Immunohistochemical observations of immunoglobulin A in the Paneth cells of germ-free and formerly-germ-free rats

The localization of secretory immunoglobulin A (IgA) in Paneth cells was immunohistochemically studied in germ-free (Gf) and ex-Gf rats that had been injected with feces obtained from specific-pathogen-free (SPF) rats. In Gf as well as SPF rats, the secretory granules of Paneth cells and the brush borders of crypt cells exhibited IgA immunoreactivity. At 12 and 24 h after inoculation, it was found that, concomitant with the occurrence of considerable degranulation, the IgA immunoreactivity in Paneth cells disappeared, except of the margin of supranuclear vacuoles. In contrast, the IgA immunoreactivity of the crypt-cell brush borders was unchanged. Four days after inoculation, secretory granules exhibiting IgA immunoreactivity reaccumulated in Paneth cells. The present study suggests that Paneth cells regulate the bacterial milieu in the intestine by releasing secretory granules containing IgA into the crypt lumen.     

Atropine inhibits the degranulation of Paneth cells in ex-germ-free mice

Summary Previous studies have shown that the secretory products of Paneth cells contain antibacterial agents (lysozyme, IgA) that are affected by the bacterial milieu in the intestine. To investigate whether Paneth-cell secretion is controlled via cholinergic mechanisms, the ultrastructure of Paneth cells was studied in four animal groups: (1) germfree (GF) control mice (Jcl: ICR [GN], male, 13 weeks old), (2) GF mice injected subcutaneously with atropine sulfate (200 mg/kg body weight, dissolved in physiological saline 20 mg/ml), (3) ex-GF mice inoculated with feces from specific-pathogen-free (SPF) mice, and (4) ex-GF mice injected with atropine and inoculated with feces from SPF mice. In ex-GF mice inoculated with feces, 70–90% of the Paneth cells showed fewer secretory granules than those from GF mice (p<0.01). Approximately 30% of the Paneth cells had a large vacuole (3–10 m diameter) in the apical cytoplasm. Exocytosed electron-dense material from secretory granules was observed in a few crypt lumens. In ex-GF mice inoculated with feces and given atropine, about 90% of the Paneth cells contained numerous secretory granules, like those in GF control mice, but vacuolated Paneth cells and exocytotic figures were rare; thus the secretion of Paneth cells was blocked by atropine. It is therefore possible that the bacterial milieu in the intestine affects the secretory activity of Paneth cells via cholinergic mechanisms.     

Immunocytochemical localization of secretory component in Paneth cell secretory granules-rat Paneth cells participate in acquired immunity

Summary With the marker of Paneth cells-lysozyme, secretory component (SC) immunoreactivity was demonstrated exclusively in Paneth cells of rat small intestine. The other types of epithelial cells (columnar, goblet, endocrine) were negative. On electron microscopic level, many SC-positive colloidal gold particles were found in rough endoplasmic reticulum, Golgi complexes, basal membrane and secretory granules of Paneth cells. These results suggest that SC is not a component of ingested immune complex, but a membrane receptor on Paneth cell. It may function as receptor for polymeric IgA and mediate its transport across the mucosal epithelium. Thus, Paneth cells are responsible for SC synthesis and participate in IgA-mediated acquired immunity in rat small intestine.     

Effect of live and heat-killed bacteria on the secretory activity of Paneth cells in germ-free mice

Summary Germ-free mice were given live or heat-killed facultative anaerobes, and the ultrastructure of ileal Paneth cells was quantitatively examined with special reference to secretory granules showing a bipartite substructure (central core and peripheral halo). After administering live or heatkilled bacteria, there was a decrease in the area occupied by the cores of secretory granules in Paneth cells, and exocytosed core material was observed in the crypt lumen. There were no changes in the area occupied by the halo of secretory granules. None of the examined Paneth cells phagocytosed bacteria. It is concluded that certain bacteria may affect the secretion of antibacterial agents contained in the secretory granules of Paneth cells.     

STUDIES ON SMALL INTESTINAL CRYPT EPITHELIUM : I. The Fine Structure of the Crypt Epithelium of the Proximal Small Intestine of Fasting Humans

Small intestinal crypt epithelium obtained from normal fasting humans by peroral biopsy of the mucosa was studied with the electron microscope. Paneth cells were identified at the base of the crypts by their elaborate highly organized endoplasmic reticulum, large secretory granules, and small lysosome-like dense bodies within the cytoplasm. Undifferentiated cells were characterized by smaller cytoplasmic membrane-bounded granules which were presumed to be secretory in nature, a less elaborate endoplasmic reticulum, many unattached ribosomes and, in some cells, the presence of glycogen. Some undifferentiated cells at the base of the crypts contained lobulated nuclei and striking paranuclear accumulations of mitochondria. Membrane-bounded cytoplasmic fragments, probably originating from undifferentiated and Paneth cells, were frequently apparent within crypt lumina. Of the goblet cells, some were seen actively secreting mucus. In these, apical mucus appeared to exude into the crypt lumen between gaps in the microvilli. The membrane formerly surrounding the apical mucus appeared to fuse with and become part of the plasma membrane of the cell, suggesting a merocrine secretory mechanism. Enterochromaffin cells were identified by their location between the basal regions of other crypt cells and by their unique intracytoplasmic granules.     

Immunohistochemical characterization of a crypt cell-specific plasma membrane protein in rat small intestine epithelium using a monoclonal antibody

Proteins of the basolateral membrane (BLM) of small intestine epithelial cells of adult rats, in the MW ranges of 50-65 KD, 85-100 KD, and over 100 KD, were obtained as follows. After isolation of the BLM and subsequent SDS-PAGE and transblotting of the proteins on nitrocellulose sheets, the bands in these MW ranges were cut out of the nitrocellulose sheet and extracted. Balb/C mice were immunized with these protein fractions and a monoclonal antibody (MAb) was then produced. MAb SI/CC1 obtained via immunization with the 50-65 KD protein fraction shows specificity for the crypt epithelium of the small intestine. It can be used to characterize, by light and electron microscopic immunohistochemical methods, a crypt cell protein (SI/CC1-Ag) with a very specific localization. Fluorescence labeling shows that the SI/CC1-Ag can be found only in the epithelium of small intestine crypts (except for the granules in eosinophilic granulocytes). The epithelium of the colon, as well as the epithelia of other organs, could not be labeled. In the small intestine crypts, SI/CC1-Ag is found only in the Paneth cells located in the basal crypt section, and in the undifferentiated cells in the middle crypt section; it is lacking in the cells of the upper crypt section. Gold labeling shows that SI/CC1-Ag in the undifferentiated cells is localized exclusively in the basolateral PM domain. On the Paneth cells, the content of the secretory granules is labeled, along with the basolateral PM domain; the labeling sometimes present on their luminal part is probably due to passively absorbed secretion from these cells. The SI/CC1-Ag in the BLM of undifferentiated and Paneth cells is found only on Days 21-23 post partum, whereas the Paneth cell granules could be labeled as early as the Day 16 post partum. With immunodetection with SI/CC1, one band at about 55 KD is specifically labeled in the protein pattern of the isolated small intestine cell BLM. In the protein pattern of the isolated crypt cells two bands were labeled, again one at 55 KD and one at about 120 KD. These findings indicate that SI/CC1-Ag is a 55 KD protein that appears on Days 21-23 post partum in the BLM of undifferentiated cells and of Paneth cells.     

Expression of a subclass of human blood group A antigenicity in A+ rabbit jejunum: specific glycosylation of the glycocalyx and a 140 K brush border glycoprotein

A monoclonal antibody (14 A4) raised against human A erythrocytes has been produced. It specifically reacts with a subclass of human blood group A determinants. Whereas all the major secreted and membrane-bound glycoproteins of A+ rabbit jejunum epithelium bear human blood group A-like determinants recognized by anti-A polyclonal serum or a monoclonal antibody with broad specificity (Cl 3.3), expression of the A-subclass recognized by 14 A4 is very restricted. The contents of secretory granules of Paneth cells but not the mucins of goblet cells were labeled by 14 A4. In the enterocytes, glycans recognized by 14 A4 were present in the glycocalyx, on an early expressed 140 K glycoprotein of brush border membranes and also on a glycoconjugate of the basolateral membrane of immature crypt cells. In the jejunal brush border membrane of blood group A secretor humans, only one glycoprotein of molecular weight 140 K bears the A-subclass recognized by 14 A4.     

Ultrastructural localization of osteopontin immunoreactivity in phagolys osomes and secretory granules of cells in human intestine

A post-embedding ultrastructural immunogold method was used to detect osteopontin in human intestinal biopsies with special emphasis on secretory and phagocytic organelles. Osteopontin immunoreactivity was localized to phagolysosomes of macrophages, fibroblasts, absorptive epithelial cells of the small intestine and Paneth cells. The mucigen secretory granules and Golgi structures of mucous epithelial cells of the small intestinal epithelium contained osteopontin, but secretory granules of numerous other cells, including Paneth cells, did not. Extracellular and phagocytosed Tropheryma whippelii within macrophage phagolysosomes also bound osteopontin. These localizations are supportive of a role for osteopontin in phagocytic and some secretory cell functions in human intestine     

Ultrastructural localization of osteopontin immunoreactivity in phagolys osomes and secretory granules of cells in human intestine

A post-embedding ultrastructural immunogold method was used to detect osteopontin in human intestinal biopsies with special emphasis on secretory and phagocytic organelles. Osteopontin immunoreactivity was localized to phagolysosomes of macrophages, fibroblasts, absorptive epithelial cells of the small intestine and Paneth cells. The mucigen secretory granules and Golgi structures of mucous epithelial cells of the small intestinal epithelium contained osteopontin, but secretory granules of numerous other cells, including Paneth cells, did not. Extracellular and phagocytosed Tropheryma whippelii within macrophage phagolysosomes also bound osteopontin. These localizations are supportive of a role for osteopontin in phagocytic and some secretory cell functions in human intestine This revised version was published online in November 2006 with corrections to the Cover Date.     

Human blood group A-like determinants as marker of the intracellular pools of glycoproteins in secretory and absorbing of A+ rabbit jejunum

Human blood group A antigenicity of glycoproteins is retained on epon-embedded jejunum sections after glutaraldehyde fixation and osmium treatment. The intracellular location of molecules bearing these determinants was visualized in the four types of epithelial cells of A+ rabbit jejunum sections with immuno-colloidal gold labeling. The brush border membrane and in particular the glycocalyx of absorbing cells as well as the secretory granules of goblet and Paneth cells were heavily labeled. In enteroendocrine cells, the membrane of secretory granules and not their content was lightly labeled. The differential labeling of secretory or membrane bound glycoproteins is accompanied by different labels of the Golgi complex as expected if labeling of the Golgi saccules was due to the presence of glycoproteins in transit. In all cases the label is primarily concentrated in only half the cisternae on the trans side of the Golgi stacks. In absorbing cells, structures have been revealed in the terminal web that could be related to the brush border membrane and consequently implicated in its biogenesis. The fibrillar material of the glycocalyx appears as highly labeled tangled structures which apparently proceed from densely stained "carrier" vesicles arising from the Golgi apparatus. Vesicles fusing at the lower part of microvilli could result of integration of this material into the lightly labeled vesicles strictly found in the terminal web. These last vesicles could also contain newly synthesized brush border hydrolases.     

Rapid expansion of intestinal secretory lineages following a massive small bowel resection in mice

Following massive small bowel resection (SBR) in mice, there are sustained increases in crypt depth and villus height, resulting in enhanced mucosal surface area. The early mechanisms responsible for resetting and sustaining this increase are presently not understood. We hypothesized that expansion of secretory lineages is an early and sustained component of the adaptive response. This was assessed in the ileum by quantitative morphometry at 12 h, 36 h, 7 days, and 28 days and by quantitative RT-PCR of marker mRNAs for proliferation and differentiated goblet, Paneth cell, and enterocyte genes at 12 h after 50% SBR or sham operation. As predicted, SBR elicited increases of both crypt and villus epithelial cells, which were sustained though the 28 days of the experiment. Significant increases in the overall number and percentage of both Paneth and goblet cells within intestinal epithelium occurred by 12 h and were sustained up to 28 days after SBR. The increases of goblet cells after SBR were initially observed within villi at 12 h, with marked increases occurring in crypts at 36 h and 7 days. Consistent with this finding, qRT-PCR demonstrated significant increases in the expression of mRNAs associated with proliferation (c-myc) and differentiated goblet cells (Tff3, Muc2) and Paneth cells (lysozyme), whereas mRNA associated with differentiated enterocytes (sucrase-isomaltase) remained unchanged. From these data, we speculate that early expansion of intestinal secretory lineages within the epithelium of the ileum occurs following SBR, possibly serving to amplify the signal responsible for initiating and sustaining intestinal adaptation.     

Zinc-secreting Paneth cells studied by ZP fluorescence.

We have used a new family of zinc-specific-responsive fluorescent dyes (ZPs) to study the sequestration and secretion of zinc from Paneth cells, which are located in the bases of the crypts of Lieberkühn within the rat small intestine. Vivid ZP fluorescence zinc staining of Paneth cell secretory granules is seen in both cryostat sections and isolated crypts, providing firm evidence for a pool of labile (rapidly exchangeable) zinc within these cells. We further demonstrate that this ionic zinc pool is secreted under physiological conditions. In vivo stimulation of the small intestine by IP injection of the secretagogue pilocarpine results in discrete zinc staining within the lumens of subsequently isolated crypts, concomitant with a decrease in the zinc staining of Paneth cell granules located within the same crypts. In contrast, the secretion of zinc into the lumens of isolated crypts stimulated in vitro with either carbachol or LPS (lipopolysaccharide) is not observed. However, a distinct change in Paneth cell morphology, suggesting attempted secretion, is seen in response to the direct application of cholinergics but not LPS. These findings suggest that zinc is coreleased with other Paneth cell anti-microbials, and that the intact intestine is necessary for secretion into the crypt lumen.     

Immunocytochemical localization of pancreatic carboxylic ester hydrolase in human paneth cells

The protein-A gold method using specific rabbit sera directed against pure human pancreatic chymotrypsinogen and carboxylic ester hydrolase was applied to locate these (pro)enzymes in human pancreatic acinar cells and intestinal Paneth cells. Quantitative evaluation of the labelling indicated that both (pro)enzymes are present in pancreatic acinar secretory granules. In Paneth cell secretory granules, only carboxylic ester hydrolase was present in significant amounts, although the labelling for this enzyme was less intense than that observed in pancreatic zymogen granules. The results obtained support the view that Paneth cells represent a "diffuse exocrine gland" scattered along the intestine, whose role is either to act as a substitute in the event of a deficient pancreas or to regulate the intestinal flora.     

Abnormal Paneth cell granule dissolution and compromised resistance to bacterial colonization in the intestine of CF mice

Paneth cells of intestinal crypts contribute to host defense by producing antimicrobial peptides that are packaged as granules for secretion into the crypt lumen. Here, we provide evidence using light and electron microscopy that postsecretory Paneth cell granules undergo limited dissolution and accumulate within the intestinal crypts of cystic fibrosis (CF) mice. On the basis of this finding, we evaluated bacterial colonization and expression of two major constituents of Paneth cells, i.e., alpha-defensins (cryptdins) and lysozyme, in CF murine intestine. Paneth cell granules accumulated in intestinal crypt lumens in both untreated CF mice with impending intestinal obstruction and in CF mice treated with an osmotic laxative that prevented overt clinical symptoms and mucus accretion. Ultrastructure studies indicated little change in granule morphology within mucus casts, whereas granules in laxative-treated mice appear to undergo limited dissolution. Protein extracts from CF intestine had increased levels of processed cryptdins compared with those from wild-type (WT) littermates. Nonetheless, colonization with aerobic bacteria species was not diminished in the CF intestine and oral challenge with a cryptdin-sensitive enteric pathogen, Salmonella typhimurium, resulted in greater colonization of CF compared with WT intestine. Modest downregulation of cryptdin and lysozyme mRNA in CF intestine was shown by microarray analysis, real-time quantitative PCR, and Northern blot analysis. Based on these findings, we conclude that antimicrobial peptide activity in CF mouse intestine is compromised by inadequate dissolution of Paneth cell granules within the crypt lumens.     

Modelling the spatio-temporal cell dynamics reveals novel insights on cell differentiation and proliferation in the small intestinal crypt

We developed a slow structural relaxation model to describe cellular dynamics in the crypt of the mouse small intestine. Cells are arranged in a three dimensional spiral the size of which dynamically changes according to cell production demands of adjacent villi. Cell differentiation and proliferation is regulated through Wnt and Notch signals, the strength of which depends on the local cell composition. The highest level of Wnt activity is associated with maintaining equipotent stem cells (SC), Paneth cells and common goblet-Paneth cell progenitors (CGPCPs) intermingling at the crypt bottom. Low levels of Wnt signalling area are associated with stem cells giving rise to secretory cells (CGPCPs, enteroendocrine or Tuft cells) and proliferative absorptive progenitors. Deciding between these two fates, secretory and stem/absorptive cells, depends on Notch signalling. Our model predicts that Notch signalling inhibits secretory fate if more than 50% of cells they are in contact with belong to the secretory lineage. CGPCPs under high Wnt signalling will differentiate into Paneth cells while those migrating out from the crypt bottom differentiate into goblet cells. We have assumed that mature Paneth cells migrating upwards undergo anoikis. Structural relaxation explains the localisation of Paneth cells to the crypt bottom in the absence of active forces. The predicted crypt generation time from one SC is 4-5 days with 10-12 days needed to reach a structural steady state. Our predictions are consistent with experimental observations made under altered Wnt and Notch signalling. Mutations affecting stem cells located at the crypt floor have a 50% chance of being propagated throughout the crypt while mutations in cells above are rarely propagated. The predicted recovery time of an injured crypt losing half of its cells is approximately 2 days.     

Protein turnover in intestinal mucosal villus and crypt brush border membranes

Relative turnover rates of intestinal brush border proteins have been studied by double labelled technique. Brush borders were isolated from cells at all levels along the villus, and from the crypts. Proteins with large molecular weight (>150,000) demonstrated more rapid turnover compared with other brush border proteins at all levels along the villus. This rapid turnover was not seen in crypt brush borders. These findings support the concept of protein turnover in intestinal brush borders, and demonstrate differences between the proteins in rapidly growing crypt cells and non growing villus cells.     

Fetal gut mesenchyme induces differentiation of cultured intestinal endodermal and crypt cells

An experimental model was designed to analyze the effect of fetal gut mesenchyme on the cytodifferentiation of crypt cells and of embryonic progenitor cells. The cells used were the rat intestinal crypt cell line, IEC-17, and primary cell cultures prepared form isolated 14-day-old fetal intestinal endoderm (EC). Both cultures prepared from isolated 14-day-old fetal rat intestinal endoderm (EC). Both types of cells were associated with 14-day-old fetal rat gut mesenchyme (Rm) and grafted under the kidney capsule of adult rats. Seventy percent of the Rm/EC and ten percent of the Rm/IEC recombinants, recovered after 9 days, exhibited well-vascularized structures in which the mesenchyme had induced morphogenesis of the cells into a villus epithelium. The four main intestinal epithelial cell types, absorptive, goblet, endocrine, and Paneth cells, were identified using electron microscopy. Biochemical determinations of enzyme activities associated with brush border membranes revealed that alkaline phosphatase, lactase, sucrase, and maltase were expressed in both types of associations. These results were confirmed by immunofluorescence staining using monoclonal antibodies to brush border enzymes. Both enzyme assays and immunocytochemistry showed that the amount of enzymes present in the brush border membrane of Rm/IEC grafts was in general lower than that of the Rm/EC recombinants. The results indicate that fetal rat gut mesenchyme enables morphogenesis and cytodifferentiation of both crypt and embryonic progenitor cells.     

Crystalloid lysozyme inclusions in Paneth cells of vitamin A-deficient rats

Summary The effect of vitamin A-deficiency on jejunal Paneth cells in rats was investigated. Crystalloid particles were observed in secretion granules of Paneth cells from 6 out of 8 rats with vitamin A-deficiency. The particles were similar to those found in Paneth cells under other experimental conditions. Using an immuno-electron-microscopic technique we demonstrated a clear lysozyme immunoreactivity of these particles. In 2 vitamin A-deficient rats tubular structures have been detected in addition to the crystalloid particles. Crystalloid particles or tubular structures were not detectable in a control group of 8 vitamin A-supplemented rats. The morphological alterations of Paneth cells may be correlated to an impaired local immunity of the intestine during vitamin A-deficiency.This work represents a portion of a doctoral thesis presented by M.J. Koch in partial fullfillment for the degree of medical doctor     

Site differences of Toll-like receptor expression in the mucous epithelium of rat small intestine

Toll-like receptors (TLRs) are known to recognize pathogen-associated molecular patterns and might function as receptors to detect microbes. In this study, the distribution of TLR-2, -4 and -9 were immunohistochemically investigated in the rat small intestine. As a result, TLR-2 was detected in the striated borders of villous columnar epithelial cells throughout the small intestine, except for the apices of a small number of intestinal villi. TLR-4 and -9 were detected in the striated borders of the villous columnar epithelial cells only in the duodenum. TLR-4-immunopositive minute granules were found in the apical cytoplasms of epithelial cells, subepithelial spaces and blood capillary lumina. TLR-2 and -4 were detected in the striated borders of undifferentiated epithelial cells and in the luminal substances of the intestinal crypts throughout the small intestine, but TLR-9 was not detected in the crypts throughout the small intestine. Only TLR-4 was detected in the secretory granules of Paneth cells in both the jejunal and ileal intestinal crypts. These findings suggest that duodenal TLRs might monitor indigenous bacteria proliferation in the upper alimentary tract, that TLR-2 might also monitor the proliferation of colonized indigenous bacteria throughout the small intestine, that the lack of TLR-2 at the villous apices might contribute to the settlement of indigenous bacteria, and that TLR-2 and -4 are secreted from intestinal crypts.     

Ulstrastructural localization of tumour necrosis factor-alpha

Summary The application of an antibody against tumour necrosis factor-alpha (TNF) to thin sections of plastic-embedded mouse tissue has identified sites of TNF activity in normal and endotoxin-treated C₃N/HeN mice. Prior to endotoxin treatment, TNF was observed in the secretory granules of the antibacterial Paneth cell and one type of crypt endocrine cell. Four hours after endotoxin treatment, these two types of intestinal cell were found to have degranulated. In addition, endotoxin treatment resulted in the appearance of TNF in the secretory granules of all eosinophils, neutrophils and monocytes in the bone marrow, spleen, lung and the proximal intestine. TNF was also observed in the internal elastic lamina (IEL) of arterioles. These results suggest that the process of TNF induction specifically targets the immune system and the vasculature. An invasive stimulus, such as circulating endotoxin, can provoke the immune cells to be armed with TNF. That same stimulus may cause arteriole smooth muscle cells to secrete TNF. TNF secretion in the presence of arteriole smooth muscle cells may play a role in the adjustment of arteriole tone. In the venules, TNF may be responsible for platelet and neutrophil accumulation which leads to embolism formation.