Postnatal development of the tubular lamina propria and the intertubular tissue in the bovine testis

Summary The postnatal development of intertubular cells and vessels and of the tubular lamina propria was studied in three locations of perfusion-fixed bovine testes from 31 animals ranging from 4 to 78 weeks. The postnatal morphological differentiation of the testis is not uniform, regional differences have to be considered. The intertubular cell population is composed of mesenchyme-like cells, fibrocytes, Leydig cells, peritubular cells and mononuclear cells. In 4 and 8-week-old testes mesenchyme-like cells are the dominating element. These pluripotent cells proliferate by frequent mitoses and are the precursors of Leydig cells, contractile peritubular cells and fibrocytes. Morphologically differentiated Leydig cells are encountered throughout the entire period of postnatal development. In 4-week-old testes degenerating fetal and newly formed postnatal Leydig cells are seen in juxtaposition to each other. From the 8th week on, only postnatal Leydig cells are present. Between 16 and 30 weeks large-scale degeneration of prepuberal Leydig cells is observed. The Leydig cells that survive this degenerative phase constitute the long-lasting adult population. 20–30% (numerically) of all intertubular cells at all ages are free mononuclear cells. These are found as lymphocytes, plasma cells, monocytes, macrophages and light intercalated cells (LIC). The latter are monocyte-derived, Leydig cell-associated typical cells of the bovine testis. The differentiation of the two main components of the tubular lamina propria, (i) basal lamina and (ii) peritubular cell sheath, seems to be effected rather independent from each other and also from hormonal signals important for the development of the germinal cells. The laminated basal lamina reaches nearly 3 m at 16 weeks and is later on continuously reduced. At 25 weeks the peritubular cells have transformed into contractile myofibroblasts. At this period the germinal epithelium is still in a prepuberal state.To Dr. E. Schilling, Mariensee, on the occasion of his 65^th birthday     

Isolation and subsequent analysis of murine lamina propria mononuclear cells from colonic tissue

Studies on colonic cells in the lamina propria (LP) of mice are important for understanding the cellular and immune responses in the gut, especially in inflammatory bowel diseases (such as morbus crohn and colitis ulcerosa). This protocol details a method to isolate LP cells and characterize freshly isolated cells by quality control experiments to obtain cells that can be used for further investigations. After different steps of digestion of the tissue using collagenase, DNase and dispase, the resulting cells are purified using Percoll gradient. The success of the isolation can be analyzed by cell viability test (Trypan Blue exclusion test) and by flow cytometric analysis to assess apoptosis. Finally, the isolated cells can be used for further investigations like comparative studies of mRNA expression, cell-proliferation assay or protein analysis. This protocol can be completed within 6-7 h.     

Immunocytochemistry of extracellular matrix in the lamina propria of the rat testis: electron microscopic localization

The distribution of laminin, type IV collagen, heparan sulfate proteoglycan, and fibronectin was investigated in the rat testicular lamina propria by electron microscopic immunocytochemistry. Distinct patterns were observed for each antigen within the extracellular matrix (ECM) layers of the lamina propria. Laminin, type IV collagen, and heparan sulfate proteoglycan all localized to the seminiferous tubule basement membrane. Type IV collagen and heparan sulfate proteoglycan, but not laminin, localized to the seminiferous tubule side of the peritubular myoid cells. All four of the antigens were localized between the peritubular and lymphatic endothelial cells. Failure to localize fibronectin in the ECM layer between the Sertoli and peritubular myoid cells tends to support the concept that adult Sertoli cells do not produce this protein in vivo. Intracellular immunostaining was insufficient to allow unambiguous identification of the cellular source of any of the ECM molecules.     

Ultrastructure of peritubular tissue in association with tubular hyalinization in human testis.

Testicular peritubular tissue, also known as the tunica propria, surrounds the seminiferous tubules and is responsible for contractile, paracrine and transport functions. The aim of the present report is to describe the pathomorphology of peritubular tissue in association with tubular hyalinization in human testis. Twenty-seven testicular biopsies from 21 subfertile and infertile men were studied with the electron microscope. Biopsies from five patients showed complete or nearly complete tubular hyalinization. In addition to changes described earlier, the following new ultrastructural features were observed: 1. loss of polarity and configuration of myoid cells; 2. protrusion of myoid cells towards the tubule and evagination of basal lamina surrounding the tubule towards the interstitial direction leading to 'bridge' formation. These 'bridges' of myoid cells often created completely separated small compartments within the tunica propria; 3. vacuolization and fragmentation of myoid cell nuclei; 4. a balloon-like swelling of myoid cell containing phagolysosomes and lipid droplets. We conclude that disorganization and loss of vital functions of the extracellular matrix and myoid cells contribute to the pathogenesis of tubular hyalinization.     

Mesenchymal entactin-1 (nidogen-1) is required for adhesion of peritubular cells of the rat testis in vitro

Epithelial-like Sertoli cells isolated from immature rat testis aggregate to form tubule-like structures when cultured on a monolayer of mesenchyme-derived peritubular cells. At the end of this morphogenetic process both cell types are separated by a basement membrane. In this study the gene expression of monocultures and direct cocultures of peritubular cells and Sertoli cells was examined using DD-RT-PCR. One of the isolated cDNA clones showed high homology to the cDNA encoding the basement membrane component entactin-1 (nidogen-1). Even though the entactin-1 (nidogen-1) gene is transcribed in peritubular cells, Sertoli cells, and in direct cocultures, the mRNA is translated only by the peritubular cells. No entactin-1 (nidogen-1) was detected in the Sertoli cells by Western blotting. Moreover, peritubular cell monocultures and cocultures showed the presence of one single band at 152 kDa in the supernatant, whereas in cell lysates two bands were detectable at 152 kDa and 150 kDa. Perturbation experiments using monoclonal antibodies directed against entactin-1 (nidogen-1) were performed with peritubular cells and Sertoli cells, respectively, and demonstrated loss of cell adhesion of the peritubular cells, while the Sertoli cells remained adherent. From these data we conclude that entactin-1 is exclusively produced and secreted by mesenchymal peritubular cells, and affects adhesion of peritubular cells in an autocrine manner.     

On the structure of intestine and presence of endocytic cells in the lamina propria of platy and black tetra (Teleostei)

The structure of the intestine in platy (Xiphophorus maculatus) and black tetra (Gymnocorymbus ternetzi) and the capability of cells within the intestinal wall to endocytose intraperitoneally injected horse-spleen ferritin, are described. The intestinal epithelial layer has about the same thickness in both species, but the width of the lamina propria and tunica muscularis in black tetra was only about 1/5 of that in platy. Ferritin was taken up by numerous cells within the lamina propria throughout the entire length of the platy intestine. The uptake was demonstrated as large and strongly coloured intracellular Prussian blue granules in sections treated with acid ferrocyanide. There was no such uptake by the lamina propria in black tetras. We suggest that the high numbers of endocytic cells within the intestinal lamina propria of platies provide a local defence against foreign cells and particles. Such a functional role may to some extent compensate for the lack of an HCl-based defence in the digestive system of this stomach-less species.     

The lamina propria of the bovine seminiferous tubule

Summary The boundary tissue of bovine testicular seminiferous tubules exhibits remarkable regional differences at the level of the seminiferous tubule proper, as compared with its terminal segment. The basal lamina of the seminiferous tubule proper is multilayered and possesses knob-like protrusions. At the level of the terminal segment the basal lamina is highly specialized; in the region of the terminal plug candelabrum-like projections of the tubular basal lamina invade the bases of the modified supporting cells up to a depth of 3.5 m. The adjoining surface of these supporting cells is densely studded with hemidesmosomes. The elongated peritubular cells are arranged in 3–5 concentric layers around the tubulus seminiferus proper but form a loose association at the level of the terminal segment. Where the terminal segment joins the testicular straight tubule, peritubular cells may assemble to constitute a contractile spiral. Elastic tissue is situated mainly subjacent to the tubular basal lamina and to a lesser degree between the peritubular cell layers. A peritubular space lined by endothelium-like cells may surround the seminiferous tubule proper and also the transitional zone of the terminal segment.Supported by a grant from the Deutsche Forschungsgemeinschaft     

A Pacinian corpuscle in the human bladder lamina propria

The fortuitous finding of a complex Pacinian corpuscle within the lamina propria of the human urinary bladder is described. It consisted of a complex of encapsulated nerve endings within the areolar connective tissue of the lamina propria immediately adjacent to the inner aspect of the detrusor muscle. It showed no structural evidence of directional sensitivity and was associated on its outer aspect with small unmyelinated axons containing small clear and dense-cored vesicles. This appears to be the first report of an encapsulated nerve ending within the lining of the adult human urinary bladder.     

Expression of immunoglobulin isotypes by lymphoid cells of mouse intestinal lamina propria

At the time of their ablation, human tonsils contained some lymphocytes which incorporated [³H]thymidine during short-term culture. The extent of proliferation seemed to be a characteristic of the individual organ pairs. Tonsil cells also secreted during culture at least three soluble factors. One factor suppressed proliferation of human PBL treated with Con A, another factor augmented the proliferation, and the third factor was mitogenic for unstimulated PBL. Mitogenic factor was demonstrable in the presence of supernatants which expressed suppressor activity, but the augmentor could not be demonstrated in such supernatants until it was physically separated from the suppressor by gel filtration or by anion-exchange chromatography. The dose-response curves for the augmentor and mitogenic factor, both of which were simultaneously present in the supernatant, were different. The expression of one of these activities, however, did not require expression of the other. Both augmentor and mitogenic factor were nondialyzable. The augmentor had a molecular weight of about 30,000 and eluted from DEAE-cellulose in 150–250 mM NaCl.     

Human colonic intraepithelial lymphocytes regulate the cytokines produced by lamina propria mononuclear cells

Using an in vitro autologous human system, the immunomodulatory function of colonic intraepithelial lymphocytes (IEL) on cytokine production by lamina propria mononuclear cells (LPMNC) has been investigated. In contrast to LPMNC, colonic IEL produced only low amounts of IL-10, interferon-gamma and interleukin-2. However, co-culture experiments (IEL + LPMNC) have shown that IEL can enhance the PHA-induced synthesis of IL-2 and interferon-gamma, but not IL-10 by LPMNC. Using a transwell filter culture system apparatus, this effect was shown not to require a cell-to-cell interaction. Thus, IEL in vitro may modulate the cytokine synthesis of LPMNC, through the production of soluble factors. This may prove highly relevant in the in vivo immune activation of the gastrointestinal mucosa.     

The interaction between walker tumour cells and mucosa cells in the lamina propria of gastric mucosa in rats

One series of 12 rats was exposed to X-irradiation (1500 R) of the stomach 19 days before implantation of Walker tumour cells in the gastric mucosa, and the frequency of tumour take and the extent of tumour growth after 10 days were compared with a second series with the same tumour implantation, but without X-ray exposure. In a third series simple gastric ulcers without tumour were produced by clamping the gastric wall with a heated (80 degrees) surgical needle holder, and the animals were killed 5-7 day later. All the rats were given injections of vinblastine sulfate 3 hours and of 3H-TDR 1 hour before sacrifice. In viewfields with diameter 180 mu the vinblastine-arrested mitoses and labelled cells on the tumour side of the tumour/mucosa border were calculated as percentages of all tumour cells. In the mucosa the total number of proliferating cells was counted at various distances from the border of the tumour or ulcer. No clear differences in the frequency of tumour take and the extent of tumour growth were found between the X-irradiated and the normal rat stomachs, and it is concluded that the X-ray exposure 3 weeks prior to tumour implantation did not reduce the normal mucosal resistance to tumour growth. The percentage of arrested mitoses and labelled cells in the tumour decreased one view field away from the mucosal border, and the number of proliferating cells in the mucosa bordering on the tumours showed a gradual fall with increasing distance up to 0.8-1.0 mm from the tumour border; within these distances, however, the numbers were much higher than at corresponding distances from edges of the ulcers. The Walker tumour thus seems to stimulate cell proliferation in mucosa to a much greater extent than a simple ulcer does. The causes of this phenomenon and the possible roles of "chalones" or "anti-chalones" are discussed.     

Changes in lamina propria dendritic cells on the oral administration of exogenous protein antigens during weaning

Two critical periods of maximum exposure to antigens occur in young mammals, immediately after birth and at weaning, as a result of colonization by commensal bacteria and the ingestion of new diets. At weaning, active immune responses of antibody production against dietary proteins are known to occur, but simultaneously, oral tolerance is acquired for harmless food proteins. However, regulated mechanisms of the immune system at weaning remain to be elucidated although its immune responses may be somewhat similar to those in adulthood. Considering that tolerogenic antigen-presenting cells (APCs) are likely to be a key factor in the acquisition of oral tolerance, in the present study, we examined the changes of dendritic cells (DCs) in the lamina propria (LP) on exposure to food proteins at weaning. C57BL/6 female mice were weaned at the age of 3 weeks and orally administered 10 mg of ovalbumin (OVA) for ten consecutive days after weaning. The administration led to a decrease in the plasma level of immunoglobulin specific for OVA, suggesting the acquisition of oral tolerance. The uptake of fluorescence-labeled OVA was significantly observed for CD11c⁺LPDCs. When we analyzed the changes of two types of LPDCs, PDCA-1⁺ MHC II⁺ DCs and CD103⁺ MHC II⁺ DCs, ten consecutive gavages of OVA marginally, but not significantly, augmented only the frequency of PDCA-1⁺ MHC II⁺ DCs. Considering that the change of APCs likely appears immediately on the response to antigen intake, we found the statistically significant increase in the frequency of PDCA-1⁺ DCs, but not in that of CD103⁺ DCs, even after two treatments, indicating PDCA-1⁺ DCs to be recruited in the LP within 2 days of exposure to food proteins. These results suggest that the behavior of tolerogenic PDCA-1⁺ DCs may change at weaning with the removal of the immunoprotective components of maternal milk.     

Isolation and differentiation of nestin positive cells from rat oral mucosal lamina propria

Despite successes in the isolation and characterization of stem cells from the oral mucosal epithelium, there have been few studies on progenitor cells from the oral mucosal lamina propria. In this study, we isolate rat oral mucosal lamina propria cells (OMLPC) using nestin as a marker in an immunomagnetic sorting technique. The OMLPCs was negative for cytokeratin. Nestin and vimentin were expressed in the OMLPCs. And CD44 and STRO-1 were expressed in a subset of the OMLPCs, which suggest that the nestin positive OMLPCs be heterogeneous. Otherwise, OMLPCs express Oct4, which is a critical gene for pluripotency. The OMLPCs proliferated actively in vitro. A colony forming study demonstrated that OMLPCs exhibited colony-generating capacity. When cultured in defined medium, OMLPCs generated cells characteristic of osteoblast, adipocyte and astrocyte-like cells. In addition, OMLPCs seeded into three dimensional scaffolds form bone-like structures in vivo after 8 weeks. All of the results demonstrate that OMLPCs are a population of mesenchymal progenitor cells existing in rat oral mucosal lamina propria. Nestin is shown to be a useful molecular marker for these cells. In certain environments, OMLPCs can form hard tissue. Thus, OMLPCs may serve as a suitable source of cells for future bone or tooth tissue engineering applications.     

Transepithelial pathogen uptake into the small intestinal lamina propria

The lamina propria that underlies and stabilizes the gut lining epithelium is densely populated with strategically located mononuclear phagocytes. Collectively, these lamina propria macrophages and dendritic cells (DC) are believed to be crucial for tissue homeostasis as well as the innate and adaptive host defense. Lamina propria DC were recently shown to gain direct access to the intestinal lumen by virtue of epithelium-penetrating dendrites. However, the role of these structures in pathogen uptake remains under debate. In this study, we report that entry of a noninvasive model pathogen (Aspergillus fumigatus conidia) into the murine small intestinal lamina propria persists in the absence of either transepithelial dendrites or lamina propria DC and macrophages. Our results suggest the existence of multiple pathogen entry pathways and point at the importance of villus M cells in the uptake of gut lumen Ags. Interestingly, transepithelial dendrites seem altogether absent from the small intestine of BALB/c mice suggesting that the function of lamina propria DC extensions resides in their potential selectivity for luminal Ags, rather than in general uptake or gut homeostasis.     

CD4+CD25bright T cells in human intestinal lamina propria as regulatory cells

It is well known that immune responses in the intestine remain in a state of controlled inflammation, suggesting that not only active suppression by regulatory T cells plays an important role in the normal intestinal homeostasis, but also its dysregulation leads to the development of inflammatory bowel disease. In this study, we demonstrate that the CD4(+)CD25(bright) T cells reside in the human intestinal lamina propria (LP) and functionally retain regulatory activities. All human LP CD4(+) T cells regardless of CD25 expression constitutively expressed CTLA-4, glucocorticoid-induced TNFR family-related protein, and Foxp3 and proliferate poorly. Although LP CD4(+)CD25(-) T cells showed an activated and anergic/memory phenotype, they did not retain regulatory activity. In LP CD4(+)CD25(+) T cells, however, cells expressing CD25 at high levels (CD4(+)CD25(bright)) suppressed the proliferation and various cytokine productions of CD4(+)CD25(-) T cells. LP CD4(+)CD25(bright) T cells by themselves produced fewer amounts of IL-2, IFN-gamma, and IL-10. Interestingly, LP CD4(+)CD25(bright) T cells with regulatory T activity were significantly increased in patients with active inflammatory bowel disease. These results suggest that CD4(+)CD25(bright) T cells found in the normal and inflamed intestinal mucosa selectively inhibit the host immune response and therefore may contribute to the intestinal immune homeostasis.