L-MBP is expressed in epithelial cells of mouse small intestine

The mannan-binding proteins (L-MBP and S-MBP, also denoted MBL-C and MBL-A), mainly produced in liver and existing in liver and serum, play important roles in the innate immunity against a variety of pathogens. Total RNA from mouse tissues were screened for MBP mRNA by RT-PCR. In addition to liver, S-MBP mRNA was detected in lung, kidney, and testis, and L-MBP mRNA was detected in kidney, thymus, and small intestine. Quantitative RT-PCR revealed that the small intestine is a predominant site of extrahepatic expression of L-MBP. Western blotting with polyclonal Abs against rat L-MBP demonstrated this protein in Triton X-100 extracts of the small intestine obtained from mice that had undergone systemic perfusion. Immunohistochemical staining with an mAb against mouse L-MBP and in situ hybridization revealed that L-MBP is selectively expressed in some villous epithelial cells of the small intestine. These findings suggest that L-MBP plays a role in mucosal innate immunity.     

Activation of Paneth cell alpha-defensins in mouse small intestine.

Paneth cells in small intestine crypts secrete microbicidal alpha-defensins, termed cryptdins, as components of enteric innate immunity. The bactericidal activity of cryptdins requires proteolytic activation of precursors by matrix metalloproteinase-7 (MMP-7; matrilysin) (Wilson, C. L., Ouellette, A. J., Satchell, D. P., Ayabe, T., Lopez-Boado, Y. S., Stratman, J. L., Hultgren, S. J., Matrisian, L. M., and Parks, W. C. (1999) Science 286, 113-117). Here, we report on the intracellular processing of cryptdin proforms in mouse Paneth cells. Peptide sequencing of MMP-7 digests of purified natural procryptdins identified conserved cleavage sites in the proregion between Ser(43) and Val(44) as well as at the cryptdin peptide N terminus between Ser(58) and Leu(59). Immunostaining co-localized precursor prosegments and mature cryptdin peptides to Paneth cell granules, providing evidence of their secretion. Extensive MMP-7-dependent procryptdin processing occurs in Paneth cells, as shown by Western blot analyses of intestinal crypt proteins and proteins from granule-enriched subcellular fractions. The addition of soluble prosegments to in vitro antimicrobial peptide assays inhibited the bactericidal activities of cryptdin-3 and -4 in trans, suggesting possible cytoprotective effects by prosegments prior to secretion. Levels of activated cryptdins were normal in small bowel of germ-free mice and in sterile implants of fetal mouse small intestine grown subcutaneously. Thus, the initiation of procryptdin processing by MMP-7 does not require direct bacterial exposure, and the basal MMP-7 content of germ-free Paneth cells is sufficient to process and activate alpha-defensin precursors. MMP-7-dependent procryptdin activation in vivo provides mouse Paneth cells with functional peptides for apical secretion into the small intestine lumen.     

Localization of vitamin A in the small intestine of mouse

Summary Localization of vitamin A in the small intestine of mice was studied with electron microscope radioautography after administration of tritiated vitamin A. The label was concentrated over lipid droplets in cells distributed in the lamina propria and the submucous layer. The cells were similar both to fibroblasts and to fat-storing cells in their morphological features. The name Vitamin A-Storing Cell is proposed for these labeled cells, including the fat-storing cell in the liver.     

Caveolin-1 knockout alters beta-adrenoceptors function in mouse small intestine

beta-Adrenoceptors are G protein-coupled receptors whose functions are closely associated with caveolae in the heart and cultured cell lines. In the gut, they are responsible, at least in part, for the mediation of the sympathetic stimulation that might lead to intestinal paralysis postoperatively. We examined the effect of caveolin-1 knockout on the beta-adrenoceptor response in mouse small intestine. The relaxation response to (-)-isoprenaline in carbachol-contracted small intestinal tissue segments was reduced in caveolin-1 knockout mice (cav1(-/-)) compared with their genetic controls (cav1(+/+)). Immunohistochemical staining showed that beta-adrenoceptor expression was similar in both strains in gut smooth muscle. Selective beta-adrenoceptor blockers shifted the concentration response curve (CRC) of (-)-isoprenaline to the right in cav1(+/+) intestine, but not in cav1(-/-), with greatest shift in case of the beta(3)-blocker, SR59230A. The CRC of the selective beta(3)-agonist BRL 37344 was also shifted to the right in cav1(-/-) compared with cav1(+/+). The cAMP-dependent protein kinase (PKA) inhibitor H-89 shifted the CRC of (-)-isoprenaline to the right in cav1(+/+) but not in cav1(-/-). H-89 reduced the relaxation due to forskolin and dibutyryl cAMP in cav1(+/+) but not in cav1(-/-), suggesting a reduction in PKA activity in cav1(-/-). In cav1(+/+), PKA was colocalized with caveolin-1 in the cell membrane, but PKA immunoreactivity persisted in cav1(-/-). Examination of PKA expression in the lipid raft-rich membrane fraction of the jejunum revealed reduced PKA expression in cav1(-/-) compared with cav1(+/+). The results of the present study show that the function of beta-adrenoceptors is reduced in cav1(-/-) small intestine likely owing to reduced PKA activity.     

Immunohistochemical analysis of neuron types in the mouse small intestine

The definition of the nerve cell types of the myenteric plexus of the mouse small intestine has become important, as more researchers turn to the use of mice with genetic mutations to analyze roles of specific genes and their products in enteric nervous system function and to investigate animal models of disease. We have used a suite of antibodies to define neurons by their shapes, sizes, and neurochemistry in the myenteric plexus. Anti-Hu antibodies were used to reveal all nerve cells, and the major subpopulations were defined in relation to the Hu-positive neurons. Morphological Type II neurons, revealed by anti-neurofilament and anti-calcitonin gene-related peptide antibodies, represented 26% of neurons. The axons of the Type II neurons projected through the circular muscle and submucosa to the mucosa. The cell bodies were immunoreactive for choline acetyltransferase (ChAT), and their terminals were immunoreactive for vesicular acetylcholine transporter (VAChT). Nitric oxide synthase (NOS) occurred in 29% of nerve cells. Most were also immunoreactive for vasoactive intestinal peptide, but they were not tachykinin (TK)-immunoreactive, and only 10% were ChAT-immunoreactive. Numerous NOS terminals occurred in the circular muscle. We deduced that 90% of NOS neurons were inhibitory motor neurons to the muscle (26% of all neurons) and 10% (3% of all neurons) were interneurons. Calretinin immunoreactivity was found in a high proportion of neurons (52%). Many of these had TK immunoreactivity. Small calretinin neurons were identified as excitatory neurons to the longitudinal muscle (about 20% of neurons, with ChAT/calretinin/± TK chemical coding). Excitatory neurons to the circular muscle (about 10% of neurons) had the same coding. Calretinin immunoreactivity also occurred in a proportion of Type II neurons. Thus, over 90% of neurons in the myenteric plexus of the mouse small intestine can be currently identified by their neurochemistry and shape.     

Class II antigen-associated invariant chain mRNA in mouse small intestine.

MHC class II antigen-associated invariant (Ii) chain mRNA appears in mouse small intestine during postnatal development. Ii chain cDNA hybridizes to RNA from epithelial sheets dissociated from the lamina propria with EDTA. Of several mouse organs tested, only bone marrow and spleen contain higher levels of Ii chain mRNA than small bowel. Ii chain mRNA is not detected in stomach, colon, duodenum, testis, liver, submandibular gland, or L-cell RNA; brain contains a cross-reactive but uncharacterized sequence. cDNA amplification using primers specific for both Ii31 and Ii41 chain mRNAs showed that both forms occur in small intestine. These results support the conclusion that regulation of the class II Ii chain gene is associated with the ontogeny of intestinal immunity.     

The kinetics of villus cell populations in the mouse small intestine

Abstract. The cell population kinetics of the villus epithelium of the mouse have been analysed with respect to the size, flux and time. Microdissection methods were employed to measure the villus cell population size and yielded reproducible, precise results. There was a proximodistal negative size gradient in villus cell population and, in those villi of normal morphology, there was a good correlation with the usual morphometric estimators such as height and row count, although correlation was improved by a product variable consisting of a height multiplied by a width parameter.
Flux onto the villus is the product of the crypt cell production rate, which was measured by a metaphase arrest method using vincristine and crypt microdissection, and the crypt:villus ratio; net villus influx was maximum proximally in the bowel, where the largest villi were found, and decreased distally. The distribution of transit times of labelled cells to the crypt: villus junction and to the villus tip was measured, allowing the measurement of the median villus transit time.
Comparison of the measured villus transit time with the theoretical transit time calculated from the villus influx and population size gave results consistent with a steady state hypothesis. It was found, at each level of the small intestine studied, that the number of epithelial cells on the villus was equivalent to the total number of crypt cells associated with the villus.     

Cell kinetics in the mouse small intestine during immediate postnatal life

The cell proliferation kinetics in the small intestine of newborn Balb/c mice were studied, from day 1 through to day 21 after birth. The size of the functional compartment and the proliferation compartment was determined as well as the cell production rate in the crypt using the micro-dissection technique combined with metaphase arrest method. The effect of weaning on cell proliferation was studied. The results suggest that: (1)There is continuous increase in cell proliferation and in the size of the functional and proliferative compartment over the 21 days. (2) The cell proliferation proceeds at a slower rate than in adult animals. (3) There is a sharp increase in cell production rate during the third week of postnatal life. (4) The cell proliferation was faster in conventionally weaned litters than the non-weaned group. (5) The intestinal mucosa in newborn mice is not in a steady-state conditions as in adults; cell production rate exceeds cell loss.     

Starvation induces phase-specific changes in the proteome of mouse small intestine

Food deprivation results in metabolic, structural, and functional changes in the small intestine that influences gut mucosal integrity, epithelial cell proliferation, mucin synthesis, and other processes. The underlying mechanisms are still unclear, which lead to the study of molecular effects of short-term and long-term starvation in the intestine of mice. A comparative proteomics approach, combining two-dimensional gel electrophoresis with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, was used to identify intestinal proteins whose expression is changed under different starvation conditions (0, 12, 24, and 72 h). In total, the expression levels of 80 protein spots changed significantly between the different groups. The results demonstrate that after 12 h of starvation, mainly proteins involved in glycolysis and energy metabolism show decreased expression levels. Starvation for 24 h results in a down-regulation of proteins involved in protein synthesis and amino acid metabolism. Simultaneously, proteins with a protective role, e.g., reg I and II, glutathione peroxidase 3, and carbonic anhydrase 3, are clearly up-regulated. The last starvation phase (72 h) is characterized by increased ezrin expression, which may enhance villus morphogenesis critical for survival. Together, these results provide novel insights in the intestinal starvation response and may contribute to improved nutritional support during conditions characterized by malnutrition.     

The distribution of endocrine cells along the mouse small intestine

The topographical distribution and incidence of endocrine cells in the crypt and villus epithelium and along the length of the mouse intestine was studied. Cells containing somatostatin and bombesin like reactivity were stained by immunocytochemical techniques using polyclonal antiserum. Most of the somatostatin cells were found in the duodenum, jejunum and ileum, and these cells were generally more frequent on the villus compared to the crypts. This may indicate that the somatostatin cells develop late in the endocrine cell lineage. Bombesin like cells were rare in occurence, and were only present in measureable numbers in the ileum, where they were observed in the crypt and villi. The application of ELISA assays to determine the specificity of the antisera for these peptides is also discussed.     

Phenotypic characterization of taste cells of the mouse small intestine

Nutrient-evoked gastrointestinal reflexes are likely initiated by specialized epithelial cells located in the small intestine that detect luminal stimuli and release mediators that activate vagal endings. The G-protein alpha-gustducin, a key signal molecule in lingual taste detection, has been identified in mouse small intestine, where it may also subserve nutrient detection; however, the phenotype of alpha-gustducin cells is unknown. Immunohistochemistry was performed throughout the mouse small intestine for alpha-gustducin, enteroendocrine cell markers 5-HT and glucagon-like peptide-1 (GLP-1), and brush cell markers neuronal nitric oxide synthase and Ulex europaeus agglutinin-1 (UEA-1) lectin binding, singly, and in combination. alpha-Gustducin was expressed in solitary epithelial cells of the mid to upper villus, which were distributed in a regional manner with most occurring within the midjejunum. Here, 27% of alpha-gustducin cells colabeled for 5-HT and 15% for GLP-1; 57% of alpha-gustducin cells colabeled UEA-1, with no triple labeling. alpha-Gustducin cells that colabeled for 5-HT or GLP-1 were of distinct morphology and exhibited a different alpha-gustducin immunolabeling pattern to those colabeled with UEA-1. Neuronal nitric oxide synthase was absent from intestinal epithelium despite strong labeling in the myenteric plexus. We conclude that subsets of enteroendocrine cells in the midjejunum and brush cells (more generally distributed) are equipped to utilize alpha-gustducin signaling in mice. Intestinal taste modalities may be signaled by these enteroendocrine cells via the release of 5-HT, GLP-1, or coexpressed mediators or by brush cells via a nonnitrergic mediator in distinct regions of the intestine.     

Response of mouse small intestine to fractionated doses of pions

The effect of single or fractionated doses of stopping pions or 200 kV X-rays on the mouse jejunal crypt cells was used to determine in vivo RBE values. For single fraction, the pion/X-ray RBE was 1.27 and it increased to about 1.31 when two fractions were applied at 3 or 24 h interval. When four fractions were given at 3 h intervals, the RBE was 1.46. This is because the fraction of "dose repaired" was always higher for X-rays than for pions and this difference was enhanced when more dose fractions were used. The data presented is, in general, consistent with the biological effects of pions reported for other in vivo end points.     

Three-dimensional organization of lymphatics and its relationship to blood vessels in rat small intestine

Summary The lymphatic organization and its relationship to the vascular system in the rat small intestine was studied by scanning electron microscopy of corrosion casts and freeze-fractured tissues, and by light microscopy of injected preparations. The villus possessed 3–10 or more central lacteals depending upon the villous width. The lacteals in each villus possessed interconnections between adjacent ones and were surrounded externally by the villous capillary network. At the villous base, the lacteals fused and formed a wide sinus, from which 2 or 3 lymphatics descended and led into the submucosal ones. In the muscularis externa there was a coarse lymphatic network which, together with the submucosal one, drained into collecting lymphatics continuous with the mesenteric ones. The central lacteals and the sinus were lined with thin endothelial cells with cytoplasmic leaves interdigitating with those of adjacent ones. There were tissue channels in the villous interstitial space, which opened through the gaps between the lymphatic endothelial cells into the central lacteals.The voluminous lacteals in the villi suggest their great potential for lymph formation. The existence of collecting lymphatics with valves in the muscularis externa suggests that contraction of the layer is involved in transporting lymph towards the efferent lymphatics.     

Development of enteropeptidase activity in mouse small intestine: influence of hormones

The postnatal development of enteropeptidase activity has been examined on mucosal scrapping of the proximal part of the mouse small intestine. The activity was present at birth and remained low during the first 15 days of life. Then it rapidly increased reaching adult level within 2 days. Daily administration of cortisone acetate (25 micrograms X g body weight (bw)-1 X day-1), insulin (12.5 mU X g bw-1 X day-1), or epidermal growth factor (4 micrograms X g bw-1 X day-1) during 3 days to 8-day-old mice induced a premature increase of enteropeptidase. The maximal increase was observed with cortisone treatment, the enzymic activity representing 70% of the adult level. Thyroxine alone (1 microgram X g bw-1 X day-1) had no significant effect on enteropeptidase activity. Hormonal interactions have been evaluated by studying the effects of different hormonal combinations. Finally, cortisone acetate which has a major effect on this activity during suckling period was unable to influence adult small intestinal enteropeptidase activity.     

Altered transit and bacterial overgrowth in the cystic fibrosis mouse small intestine

Small intestinal bacterial overgrowth (SIBO) may play an important role in the gastrointestinal complications of cystic fibrosis (CF). This work explored two potential factors in development of SIBO in the CF (cftr(tm1UNC)) mouse: impaired Paneth cell innate defenses and altered gastrointestinal motility. Postnatal differentiation of Paneth cells was followed by Defcr, Lyzs, and Ang4 gene expression, and SIBO was measured by quantitative PCR of the bacterial 16S rRNA gene. Paneth cell gene expression was low in 4-day-old CF and wild-type (WT) mice and increased similarly in both groups of mice between 12 and 16 days. Peak Paneth cell gene expression was reached by 40 days of age and was less for Defcr and Lyzs in CF mice compared with WT, whereas Ang4 levels were greater in CF mice. SIBO occurred by postnatal day 8 in CF mice, which is before Paneth cell development. With the use of gavaged rhodamine-dextran to follow motility, gastric emptying in CF mice was slightly decreased compared with WT, and small intestinal transit was dramatically less. Since antibiotics improve weight gain in CF mice, their effects on gastric emptying and small intestinal transit were determined. Antibiotics did not affect gastric emptying or transit in CF mice but did significantly slow intestinal transit in WT mice, suggesting a potential role of normal microflora in regulating transit. In conclusion, small intestinal transit was significantly slower in CF mice, and this is likely a major factor in SIBO in CF.