Systematic characterization of porcine ileal Peyer's patch, II. A role for CD154 on T cells in the positive selection of immature porcine ileal Peyer's patch B cells

We previously demonstrated that the majority (>/= 90%) of porcine ileal Peyer's patch (IPP) follicular cells are immature B cells destined to die by apoptosis, when incubated at 37 degrees. In this paper we approached the mechanisms responsible for positive selection of porcine IPP follicular immature B-cell selection, by screening for various cell types, cytokines and polyclonal and monoclonal antibodies for promoting the survival of IPP B cells. Of these reagents, only CD3 cross-linked purified T cells from mesenteric lymph nodes were able to rescue IPP follicular B cells from apoptosis, although polyclonal anti-IPP lymphocyte antibodies delayed apoptosis. This survival effect could be reproduced simply by incubating IPP follicular B cells with soluble and cell membrane-expressed CD154, an observation consistent with the demonstrated presence of CD40 and CD154 on porcine IPP follicular B cells and activated T cells, respectively. The IPP follicular B cells rescued in this manner expressed a more mature surface marker phenotype. Immunohistology and fluorescence-activated cell sorter analysis demonstrated that subpopulations of IPP follicular T cells (less than 0.5%) express CD154. Thus, perhaps unexpectedly, CD154 on T cells may play a role in the positive selection of immature B cells in the porcine IPP. The origin and control of the activated T cells identified within the porcine IPP remains to be investigated.     

Robo4 contributes to the turnover of Peyer's patch B cells

All leukocytes can get entrance into the draining lymph nodes via the afferent lymphatics but only lymphoid cells can leave the nodes. The molecular mechanisms behind this phenomenon have remained unknown. We employed genome wide microarray analyses of the subcapsular sinus and lymphatic sinus (LS) endothelial cells and found Robo4 to be selectively expressed on LS lymphatics. Further analyses showed high Robo4 expression in lymphatic vessels of Peyer's patches, which only have efferent lymphatic vessels. In functional assays, Robo4-deficient animals showed accumulation of naïve B cells (CD19⁺/CD62L^hi/CD44^lo) in Peyer's patches, whereas no difference was seen within other lymphocyte subtypes. Short-term lymphocyte homing via high endothelial venules to peripheral and mesenteric lymph nodes and Peyer's patches was also slightly impaired in Robo4 knockout animals. These results show for the first time, selective expression of Robo4 in the efferent arm of the lymphatics and its role in controlling the turnover of a subset of B lymphocytes from Peyer's patches.     

Sequence analysis of rearranged IgVH genes from microdissected human Peyer's patch marginal zone B cells.

The Peyer's patches of the terminal ileum are a source of IgA plasma cells in the intestinal lamina propria of experimental animals. They are also thought to harbour IgA memory cells. However, the microanatomical location of Peyer's patch memory cells, and whether they are also present in man is not known. Human Peyer's patches have a pronounced marginal zone (MGZ) of sIgD-negative B cells. In this study we analysed the sequence of polymerase chain reaction-amplified, rearranged IgVH genes from microdissected MGZ B cells, to determine whether this is a site of B-cell memory in Peyer's patches. We observed that the majority of Peyer's patch MGZ B cells contain heavily mutated IgVH genes and are therefore clearly memory B cells. Sequences of rearranged mutated genes in the MGZ have a pattern of replacement and silent mutations expected of selected products of the affinity maturation process. Related clones, with identical CDR3 but different patterns of mutation, are seen. This suggests that either these memory cells are formed as the germinal centre selection process proceeds, or a memory cell has re-entered the germinal centre for further rounds of mutation. Interestingly, in one patient, the MGZ in the Peyer's patches also contains a proportion of B cells with unmutated IgVH 4.21 genes.     

Monoclonal antibody-directed targeting of fluorescent polystyrene microspheres to Peyer's patch M cells.

The ability to deliver particulates to Peyer's patch M cells for uptake into gut-associated lymphoid tissue was examined by administering simultaneously fluorescent green and red polystyrene microspheres into NZW rabbit intestinal loops containing Peyer's patches. Whereas green and red microspheres were taken up by M cells at equivalent concentrations (120 +/- 17 versus 125 +/- 18/mm length of dome), particles conjugated to the anti-M-cell monoclonal antibody 5B11 (IgM, kappa) were internalized by M cells 3-3.5 times more efficiently than conjugates displaying IgM of unrelated specificity (TEPC 183) or native particles of the reciprocal colour inoculated into the same loop at a comparable load. The microspheres formed a concentration gradient from lumen to subepithelial dome, and localized on M-cell apical membranes, M-cell pockets, and subepithelial domes. The transport rate across M cells of 5B11 or TEPC 183 conjugates was similar to that of untreated microspheres. These observations show that intestinal uptake into Peyer's patches can be upregulated by targeting M-cell luminal membrane structures.     

Rescue of ileal Peyer's patch B cells from apoptosis is associated with the induction of Bcl-2 expression.

The major site of B-cell genesis in the sheep is the ileal Peyer's patch (PP). The B cells in the ileal PP undergo both extensive proliferation and massive death in association with an ongoing diversification of the immunoglobulin repertoire by somatic hypermutation. Most, if not all, the B-cell death in the ileal PP is due to apoptosis. When placed in culture, ileal PP B cells undergo rapid apoptosis. Here, we investigated the expression of the proto-oncogene bcl-2 in ileal PP cells in situ and in culture. Bcl-2 expression has been correlated with the prevention of apoptosis in many cell types. Western blotting, using anti-Bcl-2 monoclonal antibodies, revealed that a Bcl-2-reactive protein of 26,000 MW was expressed in ileal mesenteric lymph node cells, splenocytes and thymocytes from sheep, but was barely detectable in ileal PP B cells in situ or in culture. However, Bcl-2 expression could be markedly induced in ileal PP B cells cultured with phorbol ester and Ca2+ ionophore, a procedure that is known to rescue these cells from apoptosis. We hypothesize that those few B cells that survive a selection event in the ileal PP may begin to express elevated levels of Bcl-2 as they escape from the apoptotic pathway.     

An analysis of the growth and differentiation of B cells isolated from follicles of the ileal Peyer's patch of sheep.

We developed a method to isolate and culture cells from the lymphoid follicles of the ileal Peyer's (PP) patch of young sheep (6-12 weeks). These cells were 98% sIgM+ B cells and 1% T cells. Cultured follicular cells were used to investigate B-cell proliferation and differentiation. Less than 50% of B cells were viable after 24 hr of culture and this decrease in B-cell viability also occurred following co-stimulation with pokeweed mitogen (PWM) and recombinant bovine interleukin-1 (rBoIL-1) or rBoIL-2. In contrast, co-stimulation with PWM and either rBoIL-1 or rBoIL-2 induced a marked proliferative response that was maximal on Day 4 of culture. Cytokine-induced proliferation of the B cells required PWM co-stimulation and proliferation induced by rBoIL-1 and rBoIL-2 was neither additive or synergistic. This suggests that PWM bound a molecule or molecules that signalled responsiveness to both rBoIL-1 and rBoIL-2. Culture of follicular cells with PWM and both rBoIL-1 and rBoIL-2 also resulted in B-cell differentiation. This differentiation was associated with decreased proliferation, an increased number of viable B cells, and increased expression of both surface IgM and non-Ig membrane molecules. Thus, co-stimulation of ileal PP follicular cells with PWM and rBoIL-1 and rBoIL-2 resulted in both B-cell proliferation and differentiation.     

Differentiation of B lymphocytes in sheep. I. Phenotypic analysis of ileal Peyer's patch cells and the demonstration of a precursor population for sIg+ cells in the ileal Peyer's patches.

The ileal Peyer's patches (IPP) of sheep may be a primary lymphoid organ for b cells since they have a number of important features in common with the bursa of Fabricius of chickens. We have examined the surface phenotype of IPP cells. Approximately 90% to 95% of IPP cells are 'low sIgM+'; that is, they have surface IgM, but in much smaller amounts than peripheral B cells, which are 'high sIgM+'. IPP cells with sIgG or sIgA are very rare. Upon exposure to a tumour promotor, phorbol myristate acetate (PMA), in vitro, low sIgM+ cells differentiated into high sIgM+ cells. The amount of Ia-like antigens on the surface also increased after PMA treatment. Approximately 5% of IPP cells bore no identifiable markers. However, these cells could also be induced into high sIgM+ cells upon exposure to PMA; this may indicate the presence of precursors of sIgM+ cells within the IPP. While PNA (peanut agglutinin) binds strongly to the vast majority of IPP cells, it binds very little, if at all, to B cells obtained from the periphery, unless they have been treated with neuraminidase; this suggests that cells in the B lineage retain their PNA receptors, but that these become masked by sialic acid on mature B cells.     

Detection of individual Peyer's patch T cells that produce interleukin-5 and interferon-gamma.

A method was developed to detect individual interferon-gamma (IFN-gamma)- and interleukin-5 (IL-5)-producing cells among freshly isolated T cell populations and long term lines of CD4+ Peyer's patch T cells using frozen semi-thin sections of paraformaldehyde fixed-T cells and immunofluorescence techniques. Using this method, individual CD4+ Peyer's patch T cells could be shown to produce IFN-gamma, characteristic of the T helper 1 (Th1) T cell type, IL-5, characteristic of the T helper 2 (Th2) T cell type, as well as both IFN-gamma and IL-5. These data support the notion that Th1 and Th2 cells derive from a common T cell precursor.     

Confocal analysis of fluorescent bead uptake by mouse Peyer's patch follicle-associated M cells.

Latex beads coated with secretory immunoglobulin (IgA) instilled into mouse intestinal loops are taken up by M cells present in Peyer's patch follicle-associated epithelial tissue. Bead adsorption and uptake is greater at the edge compared with the apex of follicle domes. Coating beads with bovine serum albumin (BSA) causes a fourfold reduction in adsorption and a twentyfold reduction in uptake. Results demonstrate selectivity between adsorption and uptake and between the ability of different proteins to facilitate uptake.     

Molecular cloning of a beta-glucosidase-encoding gene from Sclerotinia sclerotiorum by expression in Escherichia coli

Summary Six Sclerotinia sclerotiorum genes previously described as being expressed specifically during the induction of cell-wall-degrading enzymes have been transferred to Escherichia coli. When tested for expression of beta-glucosidase activity using X-glu (5-bromo-4-chloro-3-indolyl-beta-d-glucopyranoside), one of of the the genes expressed a X-glu-degrading activity. The corresponding RNA size is reported.     

Systematic characterization of porcine ileal Peyer's patch, I. apoptosis-sensitive immature B cells are the predominant cell type

It is now apparent that the Peyer's patches of some species exhibit structural, functional and developmental heterogeneity. In sheep, for example, the ileal Peyer's patch (IPP) is the primary, antigen-independent site for the generation of the primary immunoglobulin repertoire and consequent production of the systemic B-cell pool. The pig has three distinct Peyer's patches, including an IPP, but the functional status of this organ, as primary or secondary lymphoid tissue, is not clear. Here, we have systematically characterized pig IPP follicular lymphocytes and show that about 90% B cells that are positive for surface immunoglobulin G (sIgM+) and express an immature phenotype characterized by expression of myeloid marker sWC3 (74-22-15) and two molecules recognized by IPP B-cell-specific monoclonal antibodies (F10/4, F12/35). Extensive apoptosis in vivo and in vitro was demonstrated by electron microscopy, immunohistology with TdT-mediated dUTP nick end labelling, DNA analysis and fluorescence-activated cell sorter analysis. Thus, when isolated IPP follicular cells were incubated at 37 degrees in vitro, the majority of them became apoptotic. The few that survived, however, had lost their expression of sWC3, F10/4, F12/35, but showed an increased expression of sIgM and major histocompatibility complex class II indicating that such surviving cells were of a more mature phenotype. Although more T cells were observed in porcine IPP follicles than in sheep IPP, CD3+ cells comprised less than 5% of the IPP follicular lymphocytes. Thus, the results clearly indicate that pig IPP is equivalent to sheep IPP.     

Cyclosporine affects the function of antigen-presenting cells.

The immunosuppressant drug cyclosporine (CsA) is known to affect T-cell function. We have studied the effect of CsA on the specific proliferative response of T-cell lines to antigen. In addition to blocking IL-2 release by specifically activated T-cell lines, CsA also affected the ability of irradiated spleen cells to present preprocessed antigen to T-cell lines. Irradiated spleen cells pulsed with antigen for 2 hr were able to stimulate a proliferative response in T-cell lines. Following a 2-hr pulse with CsA, antigen presentation by these irradiated spleen cells was reduced significantly, suggesting that CsA not only affects T cells, but also affects the function of antigen-presenting cells.     

Ontogeny of reticular cells in the ileal Peyer's patch of sheep and goats

The ontogeny of reticular cells in the ileal Peyer's patch of sheep from 70 days gestational age was studied by light and electron microscopy and by enzyme histochemistry. Small to medium-sized lymphocytes were seen in the lamina propria at 97 days, when the stroma was essentially still mesenchymal. By 110 days, the stromal cells in the dome/follicle primordia had differentiated into reticular fibroblasts, whose processes and fibers were seen to surround groups of lymphocytes. With advancing age the number and size of primordia increased, and proliferation was obvious among the lymphocytes. Processes of reticular cells increased in number and penetrated between individual lymphocytes of the groups. Coarser desmosome-like contacts were seen between the reticular cells from 115 days onwards. A central light area in the follicle was apparent from 130 days onwards. The fine structure of the stromal cells in this light follicle center developed towards but never became similar to that of follicular dendritic cells in a typical germinal center. The fine interdigitating end branches of the stromal cells were less numerous, and the dense homogeneous material present in between the end branches was not observed in the ileal Peyer's patch follicle. Instead, small particles and vesicles were seen between the various cell types of the light center and were not restricted to the intercellular spaces between the stromal cells. In the dark peripheral zone of the follicle, the stromal cells retained more immature features. The follicle became bordered by a capsule at an early stage. This capsule was formed by multiple layers of flattened fibroblasts separated by small amounts of intercellular material only. The alkaline phosphatase, Mg²⁺-dependent adenosine triphosphatase and 5′nucleotidase reactivities of the follicular dendritic cells in the ileal Peyer's patch were similar to those of early prenatal primary follicles of sheep lymph nodes. This study indicates that the stromal cells of the ileal Peyer's patch are mesenchymal in nature and different from those of germinal centers and the epithelial stromal cells of bursa Fabricii of birds. © 1991 Wiley-Liss, INC.     

Distribution and functional characteristics of antigen-specific helper T cells arising after Peyer's patch immunization.

Antigen-specific T-helper cells for IgA responses arise in Peyer's patches (PP) following their immunization by subserosal injection of keyhole limpet haemocyanin (KLH). These are of the W3/25 phenotype and the W3/25 receptor is shown here to be involved in their helper function. These cells originate in PP and migrate via mesenteric lymph nodes (MLN) to thoracic duct lymph, although the MLN appear to be unnecessary for the induction or maturation of antigen-specific helper cells collected in thoracic duct lymph after intra-Peyer's patch (i.p.p.) immunization. KLH-specific helper cells can be detected subsequently in the intraepithelial lymphocyte population and also among lamina propria lymphocytes. The helper cells also relocate to PP distant to their site of origin where they are retained only when antigen is present. While i.p.p. immunization is an efficient route for the induction of IgA helper cells in gut-associated lymphoid tissue, it differs from oral immunization in that concomitant induction of antigen-specific splenic suppressor cells does not occur, indicating a role for epithelial antigen processing in this phenomenon.     

Ontogeny of leukocyte populations in the ileal Peyer's patch of sheep.

Enzyme- and immunohistochemical methods were used to characterize the leukocyte populations present in the ileal Peyer's patches of sheep foetuses between 68 and 135 d of gestation and particularly in the period around 100 d of gestation, when active lymphopoiesis begins. A wide variety of leukocytes including IgM+, CD5+, CD4+, CD8+ cells, and MgATPase+ dendritic cells were present at an early stage. Groups of IgM+ cells were seen immediately beneath the epithelium as early as 70 d of gestation. Conventional morphometric and computer-assisted morphometric techniques were used to confirm the significant expansion of these cell populations from 90 d of gestation. IgM+ and CD5+ cells were responsible for the vast majority of the increase in cell numbers. It was concluded that a diverse leukocyte population was present at the initiation of active lymphopoiesis in the ileal PP of the sheep foetus and that all members of this population were associated with the emergence of the dome/follicle primordia from which the B-cell follicle develops.     

First characterization of a newly emerging phytopathogen,Sclerotinia sclerotiorum causing white mold in pea

Pea (Pisum sativum L.) is of global importance as a food crop for its edible pod and seed. A new disease causing the tan to light brown blighted stems and pods has occurred in pea (P. sativum L.) plants in Chapainawabganj district, Bangladesh. A fungus with white-appressed mycelia and large sclerotia was consistently isolated from symptomatic tissues. The fungus formed funnel-shaped apothecia with sac-like ascus and endogenously formed ascospores. Healthy pea plants inoculated with the fungus produced typical white mold symptoms. The internal transcribed spacer sequences of the fungus were 100% similar to Sclerotinia sclerotiorum, considering the fungus to be the causative agent of white mold disease in pea, which was the first record in Bangladesh. Mycelial growth and sclerotial development of S. sclerotiorum were favored at 20°C and pH 5.0. Glucose was the best carbon source to support hyphal growth and sclerotia formation. Bavistin and Amistar Top inhibited the radial growth of the fungus completely at the lowest concentration. In planta, foliar application of Amistar Top showed the considerable potential to control the disease at 1.0% concentration until 7 days after spraying, while Bavistin prevented infection significantly until 15 days after spraying. A large majority (70.93%) of genotypes, including tested released pea cultivars, were susceptible, while six genotypes (6.98%) appeared resistant to the disease. These results on identification, characterization, host resistance, and fungicidal control of white mold could be valuable to achieve improved management of a new disease problem for pea cultivation.     

Negative signaling by surface IgM on B cells isolated from ileal Peyer's patch follicles of sheep

Lymphoid follicles from the sheep ileal Peyer's patch (PP) were used to prepare a cell suspension consisting of 98% surface IgM-positive (sIgM+) B cells and 1% T cells. Co-stimulation of follicular cells with pokeweed mitogen and either recombinant bovine interleukin 1 (IL 1) or IL 2 resulted in a marked proliferative response. In contrast, the addition of soluble F(ab')2 rabbit anti-sheep Ig completely inhibited the proliferative response induced by pokeweed mitogen and IL 1 or IL 2 co-stimulation. Anti-Ig inhibition of B cell proliferation was specific for ileal PP follicular cells and was not observed with mesenteric lymph node cells or splenocytes. Furthermore, suppression of ileal PP follicular B cell proliferation required at most divalent cross-linking of sIg was independent of Fc receptors, but was dependent on the concentration of anti-Ig and required 48 h for maximal effect. Negative signaling by sIgM indicates that ileal PP follicular B cells are functionally distinct from B cells in other secondary lymphoid tissues. Also, the present observations are consistent with previous reports indicating that B cell proliferation in ileal PP follicles is antigen independent.