from E. coli O111 (Sigma, Deisenhofen, Germany) was added. Finally, 200 µl of heparinized whole blood was added and the tubes were incubated for 24 h at 37°C in 5% CO₂. After incubation, the tubes were shaken and blood cells were sedimented by centrifugation (12,000 x g, 4 min). The cell-free supernatants were stored at -80°C until the cytokine levels were measured; the cell pellets were resuspended in 0.9% NaCl and were used to prepare the RNA (4).

The protocol described above was used for superantigen-induced cytokine release, except that 1 µg of Stapylococcus enterotoxin B (SEB; Sigma) instead of LPS was added, and incubation was for 72 h instead of 24 h.

All incubations were performed in duplicate to exclude handling errors. Therefore, the results are averages of two identical experimental settings. Cytokine protein levels and gene expression were measured from unstimulated cells to exclude false-positive results. Blood samples that showed measurable cytokine levels under these circumstances were excluded from further interpretation.

Cytokine measurement. A sandwich enzyme-linked immunosorbent assay (ELISA) based on commercial antibody pairs was used to determine the cytokine levels in the plasma samples. Antibodies against IL-10 were purchased from Pharmingen (Hamburg, Germany). Antibodies against IL-2 and IL-6 were from R&D Systems (Wiesbaden, Germany), while antibodies against TNF-, IL-1ß, IL-4, IL-8, and IFN- (Biozol; Endogen, Eching, Germany) were purchased. Appropriate biotinylated secondary antibodies were used throughout. Peroxidase-coupled streptavidin was obtained from Jackson Immuno (Dianova, Hamburg, Germany), and the tetramethylbenzidine (TMB) substrate was from Sigma. Recombinant human cytokines calibrated against standards, like those used in commercial ELISA kits, served as standards. Each well of the ELISA plates (Greiner) was coated overnight at 4°C with 50 µl of antibody in 0.1 mol of NaHCO₃ (pH 8.2) per liter. After each well of the plates was blocked with 200 µl of phosphate-buffered saline (PBS) supplemented with 3% bovine serum albumin (BSA; pH 7.0; Serva, Heidelberg, Germany) for 2 h at room temperature, the plates were washed twice with PBS-0.05% Tween 20. Sample (50 µl/well) and tracer antibody (50 µl/well) in PBS-3% BSA were added, and the plates were incubated for 2 h. After six wash cycles, each well of the plates was incubated for 30 min with 100 µl of streptavidin-peroxidase (Dianova, Hamburg, Germany) at 1 µg/ml in PBS-3% BSA. After eight washes, 100 µl of TMB liquid substrate solution (Sigma) was added to each well, and the plates were incubated at room temperature for 5 to 30 min. After the addition of 50 µl of stop solution (1 mol of H₂SO₄ per liter) to each well, the absorption was measured at 450 nm by using a reference wavelength of 630 nm.

RNA preparation. RNA from whole blood was prepared with an RNeasy Blood Mini kit and by additional DNase digestion (Qiagen, Hilden, Germany), according to the instructions of the manufacturer.

Reverse transcription and competitive PCR. Reverse transcription and competitive PCR were performed as described previously (14). The Gel Doc 2000 system and Quantity One software (both from Bio-Rad, Hercules, Calif.) was used for quantification of the PCR products.

Statistical analysis. All data are given as means and standard deviations (SDs). The level of cytokine release was calculated per milliliter of blood or per leukocyte cell number. RNA products were expressed as the ratio of the amount of RNA in the sample and the amount of the control fragment. Statistical analyses were performed with SPSS software (version 10.0; SPSS GmbH, Munich, Germany). IL-10 levels and the TNF-/IL-10 ratio were analyzed prospectively. All other parameters were analyzed in a post hoc fashion. Due to the exploratory nature of the majority of results, multiple testing was not performed. Correlations between parameters were analyzed by the Spearman rank test; partial correlations were calculated where appropriate. The threshold of significance was a P value <0.05.

	RESULTS

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LPS stimulation. Thirty-two patients with a history of typical HUS (group 1) and 33 controls (group 2) were enrolled for LPS stimulation. Thirteen patients from group 1 showed proteinuria at the time of blood sampling; elevated numbers of erythrocytes were seen in the urine of two children. No patient was hypertensive or showed other residual signs from the acute episode of HUS. The groups were different according to their age distributions (mean ages, 91 months for group 1 and 125 months for group 2; P < 0.01) but not with respect to sex (group 1, 17 females and 15 males; group 2, 16 females and 16 males). Consequently, a linear regression model was used to calculate the influence of age on cytokine levels. According to the ELISA results, different ages of patients had no significant influence to most cytokines on protein and mRNA levels; the exceptions were TNF-, IL-8, and IL-10. Therefore, 23 patient pairs were matched by age and sex and evaluated for differences in the levels of these cytokine proteins. All patients were included for calculation of the other parameters. The median of the time interval between the acute episode of HUS and the time point of stimulation (t) in group 1 was 47.8 months (SD, 6.7 months).

Stimulation of whole blood with LPS was used to analyze protein (ELISA; Table 1) and mRNA (PCR; Table 2) levels. Significant differences were detected between the groups, in that the levels of the proinflammatory cytokines IL-1ß (which affected only the mRNA level) and IL-8 were elevated in group 1. In addition, the levels of all other proinflammatory cytokines evaluated (TNF-, IL-6, and IL-1ß protein levels) turned out to be increased in these patients as well, but the differences were not significant. However, the level of IL-10, the only anti-inflammatory cytokine whose levels were measured, was markedly decreased in children with a former history of HUS in comparison to those in controls, as shown in Fig. 1. The IFN- level was significantly increased. As a result, the ratios of pro- and anti-inflammatory proteins (IL-1ß/IL-10 and TNF-/IL-10) were elevated in group 1. Figure 2 demonstrates these results as box plots.

View larger version (20K): [in this window] [in a new window]   FIG. 1. Quantification of IL-10 protein and mRNA levels from plasma and leukocytes, respectively, by stimulation assays with whole blood from patients with a history of D+ HUS (group 1) and matched controls (group 2). Box plots display the means (bars), 25 and 75th quartiles (boxes), and ranges for nonextreme values (whiskers). Results are corrected for monocyte cell count. Stimulation was performed with LPS for 24 h.

View larger version (22K): [in this window] [in a new window]   FIG. 2. Quantification of TNF- and IL-10 protein and mRNA levels from plasma and leukocytes, respectively, by stimulation assays with whole blood from patients with a history of D+ HUS (group 1) and matched controls (group 2). Stimulation was performed with LPS for 24 h.

Comparison of SEB stimulation results showed decreased levels of IFN-, IL-4, and IL-2 protein production, but only the result for IL-2 was significant, as outlined in Fig. 3. It is noteworthy that the median results for patients in group 3 were even lower more than those observed previously for the other study groups (Table 4).

View larger version (19K): [in this window] [in a new window]   FIG. 3. IL-2 levels in plasma of whole blood from patients with a history of D+ HUS (group 1), matched controls (group 2), and individuals with an acute episode of D+ HUS (group 3) determined by stimulation assays. Stimulation was performed with SEB for 72 h.

	DISCUSSION

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The results of the superantigen stimulation assay (Table 4) revealed an impressively decreased IL-2 response in patients who had previously had HUS, without any hint for a T-helper-cell 1 to T-helper-cell 2 shift. Lower IL-4 and IFN- levels were also noted but were not significantly different. This is probably due to the small sample size. In accordance with the results of experiments with animals that received intravenous Stx injections (17), these data suggest an impaired capability of lymphocyte proliferation. By taking the SEB stimulation results for patients with an acute episode of HUS into consideration, it is likely that the alteration of lymphocyte proliferation occurs during the acute phase of HUS and restoration takes place over months or even years. The latter is underlined by the strong positive correlation between IFN- production and t (Table 5).

Like the TNF-/IL-10 mRNA ratio, the amount of IL-2 protein secretion secondary to SEB stimulation significantly correlates with the patients' levels of proteinuria (Table 5). Therefore, a protective effect of IL-2 secretion can be postulated.

No other clinical parameter, like erythruria or blood pressure, seemed to be linked to cytokine production in response to either SEB or LPS stimulation. We assume that proteinuria and GFR are the most sensitive parameters for impaired health status in our patients who had previously experienced typical HUS. This hypothesis is supported by the fact that other clinical parameters also tended to correlate but missed statistical significance (data not shown).

The ongoing study to test this includes patients who had previously had EHEC infections without signs of HUS in order to support our hypothesis that there is an individual inflammatory response that predisposes an individual to typical HUS when EHEC infection occurs. In addition, experiments that will evaluate other immune-stimulating pathogenic factors, like Stx1, Stx2c, Stx2d, hemolysin, and other factors, are planned.

It is noteworthy that even in children several immunological risk factors that predispose an individual to a decreased or increased severity of a certain illness have been described for other forms of infectious diseases (7, 18, 21, 23). Nevertheless, this is the first study investigating a possible individual immunologically determined risk for D+ HUS secondary to an EHEC infection. Even though the differences between the groups were small and therefore cannot represent the exclusive key to the understanding of the pathophysiology of HUS, the variations in individual cytokine profiles that were observed could be of definite importance. Studies reproducing these results in an animal model must be undertaken to support these conclusions.

	ACKNOWLEDGMENTS

 
We thank all nurses and physicians of the Department of Pediatrics, Technical University of Munich, for permanent support and critical discussions. Special thanks go to our routine laboratory for technical assistance and to K. Ulm for support with statistics. We are grateful for the excellent technical assistance of M. Klein, A. Biedermann, and G. Pinski. Special thanks go to Martin Bitzan for critically reviewing the manuscript.

	FOOTNOTES

 
* Corresponding author. Present address: Department of Neonatology and Pediatric Intensive Care Medicine, Ernst-Moritz Arndt University Greifswald, Soldmannstrasse 15, 17489 Greifswald, Germany. Phone: 49 3834 866421. Fax: 49 3834 866422. E-mail: Soeren.Westerholt{at}uni-greifswald.de.

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Westerholt, S. Articles by Oberhoffer, R. Search for Related Content PubMed PubMed Citation Articles by Westerholt, S. Articles by Oberhoffer, R.