Bacterial phagocytosis by macrophages from lipopolysaccharide responder and nonresponder mouse strains.

The phagocytic capacity of macrophages from C3H/H3J mice was assessed against lipopolysaccharide-producing (Escherichia coli) and -nonproducing (Staphylococcus aureus) bacteria. Despite their gene-coded unresponsiveness to lipopolysaccharide endotoxin and lymphokines and their defective tumoricidal activity, proteose peptone-induced C3H/HeJ macrophages did not display a defective phagocytic capacity, but rather displayed an enhanced phagocytosis of both bacterial strains compared with macrophages from closely related C3H/HeN mice. Unstimulated peritoneal resident C3H/HeJ macrophages, on the other hand, displayed a normal phagocytic activity toward E. coli and enhanced phagocytosis toward S. aureus.     

T-cell-dependent popliteal lymph node reactions to platinum compounds in mice.

The requirements for sensitization to complex salts of platinum were investigated in a mouse model by means of the popliteal lymph node (PLN) assay. A single subcutaneous injection of dissolved hexachloroplatinates without adjuvant induced a vigorous primary immune reaction in the draining PLN. Dose-dependent lymph node activation was determined by an increase in both PLN weight and cellularity. In C57BL/6 mice, peak reactions were obtained around day 6 after administration of 90-180 nmol Na2[PtCl6] or (NH4)2[PtCl6] per animal. Mice primed to [PtCl6]2- mounted an enhanced response upon local restimulation with suboptimal doses of the same but not unrelated compounds, indicating a specific secondary response. T cells were required to elicit PLN reactions to [PtCl6]2-, because athymic nude mice completely failed to respond, in contrast to their +/nu littermates. Differences between various inbred strains of mice revealed that Pt-induced PLN responses are genetically controlled. Moreover, the immunogenicity of Pt salts in mice is not confined to hexachloroplatinates, but other compounds, such as the antineoplastic agent cis-dichlorodiamine platinum, are able to induce comparable PLN reactions.     

Long-term antigen retention by dendritic cells in the popliteal lymph node of immunized mice.

Antigen retention by follicular dendritic cells (FDC) was studied in the popliteal lymph nodes (PLN) of mice actively or passively immunized against human serum albumin (HSA) or horse spleen ferritin. Electron microscopic autoradiography was used to locate a challenge dose (1 microgram) of 125I-labelled HSA in the draining PLN following injection into the hind footpad of specifically immune mice. In both actively and passively immunized mice, the radiolabelled antigen was localized to the follicles in the cortex of the draining node. In actively immunized mice, the antigen formed a crescent of label on the superficial aspect of germinal centres, while in passively immunized mice label was seen in primary follicles. In electron microscope autoradiographs, the silver grains were concentrated in areas of dendritic cell processes which emanated from a cell body containing a characteristic irregular nucleus. The size and complexity of the dendritic cell processes increased in actively immunized mice suggesting that the FDC could hypertrophy. High resolution studies using the electron-dense antigen, ferritin, showed that it was localized to the extracellular space of the FDC processes and was associated with amorphous electron-dense material; presumably immune complexes. Antigen was not present uniformly distributed in the extracellular material but rather it was in discrete patches occupying small segments of the FDC processes. Large amounts of label-free electron-dense material were present suggesting that immune complexes of various specificities were present on each DC. Ferritin was seen more than 3 months after challenge but only on the FDC. The data suggest that the antigen retaining mechanism in the lymph node of immune mice is antigen non-specific, is capable of hypertrophy in response to active immunization and provides a mechanism for stable long-term retention of antigen.     

Use of the popliteal lymph node enlargement assay to measure rat T cell function in immunotoxicologic testing

Modifications to the host vs graft response assay (HvGR) which render this technique suitable as an in vivo screen of cell-mediated immune function are described. This method utilizes mitotically inactivated spleen cells from normal heterozygous "nude" rats as stimulator population. We injected these cells into the left hind foot pad of unsensitized Charles River CD rats. Within one week, the recipients are sacrificed and both popliteal lymph nodes removed and weighed. The difference in weight between the challenged and unchallenged node represents the primary T cell response to the injected antigen sequestered in the draining lymph node. Using the immunosuppressive agents cyclophosphamide and dexamethasone we demonstrate the applicability of this assay to detect impaired cell-mediated immune function.     

Evidence for immunotoxic effects of crude Ginkgo biloba L. leaf extracts using the popliteal lymph node assay in the mouse

Allergic reactions due to contact with different parts of the ancient tree Ginkgo biloba L. have repeatedly been reported. Provocation tests in patients and animal experiments have identified alkylphenols such as ginkgolic acids as causative constituents. Leaf extracts from Ginkgo are widely used to treat peripheral or cerebral circulatory disorders and Alzheimer's disease. Since alkylphenols are also present in leaves, potential allergic and other immunological hazards of such preparations have to be carefully controlled. Thus, we have evaluated if the popliteal lymph node assay (PLNA) in the mouse may represent a suitable model for the detection of constituents with immunotoxic properties in a complex mixture of biologically active agents such as plant extracts. Subplantar injection (2 mg) of a crude aqueous-ethanolic extract from Ginkgo leaves caused a significant lymphoproliferative reaction (LPR) in the ipsilateral popliteal lymph node. PLNA-active compounds in this extract could be enriched in the lipophilic phase by liquid-liquid partition between heptane and water. Chemical analysis of the heptane extract revealed the presence of a high concentration of alkylphenols (approx. 30%) and further subfractionation indicated that the enlargement of the popliteal lymph node was mainly due to the content of ginkgolic acids. This presumption was corroborated by observing a similar LPR following injection of a purified mixture of ginkgolic or hydroginkgolic acids. Thus, our experiments confirm that Ginkgo leaf extracts may contain constituents with immunotoxic properties, underlining the need to apply adequate production procedures to guarantee the completest possible removal of these compounds. The PLNA appears to represent a simple test model for the detection, characterisation and control of ingredients with potential immunotoxic side effects in complex herbal drugs.     

Reaction of the popliteal lymph node to transcutaneous irradiation with helium-neon laser

Prolonged transcutaneous irradiation with helium-neon laser light decreases the transport ability of the popliteal and iliac lymph nodes. Laser radiation stimulates lymphocytes, predominantly T cells. Translated fromByulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 123, No. 2, pp. 237–239, February, 1997     

Presence of common idiotypes on antibodies induced by glutamic acid-lysine-containing terpolymers in responder and nonresponder mice with the Ig-1b heavy chain allotype.

A B10.A (5R) responder mouse to the random linear terpolymer, poly--(Glu, Lys, Phe), GLphi, can produce immunoglobulins which bind poly-L (Glu, Lys), GL, that share idiotypic determinants with GL-binding antibodies produced by other members of the same strain. Expression of these common idiotypic determinants, termed BGL, is independent of the H-2 halotype and closely linked to the Ig-lb heavy chain allotype. Moreover, nonresponder mice with the Ig-lb heavy chain allotype, when immunized with GLphi that has been chemically coupled to an immunogenic carrier, chicken IgG, can produce GL-binding antibodies that share BGL idiotypic specificities with anti-GLphi antibodies produced by responder animals. Also, the responses to other GL-containing polymers, such as poly-L (Glu, Lys, Ala) and poly-L (Glu, Lys, Pro), which are under the control of distinct Ir genes, can stimulate the production of GL-binding antibodies that share common BGL idiotypic determinants with antibodies induced with GLphi. These findings are discussed with respect to their implications concerning the mechanism(s) of Ir gene control.     

Structure of the sinus-lining cells in the popliteal lymph node of the rabbit

The structure of the sinus walls in the popliteal lymph node of the rabbit was studied with the electron microscope. In the marginal sinus, the endothelial cells are connected by gap junctions, puncta adherentia, and surface specializations characterized by focal approximation of the adjoining membranes without fusion. They possess large numbers of simple and compound uncoated invaginations of the plasma membrane that are closed by a diaphragm with a central thickening. The tissue strands that straddle the lumen of the sinus consist of a fibrous core containing both collagen and elastic fibers, surrounded by endothelial cells identical to those composing the outer sinus wall. Cortical sinuses that run independently of the trabeculae were identified by exploiting the fact that their endothelial cells accumulate lymph-borne ferritin, and their lumen is outlined by horseradish peroxidase administered intravenously. They are lined by a flattened, continuous endothelium and lack luminal strands. The walls of the medullary sinuses consist of endothelial cells and macrophages. The endothelial cells are interconnected by specialized junctions and contain fewer plasmalemmal vesicles than in the cortex; furthermore, dense granules are present in their cytoplasm. Macrophages adhere to the surface of the endothelial cells; typically, none of the junctional specializations that characterize the interface between endothelial cells connect endothelial cells to macrophages. However, at points along the contact region with the endothelium, the plasmalemma of the macrophage is decorated by an attachment plaque of fluffy cytoplasmic material. Sinus endothelial cells slowly accumulate lymph-borne ferritin like vascular endothelial cells elsewhere in the body, whereas macrophages contain both ferritin and engulfed erythrocytes.     

Immune responses of the ovine lymph node to Chlamydia psittaci. A cellular study of popliteal efferent lymph.

The popliteal efferent lymphatics were cannulated in sheep of two categories, seronegative or immune to Chlamydia psittaci. Following subcutaneous injection of live C. psittaci or control material into the draining area of the popliteal node, sequential samples of efferent lymph were collected and analysed. Both categories of sheep responded to C. psittaci with increased outputs of lymphocytes and blast cells. Numbers of blast cells rose both absolutely and as a proportion of the total. Plasmablasts increased in number only in seronegative sheep. Outputs of total T cells (CD5+), helper T cells (CD4+), cytotoxic/suppressor T cells (CD8+) and non-helper, non-suppressor T cells (T19) were maximal 4 and 7 days after challenge in immune and seronegative sheep, respectively. Proportionally, CD4+ T cells declined, CD8+ T cells increased and T19 cells were unaltered with time after infection. Chlamydial antigens could not be demonstrated in the cells of efferent lymph by an immunoperoxidase method. The results of this preliminary study show that both T and B cell responses are involved in immunity to C. psittaci.     

In vivo lymphoproliferation in the popliteal lymph node (PLN) assay can be inhibited by leflunomide's active metabolite A77 1726

Inflammation Research -     

Induction of popliteal lymph node enlargement and antibody production in the mouse by pyridylallylamines related to zimeldine

The role of the para-brominated phenyl ring of the allylamine antidepressant drug, zimeldine, in its immunomodulating activity was studied by comparing local lymph node responses of mice to analogues with various phenyl ring substituents. The compounds (1.0 mg) were injected into the hind footpad and weight and cell number of the popliteal lymph node (PLN) and antibody production by PLN cells were determined 7 days later. Zimeldine and the para-trimethylsilylated and ortho-para-chlorinated compounds induced a marked PLN weight gain in C57BL/6 mice. The para-chlorinated and meta-brominated analogues were significantly less effective, while the para-fluorinated and the unsubstituted analogue failed to trigger PLN weight gain. The same range of potency of the compounds was seen when increases of PLN cell number in BALB/c mice and increases of IgM production by a fixed number of BALB/c PLN cells were determined. The IgG production on a per cell basis was not increased by the fluorinated and the unsubstituted compounds, while the remaining compounds stimulated the IgG production to a similar extent. Plots of the lipophilicity of the para-substituted compounds against their ability to induce PLN weight and cell gain showed a fair correlation, but induction of antibody formation tended to be counteracted at too high lipophilicity. No correlation between pharmacologic and immunologic activity of the compounds could be found. Data indicate that lipophilicity grossly favours PLN enlargement, but the divergent responses to the equilipophilic 3- and 4-brominated compounds, suggest conformational restraints as well.(ABSTRACT TRUNCATED AT 250 WORDS)     

Female popliteal lymph node responses to H-Y antigen on male thymocytes in mice. II. H-2 restriction of secondary responses

C57BL/10 (abbreviation B10) female mice give a high primary popliteal lymph node (PLN) response to syngeneic male thymocytes. The PLN response to the H-Y antigen is suppressed if B10 females are primed by an intraperitoneal injection of syngeneic male cells. Suppression of the response can be induced by priming with not only syngeneic B10 male thymocytes but also allogeneic thymocytes which share the H-2D locus of the H-2b haplotype with the responder (Db restriction). On the other hand, B10.D2, B10.A, and HTO female mice, giving a low primary PLN response to H-Y, give high secondary PLN responses when primed intraperitoneally with syngeneic male thymocytes. A high secondary response can also be obtained by priming with allogeneic male thymocytes which share the K end loci (K, A beta, A alpha, E beta, and E alpha) of the H-2 complex with the responder. Apparently the male thymocytes used for priming must share class I (and possibly class II) H-2 loci with the female recipients to enable the recognition of the H-Y antigen and subsequent development of the genetically determined type (suppression or amplification) of the secondary PLN response.     

Different T-cell activation by streptozotocin and freund's adjuvant in popliteal lymph node (PLN)

A popliteal lymph node (PLN) test was further validated for predictive screening of autoimmunity-inducing drugs. Autoimmune-like T-cell activation of streptozotocin (STZ) was compared with the effect of Freund's complete adjuvant (FCA), injected locally into the foot pad of BALB/c mice. Early cell activation in enlarged PLN was monitored by flow cytometry. Injection of both STZ and FCA markedly increased the absolute PLN cell number as well as specific T-helper (CD4⁺), T-suppressor/cytotoxic (CD8⁺), and B (Ig⁺) subsets. However, quantitative analysis of early T-cell activation revealed important differences between STZ-induced PLN reaction and FCA-related lymphoproliferation. At 72 h, the number of cells stained with anti-early activation marker (EAM⁺; CD69⁺) increased over 10 times in STZ-enlarged nodes and only 3 times in the FCA-inflamed nodes. Furthermore, different cytometric profiles were noted for STZ-activated and FCA-activated cells stained with anti-interleukin-2 receptor (IL-2R) (CD25⁺). The data suggest the applicability of early cytometric screening of enlarged PLN for predictive analysis detection of chemicals inducing an autoimmune-like reaction.     

H-Y antigen: genetic control of the expression as detected by host-versus-graft popliteal lymph node enlargement assay maps between the T and H-2 complexes

We are using the terms 'low' and 'high' expression when the H-Y antigen on thymocytes of mouse parental strains is, respectively, weakly and strongly immunogenic for F1 hybrids in a host-versus-graft popliteal lymph node enlargement assay. Using this assay we attempted to locate the genetic factor(s) responsible for the control of H-Y expression within chromosome 17 since our previous study indicated linkage with the H-2 complex. We produced to this aim recombinants from (B10.T X C3H/Di)F1 hybrids where crossing over took place between H-2 and T complexes. In this way we were able to locate a gene denoted Hye (for H-Y expression) whose 'high' and 'low' alleles originated, respectively, from the B10 and C3H/Di strains. In the recombinants, the expression did not follow the H-2 haplotype, but obviously depended on the position of crossing over between H-2 and T. This pointed to the Hye gene mapping proximally from the H-2 complex.     

Binding of Candida albicans yeast cells to mouse popliteal lymph node tissue is mediated by macrophages.

We previously reported that Candida albicans yeast cells adhere to the macrophage-rich medullary and subcapsular sinus areas of mouse lymph node tissue. To determine whether the yeast cell-lymph node interaction is mediated by macrophages, the effect of specific elimination of macrophages on yeast cell binding was studied, and yeast cell adherence was correlated with the ingestion of India ink by lymph node cells. Macrophage elimination was done by use of liposome-containing dichloromethylene diphosphonate (L-Cl2MDP). Mice were injected in the hind footpads with the L-Cl2MDP preparation, popliteal lymph nodes were removed 5 days later, and yeast cell adherence was determined by an ex vivo binding assay. As controls, lymph nodes from mice that received footpad injections of either phosphate-buffered saline (PBS) alone or liposome-containing PBS were used. Use of macrophage- and neutrophil-specific monoclonal antibodies in tissue immunostaining showed that the L-Cl2MDP treatment eliminated macrophages but not neutrophils from the medullary and subcapsular sinus areas of the popliteal lymph nodes. A striking reduction of yeast cell adherence occurred with lymph nodes from L-Cl2MDP-treated mice compared with lymph nodes from control animals. The lymph node-yeast cell binding patterns of L-Cl2MDP-treated and control mice were the same regardless of mouse strain, sex, or T-cell competency. Results of India ink experiments, in which India ink was injected into footpads of mice and was rapidly taken up by popliteal lymph node macrophages, showed a strong correlation between yeast adherence and India ink staining of cells. In addition, the interaction of yeast cells with lymph node tissue from normal mice was not significantly affected by the addition of two extracellular matrix proteins, fibronectin and laminin, during the ex vivo adherence assay. These data indicate that medullary and subcapsular sinus lymph node macrophages express an adhesion system similar to that described for mouse splenic marginal zone macrophages.     

Popliteal lymph node reactions in mice induced by the drug zimeldine

The antidepressant drug zimeldine was screened for immune modulating properties using the popliteal lymph node (PLN) assay as a test system in mice. In immunocompetent as well as congenitally athymic nude mice, footpad injection of 1.0 mg zimeldine triggered a bimodal footswelling. A transient oedematous swelling, histologically characterized by mast cell degranulation, was followed by infiltration of polymorphnuclear cells. A dose-dependent PLN enlargement to the agent was observed, which appeared to be more pronounced in immunocompetent mice as compared with athymic nude mice, and in H-2b mice as compared with H-2d mice. After injection of 1.0 mg zimeldine into the footpad of C57BL/10 mice, significant enlargement was already observed by 3 days after injection, was optimal around day 9 and persisted for at least 30 days. Histologically, PLN reactions were characterized by blast transformation of lymphocytes and expansion of paracortical areas prior to germinal center reactions in enlarged follicles. Size of both areas gradually decreased as the medulla filled with plasma cells, 7-30 days after injection. The observed reactions could not be transferred with syngeneic lymph node cells after prior exposition to zimeldine in vivo or in vitro. We conclude that zimeldine induces strong and persistent PLN enlargement, blastogenesis and prominent germinal center reactions. Immunocompetent T-cells are apparently conducive, but not prerequisite to these reactions, which suggests involvement of multiple mechanisms including those mediated by inflammatory reactions in the foot. It is unlikely that the observed enlargement of PLN can be attributed to a direct chemical modification of leukocyte membranes by zimeldine. The protracted nature of the reaction may indicate that zimeldine somehow interferes with inhibitory feedback mechanisms.