Intestinal gamma delta T cells develop in mice lacking thymus, all lymph nodes, Peyer's patches, and isolated lymphoid follicles

Through analysis of athymic (nu/nu) mice carrying a transgenic gene encoding GFP instead of RAG-2 product, it has recently been reported that, in the absence of thymopoiesis, mesenteric lymph nodes and Peyer's patches (PP) but not gut cryptopatches are pivotal birthplace of mature T cells such as the thymus-independent intestinal intraepithelial T cells (IEL). To explore and evaluate this important issue, we generated nu/nu mice lacking all lymph nodes (LN) and PP by administration of lymphotoxin-beta receptor-Ig and TNF receptor 55-Ig fusion proteins into the timed pregnant nu/+ mice that had been mated with male nu/nu mice (nu/nu LNP- mice). We also generated nu/nu aly/aly (aly, alymphoplasia) double-mutant mice that inherently lacked all LN, PP, and isolated lymphoid follicles. Although gammadelta-IEL were slightly smaller in number than those in nu/nu mice, substantial colonization of gammadelta-IEL was found to take place in the intestinal epithelia of nu/nu LNP- and nu/nu aly/aly mice. Notably, the population size of a major CD8alphaalpha+ gammadelta-IEL subset was maintained, the use of TCR-gamma-chain variable gene segments by these gammadelta-IEL was unaltered, and the development of cryptopatches remained intact in these nu/nu LNP- and nu/nu aly/aly mice. These findings indicate that all LN, including mesenteric LN, PP, and isolated lymphoid follicles, are not an absolute requirement for the development of gammadelta-IEL in athymic nu/nu mice.     

Vimentin-positive epithelial cells in aggregated lymphoid nodules (Peyer's patches) in rabbits

Peculiarities of cytoskeleton in membranous cells and disposition of the latter in the cupola epithelium in aggregated lymphoid nodules++ (ALN) have been studied in the ileum of 5 rabbits. The material has been fixed in liquid nitrogen and in the mixture of paraformaldehyde and glutar aldehyde. Methods of immunomorphology, high resolving light and transmissive electron microscopy have been used. Monoclonal antibodies to vimentin are selectively bind with a specific population of the ALN cupola epithelial cells. These cells are regularly arranged in the epithelium of the cupola lateral part and they are absent in the epithelium of the intestinal crypts, villi and apex of the cupolas. In the lateral epithelium of the cupolas surface, nearer to their base vimentin-positive++ epitheliocytes make contacts with single interepitheliocytic lymphocytes, and nearer to the apex they surround compact groups of the interepitheliocytic lymphocytes. The vimentin-positive++ epitheliocytic cells are identified as M-cells.     

Identification of intestinal M cells in isolated lymphoid follicles and Peyer’s patches of the Angora rabbit

The presence, distribution, and localization of M cells in isolated lymphoid follicles (ILF) and in follicle-associated epithelia (FAE) covering Peyer’s patches (PP) in Angora rabbits were investigated by immunohistochemistry and electron microscopy. Although PP could macroscopically be identified along the length of the mucosal and serosal surfaces of jejunum and ileum, the presence of ILF could only be located microscopically. Typical M cells in FAE were detected within the periphery of the dome regions of the PP, and immature columnar M cells in the FAE resided in the vicinity of the crypts. M cells in the FAE of both ILF and PP showed vimentin-positive reaction. M cells in the FAE of ILF were morphologically similar to the immature M cells found in the FAE of PP. Typical mature M cells were also observed in the FAE of a few ILF. In contrast to FAE of PP, numerous goblet cells were observed in the FAE of many ILF. Moreover, among intestinal villi, we noticed villi-like solitary lymphoid structures that showed abundant lymphocytes in their lamina propria and that were surrounded with vimentin-positive cells and goblet cells. Thus, the occurrence of copious immature M cells and goblet cells, in addition to the detection of villi-like solitary lymphoid structures full of lymphocytes in the FAE of many ILF, indicate that ILF do not complete their immunological maturation in contrast to PP. Various antigenic stimulations conceivably induce the formation and maturation of ILF along the length of the small intestine. The morphological resemblance between ILF M cells and PP M cells suggests that these two types of cells perform similar or the same immunological functions.     

Enhanced secretion of IFN-gamma by activated Th1 cells occurs via reverse signaling through TNF-related activation-induced cytokine

Growing evidence has demonstrated that members of TNF superfamily transduce signals after engagement with their receptors. TNF-related activation-induced cytokine (TRANCE), a member of TNF superfamily, is preferentially expressed on the surface of activated CD4(+) Th1 cells. The soluble receptor activator of NF-kappaB (RANK).Fc fusion protein suppresses IFN-gamma secretion by activated Th1 cells, but does not affect IL-4 secretion by Th2 cells. The suppressive effect on IFN-gamma secretion is observed when Th1 cells are activated by APCs, but not by immobilized anti-TCR beta mAb. In contrast, immobilized RANK.Fc fusion protein augments IFN-gamma secretion by Th1 cells, indicating the occurrence of reverse signaling through TRANCE during T cell/APC interaction. The enhanced secretion of IFN-gamma mediated via TRANCE correlates with the activation of p38 mitogen-activated protein kinase and is blocked by SB203580, a p38 mitogen-activated protein kinase-specific inhibitor. Thus, in addition to its role in activating dendritic cells by binding to the receptor RANK, TRANCE itself can signal the augmentation of IFN-gamma secretion via a p38-dependent pathway, and this provides yet another example of reverse signaling by a member of TNF superfamily.     

Peyer's patches export lymphocytes throughout the lymphoid system in sheep

The lymphocyte output from small intestine containing either the long continuous ileal Peyer's patch (PP) or several smaller jejunal PP was examined in young lambs. Most studies were done in 2-mo-old lambs, 1 mo after removal of mesenteric lymph nodes (MLN). Extracorporeal perfusion of part of the intestine and addition of fluorescein isothiocyanate to the perfusate led to the labeling, in their normal microenvironment, of a regionally defined population of cells. One day later considerable numbers of emigrant lymphocytes were identified by fluorescence microscopy in the spleen, MLN and peripheral lymph nodes, jejunal PP, and bone marrow. In nonperfused ileal PP and thymus the labeling indexes were low. The highest labeling index was in the blood where 3.7% of the lymphocytes were labeled. A similar organ distribution of emigrant cells was found on day 3. When MLN were included in the perfused region more emigrants were identified. In some animals the intestinal lymphatic draining the perfused ileum was cannulated. Continual lymph drainage caused a dramatic decrease in the labeling indexes in other lymphoid organs. A substantial number of lymphocytes leave both ileal PP and jejunal PP via lymphatics and travel to all other lymphoid organs. However, the number of emigrant lymphocytes compared with the total number of labeled lymphocytes in the perfused tissue was about 10 times greater after perfusing gut with the jejunal PP than after ileal PP perfusion. We conclude that relatively more lymphocytes emigrate from the jejunal PP than from the ileal PP.     

Lymphotoxin and TNF produced by B cells are dispensable for maintenance of the follicle-associated epithelium but are required for development of lymphoid follicles in the Peyer's patches

Organogenesis of Peyer's patches (PP), follicle-associated epithelium, and M cells is impaired in mice lacking B cells. At the same time, lymphotoxin (LT) and TNF are known to be critical for the development of PP. To directly address the function of LT and TNF expressed by B cells in the maintenance of PP structure, we studied the de novo formation of PP in B cell-deficient mice after the transfer of bone marrow from mice with targeted mutations in LT, TNF, or their combinations. We found that although the compartmentalization of T and B cell zones and development of follicular dendritic cells were affected by the lack of B cell-derived LT and TNF, the development of follicle-associated epithelium and M cells in PP was completely independent of LT/TNF production by B cells.     

Differential effect of opioids on immunoglobulin production by lymphocytes isolated from Peyer's patches and spleen

The mucosal immune system plays an important role in blocking the penetration of invasive organisms into various mucosal surfaces. Evidence now suggests neuroendocrine peptide hormones have immunomodulatory properties, including the ability to alter mucosal immunity. The potential for opioid compounds and corticotropic hormone (ACTH) to modulate mucosal immune function was investigated. We have found beta-endorphin, ACTH, and naltrindole (delta-class opioid receptor antagonist) to significantly suppress concanavalin A-stimulated Peyer's patch lymphocyte immunoglobulin production of IgA, IgG, and IgM isotypes. Oxymorphindole, a delta class opioid receptor agonist, significantly decreased IgM but not IgA or IgG production by the mitogen-stimulated Peyer's patch lymphocytes. Both oxymorphindole and naltrindole modestly reduced interleukin-2 receptor expression of concanavalin A- (Con A)-stimulated splenic and Peyer's patch lymphocytes. Neither compound appreciably affected immunoglobulin production by lipopolysaccharide-stimulated Peyer's patch lymphocytes. Collectively, these results indicate stress-related peptides such as ACTH and opioids may be involved in the regulation of immunoglobulin synthesis by Peyer's patch lymphocytes.     

Alpha4beta7/MAdCAM-1 interactions play an essential role in transitioning cryptopatches into isolated lymphoid follicles and a nonessential role in cryptopatch formation

The alpha(4) integrins alpha(4)beta(7) and alpha(4)beta(1), and their ligands mucosal vascular addressin cell adhesion molecule 1 (MAdCAM-1) and VCAM-1, have diverse functions, including roles in the formation of secondary lymphoid tissues at early time points during the colonization and clustering of the fetal lymphoid tissue inducer (LTi) cells and at later time points during the recruitment of lymphocytes. In this study, we evaluated the role of alpha(4) integrins in the development of a recently appreciated class of intestinal lymphoid tissues, isolated lymphoid follicles (ILFs). We observed that diverse ILF cellular populations express alpha(4)beta(7) and alpha(4)beta(1), including the LTi-like cells and lymphocytes, while ILF stromal cells and vessels within ILFs express VCAM-1 and MAdCAM-1, respectively. Evaluation of adult and neonatal beta(7)(-/-) mice and adult and neonatal mice given blocking Abs to alpha(4)beta(7), MAdCAM-1, or VCAM-1 did not identify a role for alpha(4) integrins in cryptopatch (CP) development; however, these studies demonstrated that alpha(4)beta(7) and MAdCAM-1 are required for the transitioning of CP into lymphoid tissues containing lymphocytes or ILFs. Competitive bone marrow transfers demonstrated that beta(7)(-/-) LTi-like cells had a reduced but not significantly impaired ability to localize to CP. Bone marrow transfers and adoptive transfers of B lymphocytes revealed that beta(7) expression by B lymphocytes was essential for their entry into the developing ILFs. These findings demonstrate an essential role for alpha(4)beta(7)/MAdCAM-1 in ILF development corresponding to the influx of beta(7)-expressing lymphocytes and a nonessential role for beta(7)-localizing LTi-like cells to the small intestine.     

Embryonal development of grouped lymphoid nodules (Peyer's patches) in the human ileum]

Embryogenesis of Payer's patches (PP) of the ileum, has been studied in 183 human fetuses 8-40-week-old. Their anlages appear on the 8th-9th week as an accumulation of atypical villi. At first the PP are localized in the cranial part of the ileum, and then spread caudally. Their most active increase in amount takes place from the 15th up to 17th weeks of development. From the 8th up to the 40th weeks the PP amount increase from 10 up to 37. During the same time their length develops from 0.7 up to 8.3 mm, their width--from 0.3 up to 2.2. The first lymphoid nodules++ in the PP are detected on the 14th week, then their number rises from 200 up to 3,500. The superficial area of all the PP, turning into the lumen of the ileum widens from 1.4 up to 620 mm2. Their predominate form is ellipsoid. During the whole prenatal period in the lymphoid nodules++ no germinative centers are revealed. Lymphocytes in the PP are identified in 8-9-week-old fetuses. By the 29th week the whole amount of lymphocytes in them increase up to 9.6 x 10(6) cells. Lymphocytic suspension of the PP of 8-9-week-old fetuses contains 1.7% of T-lymphocytes (E = POK) and 0.1% of B-lymphocytes (EAC = POK). By the 29th week their amount increases up to 9% and 7%, respectively, but by birth it does not reach their amount in the PP of mature organisms.     

Lectin binding reveals divergent carbohydrate expression in human and mouse Peyer's patches

The nature of cell-associated carbohydrates in the human intestine that may mediate transepithelial transport of bacterial and dietary lectins and their processing by the lymphoid cells of Peyer's patches is not known. Because the cell surface carbohydrate receptors for lectins may vary in different species, the glycoconjugates of human and mouse follicle-associated epithelium and gut-associated lymphoid tissue were compared. A panel of 27, mainly recently isolated, lectins were used to identify glycoconjugate expression in M-cells, enterocytes, goblet cells, lymphocytes and macrophages in mouse and human intestine. Mouse M-cells were exclusively labelled by fucose-specific lectins but in human follicle-associated epithelium no distinct M-cell staining pattern was observed. In the human Peyer's patches,Bryonia dioica lectin bound selectively to paracortical T-lymphocytes andChelidonium majus lectin to germinal centre B-cells. Certain mannose-specific lectins (Galanthus nivalis, Hippeastrum hybrid) stained the tingible body macrophages in the germinal centre of human Peyer's patches but labelled the macrophages in the paracortical T-cell region of the mouse. The results indicate distinct differences in glycosylation between mouse and human Peyer's patches and their associated lymphoid cells. When considering cell surface glycoconjugates as target molecules for the gut immune system, care has to be taken to choose the appropriate lectin for each species.     

Identification and characterization of the precursors committed to osteoclasts induced by TNF-related activation-induced cytokine/receptor activator of NF-kappa B ligand

Osteoclasts are terminally differentiated from cells of monocyte/macrophage lineage by stimulation with TNF-related activation-induced cytokine (TRANCE) (receptor activator of NF-kappaB ligand/osteoprotegerin ligand/osteoclast differentiation factor/TNFSF11/CD254). In the present study, we attempted to determine when and how the cell fate of precursors becomes committed to osteoclasts following TRANCE stimulation. Although mouse bone marrow-derived macrophages (BMMs) were able to differentiate into either osteoclasts or dendritic cells, the cells no longer differentiated into dendritic cells after treatment with TRANCE for 24 h, indicating that their cell fate was committed to osteoclasts. Committed cells as well as BMMs were still quite weak in tartrate-resistant acid phosphatase activity, an osteoclast marker, and incorporated zymosan particles by phagocytosis. Interestingly, committed cells, but not BMMs, could still differentiate into osteoclasts even after incorporation of the zymosan particles. Furthermore, IL-4 and IFN-gamma, potent inhibitors of osteoclast differentiation, failed to inhibit osteoclast differentiation from committed cells, and blocking of TRANCE stimulation by osteoprotegerin resulted in cell death. Adhesion to culture plates was believed to be essential for osteoclast differentiation; however, committed cells, but not BMMs, differentiated into multinucleated osteoclasts without adhesion to culture plates. Although LPS activated the NF-kappaB-mediated pathway in BMMs as well as in committed cells, the mRNA expression level of TNF-alpha in the committed cells was significantly lower than that in BMMs. These results suggest that characteristics of the committed cells induced by TRANCE are distinctively different from that of BMMs and osteoclasts.     

TNF-related activation-induced cytokine (TRANCE) induces angiogenesis through the activation of Src and phospholipase C (PLC) in human endothelial cells.

Angiogenesis is an essential step for many physiological and pathological processes. Tumor necrosis factor (TNF) superfamily cytokines are increasingly recognized as key modulators of angiogenesis. In this study, we tested whether TNF-related activation-induced cytokine (TRANCE), a new member of the TNF superfamily, possesses angiogenic activity in vitro and in vivo. TRANCE stimulated DNA synthesis, chemotactic motility, and capillary-like tube formation in primary cultured human umbilical vein endothelial cells (HUVECs). Both Matrigel plug assay in mice and chick chorioallantoic membrane assay revealed that TRANCE potently induced neovascularization in vivo. TRANCE had no effect on vascular endothelial growth factor (VEGF) expression in HUVECs and TRANCE-induced angiogenic activity was not suppressed by VEGF-neutralizing antibody, implying that TRANCE-induced angiogenesis may be the result of its direct action on endothelial cells. TRANCE evoked a time- and dose-dependent activation of the mitogen-activated protein kinases ERK1/2 and focal adhesion kinase p125(FAK) in HUVECs, which are closely linked to angiogenesis. These signaling events were blocked by the Src inhibitor PP1 or the phospholipase C (PLC) inhibitor. Furthermore, these inhibitors and the Ca(2+) chelator BAPTA-AM suppressed TRANCE-induced HUVEC migration. These results indicate that the angiogenic activity of TRANCE is mediated through the Src-PLC-Ca(2+) signaling cascade upon receptor engagement in endothelial cells, suggesting the role of TRANCE in neovessel formation under physiological and pathological conditions.     

Somatic generation of diversity in a mammalian primary lymphoid organ: the sheep ileal Peyer's patches

Ileal Peyer's patches (IPPs) in the sheep are composed of tightly packed follicles in which surface IgM-positive B cells proliferate and can be exported to the periphery. We report that the light chain rearrangement pattern in a single IPP follicle is much more restricted than in the entire tissue, which indicates that, as in the chicken bursa, ongoing rearrangement does not take place in this organ. Moreover, we show that B cells extensively diversify their antigen receptor while proliferating in IPP follicles. Sequencing of part of the V lambda locus indicates that this diversification is not achieved by gene conversion, but rather by untemplated somatic mutation and intense selective pressure. These results strongly imply that sheep IPPs behave as a bursa-equivalent, primary lymphoid organ of diversification and that somatic point hypermutation, which is known to proceed during secondary immune responses, can also generate an antibody repertoire.     

Absence of Peyer's patches and abnormal lymphoid architecture in chronic proliferative dermatitis (cpdm/cpdm) mice

The chronic proliferative dermatitis (cpdm) mutation causes inflammation in multiple organs, most prominently in the skin. Examination of the immune system revealed severe abnormalities in the architecture of lymphoid tissues. Peyer's patches were absent. In contrast, the spleen, lymph nodes, and nasal-associated lymphoid tissues were present. The spleen had normal numbers of T and B cells, but the spleen, lymph nodes, and nasal-associated lymphoid tissues had poorly defined follicles and lacked germinal centers and follicular dendritic cells. The marginal zone in the spleen was absent. The total concentration of serum IgG, IgA, and IgE in cpdm/cpdm mice was significantly decreased, whereas serum IgM was normal. Fecal IgA was low to undetectable in mutant mice, and the concentration of fecal IgM was increased. The titer of DNP-specific Abs following immunization with DNP-keyhole limpet hemocyanin was significantly decreased for all IgG subclasses. In contrast, T cell function appeared normal as assessed by evaluation of the contact hypersensitivity response in cpdm/cpdm mice. The cpdm mutation causes a complex phenotype that is characterized by multiorgan inflammation and the defective development of lymphoid tissues. The cpdm/cpdm mouse may be a useful model to study the factors that control the development of lymphoid tissues, in particular the Peyer's patches, and the mechanisms that control the humoral immune response.     

Heterogeneity,position, and functional capability of the macrophages in Peyer's patches

Summary Radioenzymatic assays and light microscope radioautographic studies performed on photophores of Porichthys notatus demonstrated (1) significant amounts of catecholamines (dopamine, noradrenaline, adrenaline) and 5-hydroxytryptamine (serotonin) in these organs; (2) selective uptake and storage of [³H]noradrenaline ([³H]NA) by axon terminals innervating the photocytes, and (3) strong accumulation of [³H]5-hydroxytryptamine ([³H]5-HT) within the photocytes. Uptake and storage of [³H]NA in the nerve fibers were seemingly unaffected by the addition of ten-fold molar concentrations of unlabelled serotonin. Accumulation of [³H]5-HT by the photocytes was dose-dependent and diminished markedly in the presence of ten-fold molar concentrations of non-radioactive noradrenaline. Neither neuronal uptake of [³H]5-HT or [³H]A, nor photocytic accumulation of [³H]A were detectable under the conditions of the present experiments. This information should provide a framework for further investigations of the regulation of photophore luminescence by the biogenic amines.Supported by grants from the National Research Council and Medical Research Council of CanadaJacques de Champlain and Lise Farley provided facilities and expertise with the radioenzymatic techniques. The technical assistance of Sylvia Garcia and Marie-Hélène Parizeau was also appreciated     

Identification of M cells and their distribution in rabbit intestinal Peyer's patches and appendix

The distribution of intestinal membranous (M) cells has been studied within the follicle-associated epithelium of rabbit Peyer's patches and appendix. Vimentin expression has been assessed as a primary criterion to identify rabbit M cells in tissue sections and in whole tissue preparations. This criterion has been compared to the use of the absence of alkaline phosphatase which, due to its heterogeneous distribution within the enterocyte population, is less reliable than vimentin expression as a marker for rabbit M cells. The pattern of vimentin immunostaining revealed that the majority of M cells are located in the periphery of the follicle-associated epithelium, the dome apex being largely free of M cells. This distribution was confirmed by scanning electron microscopy. Vimentin is also expressed by follicle-associated epithelial cells in the vicinity of crypts which lack the typical lymphocyte-containing pocket of M cells. Cytoplasmic peanut agglutinin binding coincides with vimentin-expression throughout the follicle-associated epithelium but is absent from vimentin-negative enterocytes. The co-localisation of these two phenotypic markers in both M cells and epithelial cells adjacent to crypts, which lack the typical morphology of fully developed rabbit M cells, suggests that they correspond to immature M cells which by their location appear to derive directly from undifferentiated crypt stem cells and not from mature columnar enterocytes.     

Regulation of mucosal IgA production in vitro by autoreactive immunoregulatory T cells from murine Peyer's patches

In this study, we investigated whether Peyer's patch-derived T-cell subsets participate in vitro in self major histocompatibility (MHC) class II antigen (Ag)-mediated immunoregulatory circuits for gut-mucosal IgA isotype selection in the presence of Peyer's patch (PP)-derived syngeneic surface immunoglobulin M (sIgM)-bearing B cells. When fresh (in vitro unstimulated) sIgM-bearing B cells were cocultured with fresh, PP-derived L3T4+ Vicia villosa-nonadherent (VV-) T cells (T helper (Th) cells), the production of all class-specific immunoglobulins (Ig), but, in particular, IgA, was enhanced two- to sixfold. This augmented Ig production was, however, reduced by nearly 50% when fresh PP-derived Lyt2+ VV-T cells (suppressor T cells) were added. Furthermore, addition of PP-derived L3T4+ VV+ and Lyt 2+ VV+ T cells abrogated, by nearly 100%, the suppression induced by the Lyt 2+ VV-T cells (contrasuppression). When lipopolysaccharide (LPS)-stimulated, PP-derived sIgM-bearing B cells were cocultured with the Th cells, the production of each class-specific Ig was similarly enhanced, but Ig levels exceeded those obtained with cultures of the unstimulated B cells (P less than 0.001). Anti-I-A or anti-I-E monoclonal antibody (mAb) inhibited the induction of each immunoregulatory T-cell effector activity (P less than 0.001), and anti-I-A/E inhibited it synergistically. Thus, unstimulated fresh PP-derived T cells appear to be activated and then to exert T-cell effector functions in the sequential development of helper, suppressor, and contrasuppressor immunoregulatory networks in the presence of PP-derived sIgM B cells and, particularly, LPS-preactivated sIgM B cells. Based on the blocking effect of anti-I-A and/or anti-I-E mAb on the induction of each T-cell-mediated immunoregulatory function in class-specific Ig production, it appears that the autoreactive (self MHC class II Ag-reactive) activation of PP T cells evoked by Ia Ag on PP sIgM B cells largely controls mucosal IgA production by the latter cells. Furthermore, this immunoregulation by autoreactive effector T cells, especially the L3T4+ VV- helper T cell, may play a significant role in vivo in gut-mucosal IgA isotype production.