The Functional State of Neutrophils Correlates With the Severity of Renal Dysfunction in Children With Hemolytic Uremic Syndrome

0031-3998/07/6101-0123 PEDIATRIC RESEARCH Copyright © 2006 International Pediatric Research Foundation, Inc. Vol. 61, No. 1, 2007 Printed in U.S.A. The Functional State of Neutrophils Correlates With the Severity of Renal Dysfunction in Children With Hemolytic Uremic Syndrome GABRIELA C. FERNANDEZ, SONIA A. GOMEZ, MARIA V. RAMOS, LETICIA V. BENTANCOR, ROMINA J. FERNANDEZ-BRANDO, VERONICA I. LANDONI, LAURA LOPEZ, FLAVIA RAMIREZ, MARIO DIAZ, MARTA ALDUNCIN, IRENE GRIMOLDI, RAMON EXENI, MARTIN A. ISTURIZ, AND MARINA S PALERMO Division of Immunology [G.C.F., S.A.G., M.V.R., L.B., R.F.-B., V.L., M.A.I., M.S.P.], Institute of Hematological Investigations, National Academy of Medicine, C1425AUM Buenos Aires, Argentine; Department of Nephrology [M.A., I.G., R.E.], Pediatric Municipal Hospital of San Justo, B7118XAB, La Matanza, Buenos Aires, Argentine; Department of Nephrology [L.L., F.R., M.D.], Pediatric National Hospital “Juan P. Garrahan”, C1249ABN, Buenos Aires, Argentine determined by the days of anuria, a criteria proposed by Gianantonio (8). Therefore, the identification of parameters that correlate with severity may be useful as possible prognostic factors. HUS children usually show a high peripheral blood neutrophil (PMN) count at presentation, which has been correlated with a poor prognosis (9 –11). Moreover, increased levels of IL-8 have been detected in the serum of patients coursing the acute phase of HUS (12,13). This cytokine is produced by activated PMN, promotes their adhesion and migration in vivo and shows anti-apoptotic properties (14 –17). PMN are essential for host defense against microbial infections but can also be associated with the pathologic side effects of tissue destruction (18,19). Potentially PMN-mediated harmful mechanisms include the production of toxic reactive oxygen species (ROS), and the proteolytic capacity of enzymes stored in granules. These storage organelles are not only a source of proteolytic or bactericidal proteins, but are also important reservoirs of membrane proteins that become incorporated into the surface membrane of PMN when these organelles fuse with the plasma membrane and exocytose their content. It is now established that, among other important receptors, the type III receptor form the Fc portion of the IgG (CD16) is stored in secretory vesicles, the adhesion molecule CD11b is mainly found in secretory vesicles and gelatinase granules, the degranulation marker CD66b is present in the membrane of specific granules, and the bactericidal enzyme myeloperoxidase (MPO) is stored in azurophil granules (20). We have previously demonstrated that circulating PMN from HUS patients in the acute period (HUS AP) have a reduced expression of different membrane molecules and intracellular components, and also show impaired cytotoxic and cytokine-induced responses (21), indicating that PMN are partially deactivated, probably due to a postactivation exhaustion process (17,18). However, the detailed analysis of the ABSTRACT: Hemolytic Uremic Syndrome (HUS) is the main cause of acute renal failure in children. The high percentage of patients who develop long-term sequelae constitutes an important medical concern. The identification of parameters that correlate with the degree of renal failure may be useful to plan the best treatment soon after hospitalization. Here, we investigated the functional state of neutrophils (PMN) from HUS patients on admission, before dialysis and/or transfusion, in relation to the severity of renal impairment reached during the acute period (AP). We found that all PMN activation parameters measured in severe cases of HUS (HUS AP3) were statistically lower comparing to children with mild cases of HUS (HUS AP1). As HUS PMN phenotype and dysfunction is compatible with that of cells undergoing cell death, we also studied spontaneous apoptosis. Not only were HUS PMN not apoptotic, but HUS AP3 PMN showed an increased survival. Almost all phenotypic and functional parameters measured on PMN correlated with severity. Our results revealed a marked deactivation of PMN in severe cases of HUS, and suggest that studying the functional state of PMN could be of prognostic value. (Pediatr Res 61: 123–128, 2007) H emolytic uremic syndrome (HUS) is a multi-systemic disorder characterized by the triad acute renal failure, microangiopathic hemolytic anemia and thrombocytopenia (1). HUS is the most common cause of acute renal failure and the second cause of chronic renal failure and renal transplantation in children (2). HUS is developed after a prodrome of hemorrhagic diarrhea caused by infection with Shiga toxin (Stx)-producing Escherichia coli (STEC) (3), and endothelial damage appears to be an important factor in the sequence of events leading to the microangiopathic process. No specific treatment for HUS is yet available, and although supportive therapies have reduced HUS mortality considerably, different degrees of renal insufficiency can be found in up to 40% of the patients (4 – 6). It has been reported that the main long-term prognostic factor is the severity of acute renal failure (4 –7), as Received May 18, 2006; accepted August 24, 2006. Correspondence: Gabriela C. Fernández, Ph.D., Instituto de Investigaciones Hematológicas, Academia Nacional de Medicina, Pacheco de Melo 3081, C1425AUM, Buenos Aires, Argentina; e-mail: This work was supported by grants from Alberto J. Roemmers, Agencia Nacional de Promoción Cientı́fica y Tecnológica, and Consejo Nacional de Investigaciones Cientı́ficas y Tecnológicas (CONICET) Abbreviations: PMN, neutrophils; HUS, Hemolytic Uremic Syndrome; AP, acute period; HUS AP1, mild cases of HUS; HUS AP2, moderate cases of HUS; HUS AP3, severe cases of HUS; HC, healthy control; Stx, Shiga toxin; STEC, Stx-producing Escherichia coli; ROS, reactive oxygen species; MPO, myeloperoxidase; FITC, fluorescein isothiocyanate; DHR, dihydrorhodamine 123; PMA, phorbol myristate acetate DOI: 10.1203/01.pdr.0000250037.47169.55 123 124 FERNANDEZ ET AL. relationship between the functional state of PMN and the severity of the renal dysfunction of the patients has not been previously investigated. In this study, we analyzed in HUS patients on admission whether the level of deactivation of PMN correlates with the degree of renal impairment. In particular, we determined the expression of activation markers situated in different types of intracellular granules, as well as ROS generation. We also investigated if deactivation of peripheral PMN from HUS AP patients was a consequence of an increased number of cells undergoing apoptosis. METHODS Patients and samples. The study was approved by the respective Hospital Ethical Committees. Patients (n ⫽ 50) were enrolled in this study between January, 2000 and December, 2005 after informed consent from their parents had been obtained. The criteria for diagnosis were a microangiopathic hemolytic anemia with schizocytes, thrombocytopenia (platelet count ⬍15 (...truncated)