Am. J. Trop. Med. Hyg., 80(5), 2009, pp. 832–836 Copyright © 2009 by The American Society of Tropical Medicine and Hygiene 832 * Address correspondence to Márcia Marinho, Universidade Estadual Paulista–Unesp-FOA-Medicina Veterinária-Campus de Araçatuba, Rua Clóvis Pestana, 793, CEP: 16050-680, Araçatuba, São Paulo, Brazil. E-mail: mmarinho@fmva.unesp.br Pulmonary Disease in Hamsters Infected with Leptospira interrogans: Histopathologic Findings and Cytokine mRNA Expressions Márcia Marinho,* Itamar Souza Oliveira-Júnior, Cristina Maria Rodrigues Monteiro , Silvia Helena Perri, and Reinaldo Salomão Department of Animal Health and Production, Microbiology Laboratory FOA, Unesp-Estadual Paulista University, Araçatuba, São Paulo, Brazil; Inflammatory Mediators Laboratory, Geriatrics and Gerontology Division, Federal University of São Paulo, Brazil; Infectious Diseases Division, Federal University of São Paulo, Brazil Abstract. Our aim was to evaluate the pulmonary changes induced by Leptospira interrogans infection in hamsters, and the gene expression of endogenous mediators in lung fragments during 28 days of observation. The animals were euthanized on days 4, 7, 14, 21, and 28 post-inoculation. Histopathologic lung analysis showed hemorrhage, pneumonia, alveolar congestion, and infiltrated cellular areas, with increasing severity until day 21 post-inoculation. Tumor necrosis factor (TNF)-a mRNA expression enhanced in first days with peak on day 4 and slightly decreased in the final phase. The interleukin (IL)-10 remained relatively constant throughout the period, with the exceptions of days 4 and 14. The endothelial nitric-oxide synthesis (eNOS) showed an increased expression on day 4, followed by an augment on days 7 and 14, and remaining constant up to day 28 post-infection. Our results demonstrate that inoculation of L. interrogans sorovar Icterohaemorrhagiae induced pulmonary lesions, including pulmonary hemorrhage, supporting that the lung is a target organ. INTRODUCTION Leptospirosis is a globally important zoonotic disease caused by infection with pathogenic Leptospira species. 1 It affects humans in all continents, in both rural and urban areas, particularly in developing countries of warm and humid cli- mate. 2 Vihn and others 3 extracted a glycolipoprotein (GLP) present in the cell wall of a strain of Leptospira interrogans serovar Copenhageni that presented a cytotoxic effect against L929 fibroblast cells in vitro. It was later demonstrated that the GLP induced cytokine production in peripheral blood mono- cytes from healthy volunteers. 4,5 The presence of inflamma- tory cytokines has also been confirmed in sera of patients with leptospirosis. 6 Marinho and others 7,8 showed that high levels of tumor necrosis factor (TNF)-a and interleukin (IL)-6 in the supernatant of peritoneal macrophage cellular culture of genetically selected mice inoculated with a sample of Leptospira interrogans serovars Canicola was determined by immunoenzymatic assays. The lungs are an important target organ in leptospirosis presenting hemorrhagic pneumoni- tis with varying degrees of severity. 9,10 The pathophysiology of pulmonary injury in leptospirosis is poorly understood. Vascular pulmonary injury is thought to be the outcome of immunologic mechanisms with a disseminated intravas- cular coagulation-like reaction caused by Leptospira tox- ins. 11 Observation under electronic microscopy revealed that the primary lesion occurs in pulmonary capillary endothe- lial cells. 12 Andrade and others 13 concluded that leptospiro- sis profoundly influences the sodium transport capacity of alveolar epithelial cells and that inadequate handling of pul- monary fluid can affect the pulmonary function, increasing the chance of lung injury. Damage to the pulmonary endothe- lium occurs without evidence of disseminated intravascu- lar coagulation. 14–16 Nally and others 15 found that in infected guinea pigs, immunoglobulin and C3 were deposited along the alveolar basement membrane in a pattern similar to the one seen in Goodpasture’s syndrome. This finding needs to be confirmed for human leptospirosis, but it is indica- tive of the underlying autoimmune process observed in the severe pulmonary hemorrhagic syndrome. 17 The role of inflammatory mediators, such as cytokines, in the pathophysi- ology of sepsis-induced acute lung injury has been extensively studied. 18 We have previously shown that TNF-a and IL-6 were detected in high levels of bronchoalveolar lavage (BAL) in rats injected intraperitoneally with Escherichia coli. 19 In this study we aimed to evaluate the expression of tissue TNF-a, IL-10, and endothelial nitric-oxide synthesis (eNOS) mRNA levels in an experimental model of Leptospira- induced lung dysfunction, assessed by functional and histologic parameters. MATERIAL AND METHODS Animals. The animal experiments were conducted with approval from the Research Ethics Committee of the Federal University of São Paulo (São Paulo, Brazil). Male golden hamsters ( Mesocricetus auratus), weighing 120 to 150 g, were divided into five groups, each group with six ani- mals. Each experimental set was comprised of a group with five inoculated hamsters and one non-infected hamster per checking point. The hamsters were infected with 10 3 Leptospira of a virulent strain, culture grown in Ellinghausen– McCullough–Johnson–Harris (EMJH) liquid medium (Becton Dickinson, Cockeysville, MD) by intraperitoneal (IP) injection of 1 mL and observed for 4, 7, 14, 21, and 28 days. Negative control animals were injected with 1 mL of EMJH liquid medium alone. Inoculum. Leptospira interrogans serovar Icterohae- morrhagiae (strain n o 11437) was provided by a FIOCRUZ Laboratory, Brazil, and maintained in semi-solid Fletcher medium. Euthanasia and lung collections. The animals were euth- anized at the end of the respective day using a 1 mL/100 g body weight of euthanasia solution sodium pentobarbital (Tiopentax, Cristália, São Paulo, Brazil) by IP injection. At the