Induction of a rat enteric defensin gene by hemorrhagic shock

Abstract

Multicellular organisms utilize a battery of extracellular and cellular mechanisms to defend against microbial infiltration. Among the armamentarium used by the small intestine to defend against microbial invasion are antimicrobial peptides called defensins. We previously have shown that gut barrier function is impaired following hemorrhagic shock, resulting in translocation of bacteria or endotoxin. Using a rat model, we examined the effect of hemorrhagic shock on alpha-defensin expression. We utilized the anchored reverse transcriptase PCR strategy to isolate a rat enteric defensin cDNA. The cDNA is 406 bases in length and encodes a putative prepro-enteric defensin that we have named rat defensin 5 (RD-5). RD-5 expression is restricted to the small intestine and is specifically localized by in situ hybridization to the Paneth cells. A 10-fold increase in its steady state levels was observed in the distal intestine immediately after the termination of shock. This is the first study to show that enteric defensins are inducible following injury. We suggest that enteric defensins may contribute to the complex and integrated barrier function of the intestinal mucosal surface.

FIG. 1

(A) Nucleotide sequence of RD-5 cDNA and deduced sequence of the encoded immature protein. The consensus sequences for the start of translation and polyadenylation addition are in boldface and underlined. The putative methionine codon is in italics. The conserved cysteines characteristic of defensins are boxed. The predicted mature peptide of 35 amino acid residues is in italics and underlined. The nucleotide sequence is numbered on the right. The predicted amino acid sequence is numbered on the left. (B) Comparison of amino acid sequences of RD-5, mouse cryptdins 15 and 3, HD-5, rat neutrophil defensins 1 and 4, and the invariable consensus found in all α-defensins. The characteristic six conserved cysteines are indicated by solid boxes, while the three other invariant residues (arginine 6, glutamic acid 14, and glycine 18) are indicated by dashed boxes. The exact amino terminus of the putative mature RD-5 peptide cannot be predicted with certainty; therefore, several residues of the putative precursor peptide immediately proximal to the first cysteine are shown in lowercase.

FIG. 2

Tissue distribution of RD-5 expression. RNA samples (5 μg/lane) from the indicated adult rat tissues were electrophoresed, blotted, and hybridized with the RD-5 cDNA. A single band of approximately 700 nucleotides is observed only in the small intestine. Ethidium bromide staining of 18S rRNA demonstrating integrity and equality of loadings is shown in the bottom panel.

FIG. 3

Detection of RD-5 mRNA in crypt cells of the adult rat small intestine by in situ hybridization. (A) Rat ileum hybridized with [³³P]dATP-labeled RCA4A antisense oligonucleotide. Sections were counterstained with hematoxylin and eosin. The arrow indicates silver grains over a Paneth cell in the crypts. Magnification, ×200. (B) Serial section hybridized as for panel A except that the tissue was pretreated with RNase. (C and D) Selected regions shown in panels A and B, respectively. Magnification, ×400.

FIG. 4

(A) Effect of hemorrhagic shock on the level of RD-5 mRNA in the ileum of the rat small intestine. The histogram shows a quantitative representation of hybridization signals obtained from volume analysis of PhosphorImager data. Results are expressed in arbitrary units after normalization to the 18S rRNA signal. Abbreviations: IPS, immediately postshock; 2P, 2 h postshock; 24P, 24 h postshock. Error bars are standard errors of the means; the asterisk indicates statistical significance. (B) Representative Northern blot showing induction of RD-5. RNA samples (5 μg/lane) from control ileum (lanes 1 and 2) and ileum immediately after shock (lanes 3 and 4) were electrophoresed and blotted, and blots were hybridized with the RD-5 cDNA.

FIG. 4

(A) Effect of hemorrhagic shock on the level of RD-5 mRNA in the ileum of the rat small intestine. The histogram shows a quantitative representation of hybridization signals obtained from volume analysis of PhosphorImager data. Results are expressed in arbitrary units after normalization to the 18S rRNA signal. Abbreviations: IPS, immediately postshock; 2P, 2 h postshock; 24P, 24 h postshock. Error bars are standard errors of the means; the asterisk indicates statistical significance. (B) Representative Northern blot showing induction of RD-5. RNA samples (5 μg/lane) from control ileum (lanes 1 and 2) and ileum immediately after shock (lanes 3 and 4) were electrophoresed and blotted, and blots were hybridized with the RD-5 cDNA.