The antimicrobial peptide cathelicidin protects the urinary tract against invasive bacterial infection Milan Chromek 1 , Zuzana Slamova ´ 1 , Peter Bergman 2 , La ´szlo ´ Kova ´cs 3 , L’udmila Podracka ´ 4 , Ingrid Ehre ´n 5 , Tomas Ho ¨kfelt 6 , Gudmundur H Gudmundsson 7 , Richard L Gallo 8 , Birgitta Agerberth 2 & Annelie Brauner 1 The urinary tract functions in close proximity to the outside environment, yet must remain free of microbial colonization to avoid disease. The mechanisms for establishing an antimicrobial barrier in this area are not completely understood. Here, we describe the production and function of the cathelicidin antimicrobial peptides LL-37, its precursor hCAP-18 and its ortholog CRAMP in epithelial cells of human and mouse urinary tract, respectively. Bacterial contact with epithelial cells resulted in rapid production and secretion of the respective peptides, and in humans LL-37/hCAP-18 was released into urine. Epithelium-derived cathelicidin substantially contributed to the protection of the urinary tract against infection, as shown using CRAMP-deficient and neutrophil- depleted mice. In addition, clinical E. coli strains that were more resistant to LL-37 caused more severe urinary tract infections than did susceptible strains. Thus, cathelicidin seems to be a key factor in mucosal immunity of the urinary tract. Under healthy conditions, the urinary tract, except for the external part of the urethra, is free from microbial colonization. Mechanisms involved in the clearance of bacteria include mechanical factors, such as urine flow and regular bladder emptying, chemical-defense com- ponents of epithelia and, upon bacterial stimulation, epithelial shed- ding and influx of effector immune cells. Components of epithelial defense include antimicrobial substances, chemokines and cytokines. Recently, antimicrobial peptides have been shown to have an important role in the first line of mucosal immunity 1 . Two main families of antimicrobial peptides in mammals, the defensins and the cathelicidins, are expressed in immune cells and at epithelial surfaces 2,3 . Gene-deficient models have confirmed the importance of cathelicidin in protection of skin 4 and b-defensin-1 in respiratory tract 5 . Furthermore, b-defensin-1 has been suggested to be a major antimicrobial peptide in urogenital tissues 6–8 . Cathelicidins are expressed in circulating neutrophils and myeloid bone marrow cells, in epithelial cells of the skin and gastrointestinal tract, as well as in the epididymis and lungs 9–14 . In contrast to the multiple defensins, only one cathelicidin gene, CAMP , has been found in humans 12 . The gene product is synthesized as a propeptide, designated human cationic antimicrobial peptide-18 (hCAP-18) or pro-LL-37. The neutrophil propeptide is cleaved extracellularly into cathelin and the C-terminal peptide LL-37 (ref. 15). Both cleavage products have a broad antimicrobial effect with complementary action 16 . Likewise, there is only one mouse gene encoding cathelicidin, Camp (also known as CnLp), and it is very similar to the human gene. The mouse cathelicidin proform is processed to the mature bioactive peptide CRAMP (cathelin-related antimicrobial peptide) 14,17 . LL-37 and CRAMP are amphipathic, a-helical peptides that preferentially bind to negatively charged groups of the outer leaflet of the bacterial membrane. This binding results in damage to the membrane, and different models explaining the precise mechanism of the peptides’ action have been proposed 18 . In addition to antimicrobial activity, antimicrobial peptides were suggested to be multifunctional modula- tors of different immune reactions 19–21 . Notably, cathelicidins in vitro inhibited the growth of pathogens that have a major role in the urinary tract, whereas they were rather ineffective against urogenital commensal bacteria 22 . Therefore, we hypothesized that production of cathelicidin in vivo could be a natural mechanism protecting the urinary tract. Here, we show that epithelial cells of the urinary tract in humans and mice produce cathelicidins. We also provide evidence of rapid production and secretion of cathelicidin upon contact with bacteria. Moreover, we show the relevance of epithelium-derived as well as neutrophil-derived cathelicidin using CRAMP-deficient mice, neutro- phil depletion and in vitro experiments. Furthermore, our data indicate that severity of bacterial invasion is linked to bacterial resistance to cathelicidin. RESULTS Cathelicidin is present in urine To analyze the presence of the human cathelicidin LL-37/hCAP-18, we examined urine from both healthy children and children with urinary Received 27 February; accepted 7 April; published online 4 June 2006; doi:10.1038/nm1407 1 Department of Clinical Microbiology, Microbiology and Tumorbiology Center, Karolinska University Hospital and Karolinska Institutet, SE-171 76 Stockholm, Sweden. 2 Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 77 Stockholm, Sweden. 3 Department of Pediatrics, Comenius University School of Medicine, 833 40 Bratislava, Slovakia. 4 Department of Pediatrics, Pavol Jozef S ˇ afa ´ rik University School of Medicine, 040 66 Kos ˇice, Slovakia. 5 Department of Urology, Karolinska University Hospital Solna, SE-171 76 Stockholm, Sweden. 6 Department of Neuroscience, Karolinska Institutet, SE-171 77 Stockholm, Sweden. 7 Biology Institute, University of Iceland, 101 Reykjavik, Iceland. 8 Division of Dermatology, University of California, San Diego, California 92161, USA. Correspondence should be addressed to A.B. (Annelie.Brauner@ki.se). 636 VOLUME 12 [ NUMBER 6 [ JUNE 2006 NATURE MEDICINE ARTICLES © 2006 Nature Publishing Group http://www.nature.com/naturemedicine