Sites of lymph follicle formation in the draining popliteal lymph nodes of mice locally injected with antigenic and mitogenic substances

Our previous studies showed that some antigenic and mitogenic substances, when locally injected into mice, efficiently produced new lymph follicles outside pre-existing follicles in draining lymph nodes, whereas others had virtually no effect. In the present experiments, young adult male mice were injected with several antigens and mitogens in the rear footpad, and the number and development sites of newly produced lymph follicles in the draining popliteal nodes were studied using serial sections of the nodes obtained between 5 and 21 days after injection. In the unstimulated state, each popliteal node contained a limited number of lymph follicles which mostly lay in a portion of the peripheral cortex overlaying the deep cortex (this portion is referred to as the PCOU), whereas a portion of the peripheral cortex extending beyond the deep cortex (referred to as the PCBU) was underdeveloped with only occasional follicles. Mice treated with soluble PHA or fluid tetanus toxoid developed germinal centers in association with existing follicles but failed to produce new follicles. The PCBU of the draining nodes remained underdeveloped, and the number and distribution pattern of lymph follicles within a draining node were comparable to those in the control node. Animals treated with LPS (50 micrograms), Con A, alum-precipitated PHA or alum-precipitated tetanus toxoid produced significantly large numbers of new follicles outside pre-existing follicles in the draining nodes, the new follicles produced in the PCBU being generally more numerous than those in the PCOU. In these draining nodes, the peripheral cortex, comprising a number of follicles, was found to overlie the deep cortex and extend beyond the deep cortex towards the hilar region. In animals given a less effective stimulant, such as ferritin or a smaller dose of LPS (10 micrograms), the draining nodes produced a relatively small number of new follicles, most of which were formed in the PCBU. The present results indicate that in the mouse popliteal node, the PCBU is morphologically underdeveloped under normal conditions, but develops lymph follicles in response to exogenous stimuli more readily than the PCOU, and that substances efficient in inducing follicle formation can be regarded as capable of stimulating the development of the peripheral cortex.     

Interdigitating reticulum cells in the popliteal lymph node of the rat

Summary Electronmicroscopic and cytochemical studies were performed to localize interdigitating reticulum cells (IDC) in the popliteal lymph node of the rat.The morphological features of the IDC of the rat correspond to those described for other species, but also show similarities to normal macrophages in the rat. This is considered to be an argument in favour of the common origin of IDC's and macrophages.Ultrahistochemical studies with horseradish peroxidase (HRP) reveal no phagocytotic capacity of IDC's. After perfusion fixation containing ruthenium red (RR) the surface coat stains heavily: RR is also found deep in the membrane invaginations of the IDC, indicating the presence of polyanionic sialoglyco-proteins. The post-capillary-venules (PVC) are very permeable to both HRP and RR.The phosphotungstic acid-chromic acid stain (PTA-CrA) also reveals glycoproteins in the surface coat; these glycoproteins are susceptible to -neuraminidase, whereas glycoproteins in the Golgi complexes, lysosomes and in the vesicular complexes of IDC are not. The glycoproteins of the latter are susceptible to 0.1 N NaOH. These findings indicate that IDC produce different kinds of glycoprotein, one of which may be secreted and act as a factor for stimulating peripheral T-lymphocytes.Intimate contact between IDC's and PCV's could be observed. It is therefore conceivable that IDC's play an important role in the homing of T-lymphocytes.     

Ontogeny of the antigen-reactive lymph follicle-forming capacity of the popliteal lymph node in neonatal mice

The ontogenetic development of the reactive lymph follicle-forming capacity of the popliteal lymph node was investigated immunohistochemically in young mice which had received a single injection of hemocyanin (KLH) in a rear footpad at a predetermined age (between 1 and 21 days). The mice were sacrificed at various intervals after injection. In non-stimulated young mice, primary lymph follicles first appeared in the popliteal node at 11 days of age. When KLH was given to 7-day-old or older mice, each draining popliteal node showed a marked increase in B lymphocytes in the extrafollicular zone 3 days after injection and produced a number of "new" lymph follicles outside the pre-existing follicles over the next few days. In mice injected at 2-4 days of age, these nodes showed an increase in B lymphocytes in the outer cortex and had produced several lymph follicles by 8 days of age. The number of lymph follicles produced by each node tended to increase in line with age at injection. These results indicate that neonatal popliteal nodes become able to produce lymph follicles in response to exogenous antigens some time before ontogenetically developing follicles appear. The formation of new lymph follicles observed in draining popliteal nodes after KLH injection at an early postnatal age is discussed in relation to the ontogenetic development of stromal cells (precursors of follicular dendritic cells) that are capable of interacting with B lymphocytes and the extent of B lymphocyte influx into the node induced by KLH stimulation.     

The postnatal development of cell populations in the rat popliteal lymph node

Summary The postnatal development of the various cell populations in the rat popliteal lymph node was investigated applying enzyme-histochemical and immunohistochemical techniques. From birth, T-lymphocytes and interdigitating cells were demonstrable. During the development of the young lymph node, T-lymphocytes of the helper phenotype outnumbered the T-cells with a suppressor phenotype; they account for approximately 70% and 30% of all T-lymphocytes, respectively. At the very first day of postnatal life, post-capillary venules were already present. B-lymphocytes occurred later than T-cells during ontogeny; they were found on the second day after birth, most of them being IgM- or IgG-bearing lymphocytes. The first primary follicles occurred at day 18 and contained principally membrane-stained IgM cells and, to a lesser extent, membrane-stained IgG cells. The appearance of follicular dendritic cells correlated with the formation of primary follicles. With respect to the macrophages, it appeared that the ED1- and ED3-positive subpopulations were present with a similar distributional pattern as seen in adults, but in considerably lower numbers. The expression of ED2, however, showed a sudden increase in the third week of life. Findings of the present study are discussed in relation to those obtained in other investigations dealing with the ontogenetic development of lymphoid organs.Abbreviations IDC interdigitating cell - cIg cytoplasmic immunoglobulin - sIg surface immunoglobulin - FDC follicular dendritic cell - PBS phosphate-buffered saline - PCV post-capillary venule - PLN popliteal lymph node     

Immunity to D-penicillamine: genetic, cellular, and chemical requirements for induction of popliteal lymph node enlargement in the mouse

To elucidate the pathogenesis of immunological diseases induced by the drug D-Penicillamine (D-Pen) the requirements for sensitization to this drug were investigated. Mice were subcutaneously (s.c.) injected into one hind footpad with a solution of D-Pen without adjuvant, and reactivity to D-Pen was determined in the popliteal lymph node assay (PLNA) by weight increase of the draining PLN, the incorporation of 3H-thymidine, and trapping of 51Cr-labeled syngeneic lymphocytes in the draining PLN. The peak of the primary PLN response was obtained between day 7 and 10 after injecting 1 mg of D-Pen per mouse. Likewise, PLN enlargement could be induced by injecting 18 hr nonadherent spleen cells s.c. that had been pretreated overnight with D-Pen in vitro. D-Pen-induced PLN enlargement was primarily caused by cell proliferation within the lymph node, and only a minor portion was due to trapping of circulating lymphocytes. The majority of the cells in the enlarged PLN were B cells; T cells, however, were required for generation of PLN enlargement. For induction of PLN reactivity to D-Pen, the stereoisomer L-Pen, and the dimer D-Pen disulfide, it was mandatory that the respective molecules were administered in ionized form. PLN reactivity to D-Pen is controlled by at least two loci, one mapping to the I region, possibly A beta A alpha, the other(s) to the non-H-2 background. As far as studied, high responsiveness was inherited dominantly. The PLN reaction proved to be antigen-specific, since D-Pen-primed mice exhibited an enhanced reaction when challenged with a suboptimal dose of D-Pen, but not when challenged with an unrelated drug, diphenylhydantoin (DPH). The possible relationship between immunity to D-Pen and autoimmunity induced by this drug is discussed.     

Ultrastructure and permeability of lymph node microvasculature in the mouse

Summary The microvasculature of lymph nodes and Peyer's patches consists of arterioles, capillaries and venules. The postcapillary segment comprises high-endothelial venules (HE venules) as well as ordinary venules. In order to study the ultrastructure of the microvasculature, particularly with respect to the nature of intercellular junctions, lanthanum and ruthenium red were used as tracers. Furthermore, to evaluate the permeability properties of the different segments of the microvasculature, intravenously injected horseradish peroxidase (HRP; MW: 40,000) was used.All segments of the microvasculature are permeable to HRP. However, the mechanism of transport across the vascular wall varies in the different segments, apparently correlated with a gradual decrease in number of transport vesicles and a gradual attenuation in the sealing of the endothelial cells. Tight junctions are present in arterioles, and it is assumed that HRP reach the basal lamina exclusively by vesicular transport. Incomplete or focal tight junctions are present in the capillaries, and both intercellular and vesicular pathways are observed. In the venules the intercellular pathway seems to be the dominant one, while vesicular transfer is negligible. However, some micropinocytic vesicles in the HE venule endothelial cells probably represent the initial stage of an intracellular digestion.     

The early postnatal development of the primary immune response in TNP-KLH-stimulated popliteal lymph node in the rat

Summary The popliteal lymph nodes were removed from young rats of various ages five days after a single immunization with TNP-KLH in the hind footpads. Cryostat sections of the lymph nodes were investigated by means of enzyme and immunohistochemical techniques at the light-microscopical level.The presence and localization of anti-TNP antibody-containing cells were examined using a new technique to visualize specific antibodies. Moreover, the development of the lymph nodes following exogenous antigenic stimulation was compared with that of unstimulated lymph nodes.Specific antibody-containing cells could not be found before day 15 after birth, in rats immunized at day 10. From that time these lymphoid cells were located primarily at the border between cortex and medulla. Younger popliteal lymph nodes showed only aspecific immunoglobulin-containing lymphoid cells. With age, the number of specific antibody-containing cells tended to increase. These cells were more mature, according to morphological criteria and were located nearer the medulla.The first primary follicles were seen at day 19, as was the case in unstimulated animals. The first secondary follicles, containing germinal centers, were detected at day 23, whereas in unstimulated popliteal lymph nodes they were never found.Trapping of immune complexes could not be demonstrated before day 33 after birth. The later appearance of this phenomenon might be a consequence of the techniques applied to demonstrate specific antibody-containing cells.Abbreviations PLN popliteal lymph node - FDC follicular dendritic cell - IDC interdigitating cell - HEV high endothelial venule - TNP trinitrophenyl - KLH keyhole limpet hemocyanin - PBS phosphate-buffered saline - GCPC germinal center precursor cell - sIg surface immunoglobulin - cIg cytoplasmic immunoglobulin     

Induction by cells of immune responses to intestinal epithelial cell-associated components (ECAC): transfer with cultured murine mesenteric and popliteal/axillary lymph node cells

Although systemic and mucosal immune responses to intestinal epithelial self-antigens occur in several human disorders, there is no model system with which to study the physiology and regulation of the underlying cellular events. Therefore, we undertook to induce an immune response to purified epithelial macromolecules in the Lewis rat; characterize in vitro the reactive cells; and then transfer with immunocytes this antiepithelial reactivity to naive syngeneic rats, identifying the fine specificity and site of humoral and cell-mediated immunity induced in the cell recipient. Donor animals sensitized systemically (via footpad) or locally in gut mucosa (via Peyer's patches) to syngeneic or xenogeneic epithelial antigens generated specific immunoglobulin and were found to have T lymphocytes in the draining nodal areas (including the mesenteric nodes) which were (a) antigen-specific, having a [3H]thymidine uptake in the presence of antigen 30-fold the control; (b) generally of the Thelper/inducer subclass (W3/25+) which, upon further culture, developed phenotype surface markers for activation (IL-2R+); (c) able to induce an antigen-specific humoral and cell-mediated responses upon intravenous injection into naive syngeneic hosts; and (d) demonstrable in gut-associated lymphoid tissue (mesenteric lymph nodes) and, to a lesser extent in spleen, of the cell recipient. Further, lymphocytes cloned from reactive mesenteric lymph node cells demonstrated specificity for a gel-purified subfraction of epithelial antigen, designated P1, containing highly conserved organ-specific macromolecules thought to be autoantigenic for gut.     

KIT signaling regulates primordial follicle formation in the neonatal mouse ovary

The pool of primordial follicles determines the reproductive lifespan of the mammalian female, and its establishment is highly dependent upon proper oocyte cyst breakdown and regulation of germ cell numbers. The mechanisms controlling these processes remain a mystery. We hypothesized that KIT signaling might play a role in perinatal oocyte cyst breakdown, determination of oocyte numbers and the assembly of primordial follicles. We began by examining the expression of both KIT and KIT ligand in fetal and neonatal ovaries. KIT was expressed only in oocytes during cyst breakdown, but KIT ligand was present in both oocytes and somatic cells as primordial follicles formed. To test whether KIT signaling plays a role in cyst breakdown and primordial follicle formation, we used ovary organ culture to inhibit and activate KIT signaling during the time when these processes occur in the ovary. We found that when KIT was inhibited, there was a reduction in cyst breakdown and an increase in oocyte numbers. Subsequent studies using TUNEL analysis showed that when KIT was inhibited, cell death was reduced. Conversely, when KIT was activated, cyst breakdown was promoted and oocyte numbers decreased. Using Western blotting, we found increased levels of phosphorylated MAP Kinase when KIT ligand was added to culture. Taken together, these results demonstrate a role for KIT signaling in perinatal oocyte cyst breakdown that may be mediated by MAP Kinase downstream of KIT.     

Kinetics and morphology of chemically induced popliteal lymph node reactions compared with antigen-, mitogen-, and graft-versus-host-reaction-induced responses

Changes in the popliteal lymph node (PLN) in mice evoked by a local graft-versus-host (GVH) reaction and by a single injection of various agents into the hind footpad were compared. The drug diphenylhydantoin induced similar weight changes in time as the GVH reaction. More vigorous and protracted reactions were induced by the drug nitrofurantoin and the contact sensitizer dinitrochlorobenzene, whereas the antigens lipopolysaccharide and sheep erythrocytes caused very moderate and short-lasting weight changes. Alterations of lymph node architecture upon injection of diphenylhydantoin resembled those observed during the GVH response. Some quantitative and qualitative differences were noted for nitrofurantoin, but clearly deviant morphological alterations were seen in response to lipopolysaccharide and sheep erythrocytes. The PLN reaction to dinitrochlorobenzene had features of both the GVH reaction and the antigen-induced responses. These findings support the concept that some drugs and chemicals may induce or exacerbate lymphoproliferative disorders by GVH-like mechanisms.     

Kinetics and morphology of chemically induced popliteal lymph node reactions compared with antigen-, mitogen-, and graft-versus-host-reaction-induced responses

Changes in the popliteal lymph node (PLN) in mice evoked by a local graft-versus-host (GVH) reaction and by a single injection of various agents into the hind footpad were compared. The drug diphenylhydantoin induced similar weight changes in time as the GVH reaction. More vigorous and protracted reactions were induced by the drug nitrofurantoin and the contact sensitizer dinitrochlorobenzene, whereas the antigens lipopolysaccharide and sheep erythrocytes caused very moderate and short-lasting weight changes. Alterations of lymph node architecture upon injection of diphenylhydantoin resembled those observed during the GVH response. Some quantitative and qualitative differences were noted for nitrofurantoin, but clearly deviant morphological alterations were seen in response to lipopolysaccharide and sheep erythrocytes. The PLN reaction to dinitrochlorobenzene had features of both the GVH reaction and the antigen-induced responses. These findings support the concept that some drugs and chemicals may induce or exacerbate lymphoproliferative disorders by GVH-like mechanisms.