Paneth cells in mouse small intestinal crypts secrete granules rich in microbicidal peptides when exposed to bacteria or bacterial antigens. The dose-dependent secretion occurs within minutes and alpha-defensins, or cryptdins, account for 70% of the released bactericidal peptide activity. Gram-negative bacteria, Gram-positive bacteria, lipopolysaccharide, lipoteichoic acid, lipid A and muramyl dipeptide elicit cryptdin secretion. Live fungi and protozoa, however, do not stimulate degranulation. Thus intestinal Paneth cells contribute to innate immunity by sensing bacteria and bacterial antigens, and discharge microbicidal peptides at effective concentrations accordingly.

Precursors of alpha-defensin peptides require activation for bactericidal activity. In mouse small intestine, matrilysin colocalized with alpha-defensins (cryptdins) in Paneth cell granules, and in vitro it cleaved the pro segment from cryptdin precursors. Matrilysin-deficient (MAT-/-) mice lacked mature cryptdins and accumulated precursor molecules. Intestinal peptide preparations from MAT-/- mice had decreased antimicrobial activity. Orally administered bacteria survived in greater numbers and were more virulent in MAT-/- mice than in MAT+/+ mice. Thus, matrilysin functions in intestinal mucosal defense by regulating the activity of defensins, which may be a common role for this metalloproteinase in its numerous epithelial sites of expression.

The release of endogenous antimicrobial peptides by mammalian epithelial cells contributes to innate mucosal immunity (1, 2). The crypts of Lieberkü hn in the small intestine of most mammals contain Paneth cells that secrete ␣-defensins (cryptdins), lysozyme, secretory phospholipase A 2 , xanthine oxidase, CD95 ligand, CD15, and tumor necrosis factor-␣ as components of apically oriented secretory granules (3-10). Although certain Paneth cell ␣-defensins have been detected in mouse skin and testis (11,12) and in human oropharyngeal and urogenital mucosa (13,14), in the small intestine, ␣-defensins are specific to Paneth cells (9). Exposure of Paneth cells to cholinergic agonists or bacterial stimuli elicits granule discharge into the crypt lumen (15), and carbamylcholine mediates secretion via increased cytosolic Ca 2ϩ (16). Regardless of how mouse Paneth cell secretion is stimulated, cryptdins constitute ϳ70% of the released bactericidal activity, and the concentration of cryptdins is estimated to be 25 mM at the point of secretion in the crypt lumen (15).␣-Defensins are processed from inactive proforms by specific proteolytic cleavage steps. Both neutrophil and Paneth cell ␣-defensins derive from ϳ10-kDa prepropeptides that contain canonical signal sequences, acidic proregions, and an ϳ3.5-kDa mature ␣-defensin peptide in the C-terminal portion of the precursor. For example, maturation of myeloid pro-␣-defensins appears to involve two primary cleavage steps, and most ␣-defensins in mature phagocytic leukocytes are completely processed (17)(18)(19)(20). In a heterologously expressed human neutrophil pro-␣-defensin, deletions in the prosegment adjacent to the proregion-defensin junction impairs post-translational processing in 32DCL3 cells (19).In mouse Paneth cells, matrix metalloproteinase-7 (MMP-7 1 ; matrilysin) mediates the processing and activation of ␣-defensins from 8.4-kDa proforms (21). MMP-7 gene disruption ablates procryptdin processing, resulting in accumulation of cryptdin precursors and the absence of activated mature cryptdin peptides in the small intestine (21). Lacking functional cryptdin peptides, MMP-7-null mice have a defect in clearance of intestinal infections, and they succumb more rapidly and to lower doses of virulent Salmonella typhimurium compared with control mice (21). Thus, the cryptdin deficiency resulting from defective procryptdin activation is associated with a measurable deficit in mucosal immunity and increased risk of systemic disease.In this study, cryptdin biosynthesis was investigated by characterizing details of intracellular procryptdin processing in

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The bactericidal activity of mouse ␣-defensins (cryptdins) requires proteolytic activation of inactive precursors by matrix metalloproteinase-7 (matrilysin, EC 3.4.24.23, MMP-7 a ). To investigate mechanisms of cryptdin-4 (Crp4) peptide interactions with membrane bilayers and to determine whether MMP-7-mediated proteolysis activates the membrane disruptive activity of Crp4, associations of Crp4 and melittin with biomimetic lipid/ polydiacetylene chromatic vesicles were characterized. The peptides differ in their sensitivity to vesicle lipid composition and their depth of bilayer penetration. Crp4 undergoes strong interfacial binding onto lipid bilayers with disruption of the bilayer head group region, unlike melittin, which inserts more deeply into the hydrophobic core of the bilayer. Colorimetric and tryptophan fluorescence studies showed that Crp4 insertion is favored by negatively charged phospholipids and that zwitterionic and Escherichia coli phospholipids promote stronger interfacial binding; melittin-membrane interactions were independent of either variable. In contrast to the membrane disruptive activity of Crp4, pro-Crp4 did not perturb vesicular membranes, consistent with the lack of bactericidal activity of the precursor, and incubation of Crp4 with prosegment in trans blocked Crp4 and G1W-Crp4 membrane interactions at concentrations that inhibit Crp4 bactericidal activity. CD measurements showed that Crp4 has an expected ␤-sheet structure that is not evident in the pro-Crp4 CD trace or when Crp4 is incubated with prosegment, indicating that the ␤-sheet signal is attenuated by proregion interactions or possibly disrupted by the prosegment. Collectively, the results suggest that the prosegment inhibits Crp4 bactericidal activity by blocking peptidemediated perturbation of target cell membranes, a constraint that is relieved when MMP-7 cleaves the prosegment.Antimicrobial peptides have been identified as components of innate immunity in every living organism investigated (1), and the mammalian ␣-defensins were among the first antimicrobial peptide families to be recognized and characterized (2, 3). ␣-Defensins are major constituents of both azurophilic granules in mammalian phagocytic leukocytes and secretory granules of mammalian Paneth cells, where they are termed cryptdins (Crps) 1 in mice (4, 5). In contrast to ␣-defensins in cells of myeloid origin, which provide a nonoxidative means for killing microorganisms after phagocytosis (6), Paneth cell ␣-defensins are secreted to function in the extracellular compartment (7,8). These cationic peptides with molecular masses of 3-4 kDa contain six cysteine residues that form a distinctive tridisulfide array to produce an amphipathic peptide, a feature that is essential for its microbicidal activity (6). In mouse Paneth cells, the production of mature bactericidal ␣-defensins requires proteolytic activation by matrix metalloproteinase-7 (9), a process that precedes secretion (10).Human and rabbit neutrophil ␣-defensins are structurally and functionally disti...