What Keeps the Urinary Tract Sterile?

Despite the colonization of the external part of the urethra by bacteria, the urinary tract is usually free from microbial colonization for reasons that today become increasingly clear. In the past it had been assumed that the absence of bacterial colonization resulted from mechanical clearance of organisms by voiding, by shedding of colonized epithelial cells, by microbe-binding proteins such as Tamm-Horsfall protein, by influx of and phagocytosis by neutrophils, as well as by adaptive immunity, i.e., bactericidal antibodies and effector immune cells (1). The classic experiments of Norden et al. (2), however, showed that when phosphorous-labeled Escherichia coli bacteria were introduced into the urinary bladders of female guinea pigs, >99.9% of the bladder inoculum was rapidly excreted but 0.1% of E. coli remained attached to the bladder wall, a quantity sufficient to perpetuate infection if bacterial growth were uninhibited (3). In a series of brilliant experiments, Norden et al. documented that bacteria attached to the bladder mucosa were rapidly killed as a result of intrinsic antibacterial activity of the epithelial cells within minutes, independent of polymorphonuclear leukocytes. With clairvoyance the authors postulated “that a substance (yet unidentified) exists in the bladder wall which is used… as the intact bladder exerts its antibacterial activity.” Recently it has become clear that, apart from adaptive immunity, i.e. bactericidal antibodies and effector immune cells, the antibacterial defense by epithelial cell layers (skin, gastrointestinal, respiratory, and urogenital tracts) is the task of a phylogenetically ancient system that is found in plants, insects, and vertebrates (but obviously not in bacteria), namely inducible antimicrobial peptides, which were first detected in silk moths (3) and Drosophila melanogaster (4), and later in mammalian species (5). These molecules are characterized by clusters of hydrophobic and cationic amino acids organized in what has been called an “amphipathic design” (6), which disrupts the bacterial membranes, the outermost leaflet of which differs strikingly from that of plants and animals. It is disrupted by insertion of inducible antibacterial peptides at micromolar concentrations. Amazingly, bacteria have not developed resistance to these peptides. This observation gave rise to great hopes that the golden bullet against multiresistant bacteria was around the corner, hopes that so far have not materialized. In humans, there are two major families of inducible antibacterial substances. The first class is the cationic β-defensin family coded for by a number of genes (7,8). Human β-defensin1 (HBD1) has been demonstrated in urogenital tissues (9,10), including the epithelial cells of the loop of Henle, of distal tubules, and of collecting ducts (9). The molecule, interestingly, had originally been isolated from human hemodialysate of patients with renal failure (11). During human kidney infections, the inducible defensin is strikingly elevated (12) and, conversely, mice genetically manipulated to lack the murine homolog have bacteria in their urines (10). The second class, investigated in this study by Chromek et al., are the cathelicidins. In contrast to the β-defensin family, only one single gene codes for cathelicidin in humans, CAMP (cathelicidin antimicrobial peptide), and in mice, CRAMP (cathelicidin rodent antimicrobial peptide). The human cathelicidin precursor (hCAP18) contains a C-terminal cationic antimicrobial peptide domain that is activated by cleavage from the N-terminal cathelin portion of the propeptide. The precursor is synthesized and stored in secondary granules of neutrophils, but also is found in other cells exposed to microbes, such as epithelia of the mouth, tongue, esophagus, intestine, cervix, vagina (13), lung (14), as well as salivary, sweat, or mammary glands (15,16). The precursor is processed to release the active antimicrobial peptide LL37, which also attracts neutrophils, monocytes, and T cells through a G-protein–coupled receptor for formyl peptide receptor-like 1 (FPRL1) (17). In addition to their bactericidal activity, LL37 also modulates several aspects of immune function (18,19). What is the evidence that cathelicidin is involved in the defense against urinary tract infection in humans? The authors examined the cathelicidin concentration in the urine of healthy children and children with acute urinary tract infections. Low concentrations were found in normal urine; in children with urinary tract infections the concentration was higher but not tightly correlated to the number of leukocytes, suggesting another source of cathelicidin. In a next step, the authors therefore examined noninfected human kidney tissue by PCR and ELISA. They found both CAMP mRNA and cathelicidin peptide. The peptide was, however, in the tubule lumen and the parenchymal cells did not stain for cathelicidin. To assess the response of renal tissue to infection, the authors incubated human renal cortex with uropathogenic E. coli; 5 min after exposure to the bacteria a rapid increase of CAMP mRNA was seen, suggesting that cathelicidin is the acute emergency reaction postulated by Kass (2). The increase in CAMP mRNA lessened after 135 min, either as a result of less synthesis or increased breakdown of the mRNA. In contrast, the active peptide LL37 continued to be released into the media. Secretion of cathelicidin during urinary tract infection could be confirmed in mice; this model showed that both leukocytes and cells other than leukocytes are the source of the peptide. To prove that cathelicidins can indeed kill uropathogenic E. coli, the bacteria were exposed to micromolar concentrations of LL37 and its murine homolog, both of which were bactericidal. The role of cathelicidin to protect against urinary tract infection was assessed in mice with an intact or deleted CAMP gene. The end point was the number of bacteria attached to the bladder 1 h after infection, a time point before neutrophils had entered the urinary space. The number of adhering bacteria was greater in the CAMP knockout mice. It was not influenced by whether the mice had normal neutrophil counts or neutropenia after treatment with neutrophil-specific monoclonal antibody, excluding a major role of neutrophils in this early response. A final observation arguing for an important in vivo role of cathelicidin was that, in children with more invasive upper urinary tract infections, the E. coli in their urine were more resistant against LL37. These observations are strong arguments that, by increased synthesis and secretion of cathelicidin, resident uroepithelial cells play an important neutrophil-independent role in the protection against uropathogenic bacteria. In later stages, neutrophils appear to be an additional source of cathelicidin. The relative roles of the two inducible antibacterial peptides (i.e., β-defensin and cathelicidin) in the protection against bacterial invasion of the urinary tract require further studies. Presumably both are acute emergency systems to ward off bacterial invasion before the above-mentioned slower mechanisms take over (1). Interestingly, the authors noted that the early release of cathelicidin did not depend on an increase in mRNA, possibly the result of unblocking translation of mRNA into peptide. The assumption that translation is not tightly linked to transcription would also be compatible with the observation that the peptide could not be detected in the renal cells despite the presence of mRNA. The same has also been observed in the gut (20) and may be a useful adaptive mechanism, as antimicrobial peptides at high concentrations are cytotoxic for eukaryotic cells (21). In retrospect, these experiments are an impressive confirmation of the hypothesis of Kass (2) that uroepithelial cells play an important sentinel role in the defense against urinary tract infection.