IgE formation in the rat following infection with Nippostrongylus brasiliensis: I. Proliferation and differentiation of IgE-bearing cells

Sprague-Dawley rats were infected with Nippostrongylus brasiliensis larvae, and IgE formation was studied. Before infection, the serum IgE level was less than 0.4 μg/ml. The IgE level began to increase from the 10th day of infection, reached its maximum (50–100 μg/ml) at the 14th day and gradually declined. Reinfection of the rats resulted in an increase of the serum IgE level within 7 days. The IgE antibody response to N. brasiliensis antigens did not parallel the increase of IgE synthesis. In most animals, the antibody became detectable in the serum at the 21st day when the total IgE level already began to decrease. The animals showed a secondary IgE antibody response upon reinfection. Both mesenteric lymph nodes and spleen cell suspensions were examined for the presence of IgE-bearing cells (IgE-B cells) and IgE-forming cells by fluorescent antibody technique. The IgE-bearing lymphocytes became detectable in the mesenteric lymph nodes and spleen at the 8th day of infection. The proportion of the IgE-B cells in nonadherent cell population gradually increased and reached maximum at the 14th day; about 20% of immunoglobulin (Ig)-bearing cells in the mesenteric lymph nodes and 10% of Ig-bearing cells in spleen bore IgE on their surface. Evidence was obtained that these lymphocytes synthesized IgE. The IgE-forming cells were detected in both mesenteric lymph nodes and spleen of the infected animals. The number of IgE-forming cells was greater in the mesenteric lymph nodes than in spleen, indicating that the regional lymph nodes are the major source of serum IgE in the N. brasiliensis-infected animals.     

Differentiation of Langerhans Cells from Interdigitating Cells Using CD1a and S-100 Protein Antibodies

The present study shows that Langerhans cells can be differentiated from Interdigitating cells at the light microscopic level. Superficial lymph nodes and skin taken from necropsies and the lymph nodes of dermatopathic lymphadenopathy (DPL) were used for this experiment. Sections of lymph node and skin were embedded using the acetone, methyl benzoate and xylene (AMeX) method and dendritic cells were immunostained with anti S-100 protein antibody (S-100, and OKT-6 (CD1a) using the restaining method. Langerhans cells in the skin were positive for both CD1a and S-100. Dendritic cells positive for both CD1a and S-100, and dendritic cells positive for S-100, but not for CD1a were observed in superficial lymph nodes. In normal superficial lymph nodes, there were more interdigitating cells than Langerhans cells. The majority of the dendritic cells in the DPL were Langerhans cells. We conclude that the S-100 and CD1a positive cells are Langerhans cells, and the S-100 positive-CD1a negative cells are interdigitating cells.     

Differentiation of Langerhans Cells from Interdigitating Cells Using CD1a and S-100 Protein Antibodies

The present study shows that Langerhans cells can be differentiated from Interdigitating cells at the light microscopic level. Superficial lymph nodes and skin taken from necropsies and the lymph nodes of dermatopathic lymphadenopathy (DPL) were used for this experiment. Sections of lymph node and skin were embedded using the acetone, methyl benzoate and xylene (AMeX) method and dendritic cells were immunostained with anti S-100 protein antibody (S-100, and OKT-6 (CD1a) using the restaining method. Langerhans cells in the skin were positive for both CD1a and S-100. Dendritic cells positive for both CD1a and S-100, and dendritic cells positive for S-100, but not for CD1a were observed in superficial lymph nodes. In normal superficial lymph nodes, there were more interdigitating cells than Langerhans cells. The majority of the dendritic cells in the DPL were Langerhans cells. We conclude that the S-100 and CD1a positive cells are Langerhans cells, and the S-100 positive-CD1a negative cells are interdigitating cells.     

Activated CD8 T cells redistribute to antigen-free lymph nodes and exhibit effector and memory characteristics

Exogenous dendritic cells display restricted trafficking when injected in vivo and stimulate CD8 T cell responses that are localized to a small number of lymphoid compartments. By examining these responses in the presence and absence of FTY720, a drug that causes sequestration of T cells in lymph nodes, we demonstrate that a significant fraction of divided CD8 T cells redistribute into Ag-free lymph nodes within 3 days of activation. Despite variation in the level of expression of CD62L, redistribution of these cells is CD62L-dependent. Redistributed CD8 T cells exhibit characteristics of differentiated effectors. However, when re-isolated from Ag-free lymph nodes 3 days after activation and transferred into naive mice, they persist for at least 3 wk and expand upon Ag challenge. Thus, CD8 T cells that redistribute to Ag-free lymph nodes 3 days after immunization contain memory precursors. We suggest that this redistribution process represents an important mechanism for establishment of lymph node resident central memory, and that redistribution to Ag-free nodes is an additional characteristic to be added to those that distinguish memory precursors from terminal effectors.     

Fibroblast-type reticular stromal cells regulate the lymph node vasculature

The lymph node vasculature is essential to immune function, but mechanisms regulating lymph node vascular maintenance and growth are not well understood. Vascular endothelial growth factor (VEGF) is an important mediator of lymph node endothelial cell proliferation in stimulated lymph nodes. It is expressed basally in lymph nodes and up-regulated upon lymph node stimulation, but the identity of VEGF-expressing cells in lymph nodes is not known. We show that, at homeostasis, fibroblast-type reticular stromal cells (FRC) in the T zone and medullary cords are the principal VEGF-expressing cells in lymph nodes and that VEGF plays a role in maintaining endothelial cell proliferation, although peripheral node addressin (PNAd)(+) endothelial cells are less sensitive than PNAd(-) endothelial cells to VEGF blockade. Lymphotoxin beta receptor (LTbetaR) blockade reduces homeostatic VEGF levels and endothelial cell proliferation, and LTbetaR stimulation of murine fibroblast-type cells up-regulates VEGF expression, suggesting that LTbetaR signals on FRC regulate lymph node VEGF levels and, thereby, lymph node endothelial cell proliferation. At the initiation of immune responses, FRC remain the principal VEGF mRNA-expressing cells in lymph nodes, suggesting that FRC may play an important role in regulating vascular growth in stimulated nodes. In stimulated nodes, VEGF regulates the proliferation and expansion of both PNAd(+) and PNAd(-) endothelial cells. Taken together, these data suggest a role for FRC as paracrine regulators of lymph node endothelial cells and suggest that modulation of FRC VEGF expression may be a means to regulate lymph node vascularity and, potentially, immune function.     

Phenotypic and functional differences between NKT cells colonizing splanchnic and peripheral lymph nodes

NKT cells are considered unconventional T cells. First, they are restricted by a nonclassical MHC class I molecule, CD1d, which presents glycolipids; second, their TCR repertoire is very limited. After stimulation by their TCR, NKT cells rapidly release large amounts of cytokines, such as IL-4 and IFN-gamma. Little is known about NKT cells present in lymph nodes. In the present report we show that NKT cells are differently distributed in various lymph nodes and are, for instance, abundant in pancreatic and mesenteric lymph nodes of C57BL/6 mice and nonobese diabetic mice. The high frequency of NKT cells in splanchnic lymph nodes is not simply a consequence of inflammatory signals, as draining lymph nodes still contain low frequencies of NKT cells after IFA or CFA injections. NKT cells from splanchnic lymph nodes harbor a Vbeta repertoire similar to that of splenic and liver NKT cells, in contrast to peripheral NKT cells that are not biased toward Vbeta8 segments. Analysis of cytokine production by NKT cells from splanchnic lymph nodes reveals that they produce at least as much IL-4 as IFN-gamma, in contrast to NKT cells from other organs (spleen, liver, and peripheral lymph nodes), which produce much more IFN-gamma than IL-4. These specific features of NKT cells from splanchnic lymph nodes might explain their protective action against the development of pathogenic Th1 cells in type 1 diabetes.     

Chronic alcohol consumption decreases the percentage and number of NK cells in the peripheral lymph nodes and exacerbates B16BL6 melanoma metastasis into the draining lymph nodes

NK cells in the lymph nodes play important roles in inhibiting tumor metastasis into draining lymph nodes. Previously, we reported that chronic alcohol consumption interferes with NK cell trafficking from the bone marrow to the spleen. Herein, we found that alcohol consumption decreases the numbers of NK cells in lymph nodes. Adoptive transfer experiments indicated that continued exposure of donor splenocytes to alcohol inhibits NK but not T cell trafficking to lymph nodes. Alcohol did not negatively affect CCR7⁺ and CXCR3⁺ NK cells, but decreased the percentage and number of CD62L⁺ NK cells in the spleen, which are an important source of NK cell trafficking into the lymph nodes. These data suggest that modulation of the microenvironment associated with alcohol consumption impairs the trafficking of NK cells to lymph nodes. The decreased number of NK cells in the lymph nodes was associated with increased melanoma metastasis into the draining lymph nodes.     

Interaction of the lymph node and splenic lymphocytes of mice in inactivation of allogeneic hematopoietic stem cells

A study was made of interaction of mouse spleen and lymph node lymphocytes in inactivation of allogeneic stem cells. It was established that T lymphocytes of the lymph nodes and spleen lymphocytes do not interact on combined administration; their action is of additive nature. B lymphocytes of the lymph nodes have a regulating activity both in respect to T lymphocytes of the lymph nodes and lymphocytes of the spleen. The stem cells serve as target. Depending on the stem cells/B lymphocytes ratio B lymphocytes are capable of exerting either helper or suppressor action.     

Distribution of primed T cells and antigen-loaded antigen presenting cells following intranasal immunization in mice

Priming of T cells is a key event in vaccination, since it bears a decisive influence on the type and magnitude of the immune response. T-cell priming after mucosal immunization via the nasal route was studied by investigating the distribution of antigen-loaded antigen presenting cells (APCs) and primed antigen-specific T cells. Nasal immunization studies were conducted using the model protein antigen ovalbumin (OVA) plus CpG oligodeoxynucleotide adjuvant. Trafficking of antigen-specific primed T cells was analyzed in vivo after adoptive transfer of OVA-specific transgenic T cells in the presence or absence of fingolimod, a drug that causes lymphocytes sequestration within lymph nodes. Antigen-loaded APCs were observed in mediastinal lymph nodes, draining the respiratory tract, but not in distal lymph nodes. Antigen-specific proliferating T cells were first observed within draining lymph nodes, and later in distal iliac and mesenteric lymph nodes and in the spleen. The presence at distal sites was due to migration of locally primed T cells as shown by fingolimod treatment that caused a drastic reduction of proliferated T cells in non-draining lymph nodes and an accumulation of extensively divided T cells within draining lymph nodes. Homing of nasally primed T cells in distal iliac lymph nodes was CD62L-dependent, while entry into mesenteric lymph nodes depended on both CD62L and α4β7, as shown by in vivo antibody-mediated inhibition of T-cell trafficking. These data, elucidating the trafficking of antigen-specific primed T cells to non-draining peripheral and mucosa-associated lymph nodes following nasal immunization, provide relevant insights for the design of vaccination strategies based on mucosal priming.     

Development of follicular dendritic cells in lymph nodes of B-cell-depleted mice

Summary We have studied follicular dendritic cells (FDC) in lymph nodes of normal and thymus dysgeneic nude mice depleted of B-cells by chronic treatment with anti-IgM antibodies. We found that B cell depletion was accompanied by the absence of mature FDC as defined morphologically at the ultrastructural level. Only precursor FDC (p-FDC) could be demonstrated. Upon release of B-cell suppression, the repopulation of lymph nodes with B-cells was associated with the reappearance of fully differentiated FDC in primary follicles of nude mice and in secondary follicles of T-cell competent mice. We conclude that mature B-cells and/or B-cell products are required for the development of mature follicular dendritic cells in the mouse lymph node.     

Morphocytochemical reaction of the lymph nodes in dogs to the administration of lysozyme

By means of morphological, morphometrical and histochemical methods pelvic and tracheobronchial lymph nodes have been studied in dogs and concentration of lysozyme has been estimated in blood serum, in lymph and the lymph nodes after a single intramuscular injection of lysozyme (2 mg/kg of body mass). In the material investigated total concentration of lysozyme reaches its maximal values in 6 h after injection, then it gradually decreases and in 48 h reaches its control level. Morphometrically changes in cell composition are revealed predominantly of immune-competent cells in T- and B-dependent zones of the lymph nodes. Thus, the volumetric part of lymphoblasts in the germinative centers of the lymphoid nodules reaches its maximal indices by 48 h after lysozyme injection, while plasmatization of the paracortical zone and of medullary cords increases up to the 7th day. By the 14th day the volumetric part of lymphoblasts, immunoblasts and plasmocytes decreases gradually, and in 21 days after injection of the drug contents of the blast forms of the cells in the structural-functional zones of the lymph nodes does not differ from that in the control. The data obtained demonstrate the immunomorphological rearrangement of the lymph nodes in response to the exogenic lysozyme administration.     

Subset-specific regulation of the lymphatic exit of recirculating lymphocytes in vivo

The blood-to-lymph recirculation of lymphocytes is required for the maintenance of immune surveillance and the dissemination of memory. Although the ability of lymph-borne cells to recirculate has been well documented, relatively less is known about the migration capacity of PBLs. We have found a clear preference for PBLs to recirculate through s.c. rather than intestinal lymph nodes. This preference could be directly attributed to the migratory characteristics of gammadelta-T cells. gammadelta-T cells were found to express significantly higher levels of L-selectin than other subsets, suggesting that at least some of this preferential migration could be attributed to their interaction with ligands on vascular endothelium. More detailed experiments showed that gammadelta-T cells migrated through lymph nodes with greater efficiency than alphabeta T cells or B cells, which clearly indicated an enhanced ability of gammadelta-T cells to exit lymph nodes in the efferent lymph independent of entry from the blood. This hypothesis was supported by histological examination, where gammadelta-T cells were found almost exclusively in the interfollicular traffic areas within lymph nodes. These data indicate that gammadelta-T cells are the most active recirculating lymphocyte subset in ruminants and suggest new mechanisms to regulate the traffic of lymphocyte subsets through normal lymph nodes.     

Catecholamines in adrenergic nerve fibers of the lymph nodes after sympathectomy

Using Falck method in combination with microfluorimetry, catecholamine level in adrenergic nervous fibers has been measured in the canine popliteal lymph nodes, normal and in 12 h, 7, 30, and 90 days after unilateral lumbar sympathectomy. During first 24 h after the operation the level of catecholamines is for certain increased in the nervous fibers of the lymph node of the sympathectomized extremity. In 30 days after the sympathectomy their content drops at the side of the operation and increases in the contralateral extremity. By 3 months the equilibrium of the catecholamine content is restored in the nervous fibers of the lymph nodes in the homo- and contralateral extremities at the level higher than in the control. A conclusion is made that under conditions of unilateral sympathectomy only a partial sympathectomy of the popliteal lymph node is reached. All luminiscent adrenergic nervous fibers of the sympathectomized lymph node are processes of neurons, situating in the contralateral sympathetic trunk, or neurites of cells in sacral nodes, getting their preganglionic fibers from the contralateral trunk. The changes in catecholamine concentrations mentioned are considered as a compensatory reaction, directed to maintenance of general homeostatic equilibrium under conditions, when the nervous system transfers to a new level, ensuring the partly desympathized tissue by mediators.     

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     

Study of Response of Thymic and Submaxillary Lymph Node Lymphocytes to Administration of Lead by Different Routes

A number of studies have reported that heavy metals are not only toxic for the organism but they may modulate immune responses. In the current study, the effect of 4-week administration of 200 ppm of PbAc₂, using different routes of administration (orally and intraperitoneal injection), on lymphatic organs was evaluated. In the thymus, the number of lymphocyte cells and the cellularity diminished significantly for both routes of treatment. Regarding the submaxillary lymph nodes, no significant variations took place. Cell-mediated immune response is commonly evaluated by cell proliferation assays. Mitogens are known to induce a vigorous proliferative response in lymphoid cells from mammals. An increase in the proliferation of T lymphocytes stimulated by concanavalin A and the proliferation of B lymphocytes stimulated with lipopolysaccharides was found in thymus for both routes of administration, whereas in the lymph nodes, there was a decrease in proliferation of T lymphocytes. Furthermore, lead administration by intraperitoneal route caused an effect on B and T lymphocyte subpopulations. Thus, there was an increase in B+ cells and a decrease in T+ cells. Regarding CD4+ and CD8+ T cells, there were only variations, concretely a drop in both subpopulations, in lymph nodes when lead was administered intraperitoneally. It is important to emphasize that an increase in apoptosis was found in this tissue. At the histological level, evident alterations were described in thymus both for the oral and for the intraperitoneal route. Therefore, it is possible to show that lead administered by both routes generated effects on an immunological level.     

Morpho-functional status of the lymph nodes of rats during rehabilitation after exposure to vapors of dimethyl sulfate]

By means of histological methods inferior tracheobronchial lymph nodes have been investigated in 48 Wistar rats in 2 weeks and 3 months after cessation of inhalation of DNSV at maximum possible dose (0.1 mg/m3) for 2 and 14 days. Cell composition of the lymph nodes and cross sections of structural components have been compared. The cross section of the cortex decreases, while that of the medulla increases in comparison with corresponding parameters of the acute experiment. In 2 weeks and 3 months after DMSV effect for 2 days the part (%) of poorly differentiated cells and middle lymphocytes increases, while content of plasma cells in the medullary cords decreases. During rehabilitation after DMSV effect for 14 days a low level plateau is noted (as in the acute experiment). However, in lymphoid nodules++ within content of the poorly differentiated cells and middle lymphocytes in the cortical germinative centers in 2 weeks and 3 months the number of the poorly differentiated and reticular cells increases essentially, as well as mitotically dividing cells (in comparison to the acute experiment). In the medullary cords of the lymph nodes after 3 months of rehabilitation content of immature plasma cells is essentially higher than in 2 weeks of rehabilitation and than in the acute experiment. During rehabilitation the level of cells destruction in the lymph nodes noticeably decreases in comparison to the acute experiment.     

Heterogeneous expression of toll-like receptors in lymphatic endothelial cells derived from different tissues

As lymphatic endothelial cells (LECs) express different lymphatic and vascular markers depending on the organ they are derived from, we analysed whether they also show a heterogeneity of response against pathogens. To this end we analysed, for the presence of mRNA encoding for all human toll-like receptor (TLR), LECs isolated from lymph nodes and thymuses. RNA for TLR1-6 and 9 was identified in thymus-derived cells, whereas cells derived from lymph nodes contained mRNA for TLR1-4, 6 and 9, but failed to express mRNA specific for TLR5. The differential expression of TLRs was confirmed by the phosphorylation of nuclear factor-κB p65 only when the two types of LECs were incubated with the appropriate TLR agonists. The stimulation with specific agonists gives rise to a heterogeneous pattern of cytokine and chemokine secretion: thymus-derived LECs produced preferentially interleukin-6, interferon-inducible protein (IP)-10 and tumour necrosis factor-α, whereas cells prepared from lymph nodes mainly released interleukin-8, monocyte chemotactic protein-1, RANTES and (IP)-10. Finally, cells purified from lymph nodes expressed a higher level of intercellular adhesion molecule-1 than did cells prepared from the thymus when stimulated with several TLR agonists. The expression of a large set of TLRs and the responsiveness to specific agonists suggest that LECs are able to respond to pathogens, and the observed differences reflect specialized functions, redundancy and/or roles of LECs of different origin.     

Comparison of local and systemic responsiveness of lymphocytes in vitro to Bovicola ovis antigen and Concanavalin A in B. ovis-infested and naive lambs

The in vitro proliferation assay was used to determine lymphocyte responsiveness to soluble antigen of B. ovis and to Concanavalin A (Con A) in peripheral blood, spleen and various lymph nodes from B. ovis-infested and naive lambs. From March to July, an assay of monthly blood samples showed generally higher proliferative responses to antigen and Con A in B. ovis-infested than naive lambs. The proliferative response of cells from the skin-draining prescapular lymph nodes to B. ovis antigen was significantly higher in B. ovis-infested than naive lambs. Responses of cells from the medial iliac, mediastinal and mesenteric lymph nodes (which do not receive lymph from the skin) and spleen showed no significant differences between groups. Within the B. ovis-infested lambs, the response of cells from the prescapular lymph node was significantly higher than that from any other lymphoid organ examined. Responsiveness of the prescapular, medial iliac and mesenteric lymph node and spleen cells to Con A was not significantly different between groups, while mediastinal lymph node cells showed a significantly higher response in B. ovis-infested lambs. The data indicate that the antigen-specific cellular immune response is operating mainly locally, at the level of the skin and draining lymph nodes. Responses to the T cell mitogen Con A did not support non-specific immunodepression as reported in other ectoparasite/host systems.     

Lymphocyte function in experimental African trypanosomiasis. III. Loss of lymph node cell responsiveness

The relationship between immunosuppression and suppressor cell activity in the lymphoid organs of animals with experimental African trypanosomiasis has been examined further. In the present study we measure the primary in vitro PFC response to SRBC by spleen and lymph node cells from Trypanosoma rhodesiense infected or drug-cured C57BL/6 mice. Passive transfer experiments with this culture system tested for the presence or absence of suppressor cells. We demonstrate that infected mice exhibit immunosuppression in the spleen cell population several weeks before becoming suppressed at the level of the lymph node cell populations. Although suppressor cells are present in immunosuppressed spleen cell populations, suppression of lymph node cell responsiveness was not attributable to suppressor cells detectable withi, lymph nodes. After Berenil treatment of terminally infected mice immunocompetence was restored gradually, first to the lymph node cells and subsequently to the spleen cell population. Recovery of spleen cell responsiveness was attributable to the loss of detectable suppressor cell activity within spleens. These results demonstrate that there is anatomical restriction of the suppressor cell population to trypanosome-infected mouse spleen and that loss of immunocompetence in the lymph nodes may be due to factors unrelated to suppressor cell effects.     

Structure of the non-lymphoid cells during the postnatal development of the rat lymph nodes

This study describes the postnatal development of the nonlymphoid cells with special reference to the fibroblastic reticulum cells (FRCs) and interdigitating cells (IDCs). The first lymphocytes of the neonatal lymph nodes are located in the developing deep cortex units (DCUs) identified by the Gomori's technique for reticulin fibres. Ultrastructural studies demonstrate that FRCs form the stroma of the DCUs. By light and electron microscopy, it is demonstrated that FRCs occupy the outer cortex in the following stages of development of the lymph nodes. Thus, FRCs form the stroma of the primary follicles and, later, are transformed in follicular dendritic cells (FDCs) of the germinal centres. Immature or pro-IDCs appear as migrating elements in the deep cortex of lymph nodes of the neonatal rats. The ultrastructure of the pro-IDCs resembles that of the mature IDCs but not that of the phagocytic cells. Pro-IDCs are transformed into mature IDCs whose cytoplasmic expansions contact lymphocytes via tight junctions. Some of these lymphocytes are likely apposed to FRCs of the DCUs. No cells containing Birbeck granules were found in the parenchyma of the lymph nodes during the postnatal development. The role of these nonlymphoid cells is discussed with respect to the immunologic function of mammalian lymph nodes.