Chapter 12 : Antimicrobial Peptide Effectors of Small Intestinal Innate Immunity

Structures of α-, β-, and θ-defensins. The backbone and disulfide structures are shown of RK-1 (upper left), a monomeric rabbit α-defensin ( 169 , 262 ); HNP-3 (upper right), a dimeric human β-defensin; hBD-1 (lower left), a monomeric human α-defensin; and RTD-1 (lower right), a α-defensin from rhesus macaque.

Primary structures of Paneth cell α-defensins. The single letter notation amino acid sequences of mouse cryptdins 1 to 6, human Paneth cell α-defensins HD-5 and HD-6, and rat Paneth cell α-defensin RD-5 were positioned manually to align conserved Cys, Glu, and Gly residues. Dash characters denote spaces introduced to maintain the alignment of conserved amino acids (bolded typeface) that are conserved in all known α-defensin peptides. The cysteine connectivities that characterize the α-defensin tridisulfide array are shown. The HD-5 and HD-6 peptides have been purified from ileal neobladder urine ( 176 ), and the RD-5 peptide sequence was deduced from the cDNA sequence and by homology with mouse cryptdin N termini ( 34 ).

Recognition and cleavage of mouse procryptdins by MMP-7. In A, samples of procryptdins A, B, and C were incubated overnight with (+) or without (–) MMP-7, and samples of those digests were resolved electrophoretically and stained. Electrophoretic mobilities of individual components are noted at left in descending order as follows: MMP-7, matrilysin; PC, purified procryptdins; Crp, MMP-7-activated cryptdin peptides. As described in the text, MMP-7 is the activating metalloproteinase for all mouse Paneth cell α-defensins. Dashes at right denote, in descending order, the position of 28-, 18-, 15.6-, and 7.6-kDa molecular size markers. In B, the consensus cleavage sites disclosed by protein sequencing of MMP-7 digests of procryptdins A to C are noted by asterisks (☆) that interrupt the procryptdin-1 sequence, and the pound character (#) shows the N terminus of procryptdin intermediates purified from mouse small bowel by Putsep et al. ( 179 ) that were not evident in the procryptdin A to C digests. Numerals above the primary structure refer to residue positions, with the initiating Met residue in preprocryptdin-1 as residue #1. Reprinted from reference 10 with permission.

Release of microbicidal peptide activity by crypt Paneth cells in response to live bacteria. Crypts (solid circles) or villi (shaded diamonds) were isolated from adult mice and incubated in numbers shown with 103 of S. enterica serovar Typhimurium CFU for 1 h at 37°C in 50 µl of isotonic buffer. Viable CFU were quantitated by plating the entire mixtures on semisolid nutrient media overnight. Data points show surviving bacteria from individual replicate mixtures. Open circles represent surviving bacteria after incubation of 103 CFU for 1 h at 37°C in 50 µl of isotonic buffer in the absence of crypts or villi. As noted in the text, bacteria exposed to crypts die in response to Paneth cell secretions ( 11 ), but equivalent exposure to villus epithelium does not affect S. enterica serovar Typhimurium viability. Reprinted from reference 11 with permission.

Role of mIKCa1 in Paneth cell secretion. Exposure of Paneth cells to pharmacologic agents ( 197 ) or bacterial antigens results in an initial increase in cytosolic Ca2+ ([Ca2+i]) by mobilization of intracellular stores. mIKCa1 channels in the Paneth cell membrane would be predicted to open as cytosolic [Ca2+i] approaches 300 nM, providing the counterbalancing K_ efflux necessary to sustain Ca2+ entry from the external milieu ( 12 ). As noted in detail in the text, blockade of mIKCa1 would depolarize the membrane and attenuate the calcium-signaling response required to generate a complete Paneth cell secretory response. Electron micrograph was generously provided by Susan J. Hagen, Beth Israel Deaconess Medical Center, Boston, Mass.

Dense granules in mouse small intestinal intermediate granulomucous cells during T. spiralis infection. (A) This transmission electron micrograph shows evidence of extensive Paneth cell degranulation into the crypt lumen (L). Cells at the top of the field display large electron dense granules within electron-lucent mucus-containing granules, the hallmark of intermediate granulomucous cells. (B) Two representative intermediate cells that were detected above the crypt villus junction. Such cells are immunopositive for mouse Paneth cell α-defensins and are not found after clearance of the infection ( 101 ). Reprinted from reference 101 , with permission.

Paneth cells in the context of the small intestinal villus-crypt axis ( 56 ). Stem cells in small intestinal crypts divide, and their progeny migrate upward toward the villi or descend toward the base of the crypt. Migration toward the villus tips is accompanied by cellular differentiation into absorptive enterocytes, goblet cells, or enteroendocrine cells. The life span of these villus cells from their origin in the crypt, through migration and differentiation, until apoptotic death and exfoliation into the lumen is approximately 2 to 5 days. Stem cell progeny that migrate to the crypt base differentiate into Paneth cells that live for several weeks. (Inset) Paneth cells release secretory vesicles into the narrow lumen of the crypt ( 56 ). The secretory responses are mediated by Ca2+, and the secretions contain α-defensins, lysozyme, and secretory phospholipase A2. Illustration by D. Schumick, Department of Medical Illustration, Cleveland Clinic Foundation, ©2000, Cleveland Clinic Foundation. Reprinted from reference 56 , with permission.