Popliteal Lymph Node Response Research Articles

Cellular immune response in popliteal lymph nodes and liver of dogs with visceral leishmaniasis

Cellular immune response in popliteal lymph nodes and liver of dogs with visceral leishmaniasis

Norfloxacin, a Fluoroquinolone Antibiotic, Inhibits Langerhans Cell-Mediated Th1 and Th2 Cell Development.

It is widely acknowledged that Langerhans cells (LCs) play a primary role in the polarization of T helper type 1 (Th1) or T helper type 2 (Th2) immune responses. Our aim was to find fluoroquinolone ("new quinolone") antibiotics that would inhibit LC-mediated Th2 cell development. Expression of LC surface molecules was investigated using the reverse transcriptase polymerase chain reaction. The effects of fluoroquinolone antibiotics on T-cell immunoglobulin and mucin domain-containing protein (TIM)-4 expression in LCs were examined to predict whether they would inhibit Th2 cell development. Mice were primed via the hind footpad with ovalbumin (OVA) peptide-pulsed LCs that had been treated with a selected fluoroquinolone antibiotic, then 5 days later the cytokine response in popliteal lymph nodes was examined by enzyme-linked immunosorbent assay. Norfloxacin was selected as a candidate inhibitor of Th2 cell development. As expected, OVA peptide-pulsed LCs that had been treated with norfloxacin and injected into the hind footpads of mice inhibited Th2 cell development, as represented by down-regulation of interleukin (IL)-4 production, as well as Th1 cell development, as represented by down-regulation of interferon (IFN)- g production. This additional inhibition of Th1 cell development was accompanied by suppression of CD40 expression in LCs. In addition, Staphylococcus aureus strains isolated from skin lesions of patients with atopic dermatitis (AD) were more susceptible to norfloxacin than to gentamicin. Topical treatment with norfloxacin significantly suppressed the increase in the skin severity score in NC/Nga mice with AD-like skin lesions. This suppressive effect was associated with a decrease in the production of IFN-g and IL-4 in auricular lymph node cells. The present results show that topical application of norfloxacin inhibits the development of AD-like skin lesions in NC/Nga mice. This suggests that topical application of norfloxacin to AD lesions colonized with S. aureus would act on both superficial S. aureus and epidermal LCs, thus possibly inhibiting the development of Th1 and Th2 cells in vivo, and controlling the severity of AD.

Cellular immune response in popliteal lymph nodes and liver of dogs with visceral leishmaniasis

Cellular immune response in popliteal lymph nodes and liver of dogs with visceral leishmaniasis

The role of properdin in murine zymosan-induced arthritis

Using properdin-deficient and wild-type mice, we have investigated the role of properdin in development and progression of zymosan-induced arthritis. At the initial phase of local, zymosan-induced inflammation, properdin-deficient and wild-type mice showed bone erosion, proteoglycan loss and cell infiltration. Compared to wild-type, properdin-deficient mice had reduced C5a and IL-6 but similar synovial TNF-α and sRANKL levels. Both groups showed a systemic immune response. Elevated IFN-γ production and STAT1 signaling in splenocytes and a shift to Th1 response in popliteal lymph nodes were observed in properdin-deficient mice. Properdin-deficient mice had significantly less circulating zymosan-specific IgG antibodies than wild-type. In the chronic phase, the lack of properdin resulted in significant proteoglycan loss in the joints and lower cartilageneous STAT1 expression. The level of synovial C5a on day 30 was comparable in both groups, but C5aR staining was more apparent in the joints of properdin-deficient mice. Our data show that properdin is an important player in processes involved in inflammatory joint degradation.

Fine Ambient Particles from Various Sites in Europe Exerted a Greater IgE Adjuvant Effect than Coarse Ambient Particles in a Mouse Model

In the European Union (EU)-funded project Respiratory Allergy and Inflammation due to Ambient Particles (RAIAP), coarse and fine ambient particulate matter (PM) was collected at traffic dominated locations in Oslo, Rome, Lodz, and Amsterdam, in the spring, summer, and winter 2001/2002. PM was also collected in de Zilk, a rural seaside background location in the Netherlands. The aim of this study was to screen the ambient PM fractions for allergy adjuvant activity measured as the production of allergen- (ovalbumin-) specific immunoglobulin (Ig) E following subcutaneous (sc) injection into the footpad of mice. A second aim was to determine whether the 6-d popliteal lymph node (PLN) assay can be used to detect an allergy adjuvant activity. Allergy screening for IgE adjuvant activity showed that in the presence of ovalbumin (Ova) 12 out of 13 of the fine ambient PM fractions exerted a significant IgE adjuvant activity. In contrast, only 3 out of 13 of the coarse PM fractions had significant adjuvant activity. Overall, fine ambient PM exerted significantly greater IgE adjuvant activity per unit mass than coarse PM. No significant differences were observed between locations or seasons. Substantial higher levels of specific components of PM such as vanadium (V), nickel (Ni), zinc (Zn), ammonium (NH4), and sulfate (SO4) were present in the fine compared to coarse PM fractions. However, differences in the content of these components among fine PM fractions did not reflect the variation in the levels of IgE anti-Ova. Still, when comparing all seasons overall, positive correlations were observed between V, Ni, and SO4 and the allergen specific IgE levels. The PLN responses (weight and cell number) to Ova and ambient PM in combination were significantly higher than to Ova or PM alone. Still, the PLN assay appears not to be useful as a quantitative assay for screening of allergy adjuvant activity since no correlation was observed between PLN responses and allergen specific IgE levels. In conclusion, fine ambient PM fractions consistently were found to increase the allergen-specific IgE responses more than the coarse ones. Our finding is in agreement with the notion that traffic-related air pollution contributes to the disease burden in asthma and allergy, and points to fine and ultrafine ambient PM as the most important fractions in relation to allergic diseases.

Experimental envenomation with Crotalus durissus terrificus venom in dogs treated with antiophidic serum - part II: laboratory aspects, electrocardiogram and histopathology

The present work shows laboratory aspects, electrocardiogram and histopathology results during experimental envenomation by Crotalus durissus terrificus in dogs treated with antiophidic serum. Twenty-one dogs were divided into three groups of seven animals each. Group I received 1mg/kg venom (sc); Group II received 1mg/kg venom (sc), 50mg antiophidic serum (iv) and fluid therapy including 0.9% NaCl solution (iv); and Group III received 1mg/kg venom (sc), 50mg antiophidic serum (iv) and fluid therapy including 0.9% NaCl solution containing sodium bicarbonate diluted to the dose of 4mEq/kg. Urinalysis showed brown urine, proteinuria, occult blood and myoglobinuria. Respiratory acidosis and hypotension were also observed. At the venom inoculation site, there was discreet edema, popliteal lymph node response, musculature presenting whitish areas and necrotic myositis with myoregenerative activity. There was not evidence of electrocardiographical and biochemical alterations.

Investigating the Role of 2-Phenylpropenal in Felbamate-Induced Idiosyncratic Drug Reactions

Felbamate (2-phenyl-1,3-propanediol dicarbamate, FBM) can cause aplastic anemia and hepatotoxicity. The mechanism of FBM-induced toxicities is unknown; however, it has been proposed that 2-phenylpropenal, a reactive metabolite of FBM, is responsible. The pathway leading to this metabolite involves hydrolysis of FBM to 2-phenyl-1,3-propandiol monocarbamate (MCF), oxidation to 3-carbamoyl-2-phenylpropionaldehyde (CBMA), and spontaneous loss of carbon dioxide and ammonia. We made a polyclonal antibody against 2-phenylpropenal bound to protein and confirmed its specificity using ELISA. We attempted to develop an animal model of FBM-induced aplastic anemia and/or hepatotoxicity, and we also used the antibody to try to detect covalent binding of 2-phenylpropenal using immunoblotting. However, none of the animals developed evidence of bone marrow or liver toxicity, and we were unable to detect covalent binding, possibly because significantly less 2-phenylpropenal is formed in rodents than in humans. As this type of idiosyncratic drug reaction is believed to be immune-mediated, we also studied the potential of FBM and its metabolites to stimulate an immune response using the reporter antigen popliteal lymph node assay in female Balb/c mice. We found that neither FBM nor MCF induced an immune response in popliteal lymph nodes (PLNs). However, CBMA treatment appeared immunogenic, causing footpad inflammation, hardening, scab formation, and an increase in thickness. The PLN cell count in CBMA-treated mice increased 8-fold as compared to control, FBM-, or MCF-treated mice. Immunohistochemical analysis of the CBMA-exposed PLNs revealed germinal center formation, indicating B cell proliferation, later confirmed by flow cytometry. Most of the cells expressing the activation surface marker CD54 were B cells. We also found that CBMA treatment caused an increase in the production of IgM and IgG1 antibodies as well as IL-4 and IFN-gamma cytokines. Our findings indicate that 2-phenylpropenal is a very potent immunogen, supporting its possible involvement in the FBM-induced hepatotoxicity and aplastic anemia.

Positive responses to imipramine in the popliteal lymph node assay are due to primary irritation.

The popliteal lymph node (PLN) assay has long been proposed as a tool to detect immunotoxicants with the potential to induce systemic autoimmunity. A major problem hampering the further validation of this assay is the need to rule out irritants that cause false-positive PLN responses. The anti-depressant, imipramine, has not been reported to induce systemic autoimmune reactions in treated patients, but has been repeatedly found positive in the PLN assay, suggesting that this is a false-positive response. To test this hypothesis, the effects of imipramine were compared to those of 50% ethanol in C57B1/6 mice. Footpad edema was evidenced in the few days after injection of both ethanol and imipramine. T-cell depletion using monoclonal antibodies against either CD4+ or CD8+ T-lymphocytes prior to the PLN assay did not influence the responses to either ethanol or imipramine. Cytokine (TNFalpha, IL-1alpha, IL-1beta, IL-2R, IL-6, IL-12 and IFN-gamma) fingerprinting of the PLNs after injection of ethanol and imipramine evidenced the same pattern of responses. These results indicate a closely similar pattern of responses following the footpad injection of either imipramine or ethanol. The conclusion can be drawn that imipramine induces positive responses in the PLN assay via primary (nonspecific) irritation.

T cells ignore aniline, a prohapten, but respond to its reactive metabolites generated by phagocytes: possible implications for the pathogenesis of toxic oil syndrome.

The most basic arylamine, aniline, belongs to a class of compounds notorious for inducing allergic and autoimmune reactions. In 1981 in Spain, many people succumbed to toxic oil syndrome (TOS), a disease caused by ingestion of cooking oil contaminated with aniline. Indirect evidence points toward an immune pathogenesis of TOS driven by T lymphocytes, but it is unclear to which antigens these cells could react. Here, using the popliteal lymph node (PLN) assay in mice, we analyzed the sensitizing potential of aniline, its metabolites, and some of the aniline-coupled lipids detected in the contaminated cooking oil. Whereas aniline itself and its non-protein-reactive metabolites nitrobenzene, p-aminophenol and N-acetyl-p-aminophenol, failed to elicit PLN responses, its reactive metabolites nitrosobenzene and N-hydroxylaniline did. The aniline-coupled lipids, namely, linoleic anilide and linolenic anilide, and a mixture of fatty acid esters of 3-(N-phenylamino)-1,2-propanediol, all implicated in TOS, induced significant PLN responses, whereas the respective aniline-free lipids, linoleic acid, linolenic acid, and triolein, did not. Hence, the aniline moiety plays a crucial role in the immunogenicity of the aniline-coupled lipids of TOS. PLN responses to the reactive aniline metabolites and the one aniline-coupled lipid that was tested, linolenic anilide, were T-cell-dependent. Secondary PLN responses to nitrosobenzene were detectable not only after priming with nitrosobenzene but, in some experiments, also after priming with linolenic anilide. This suggests that the aniline moiety was cleaved from the aniline-coupled lipid and metabolized to the intermediate nitrosobenzene that generated the prospective neoantigens. Consistent with this, in lymphocyte proliferation tests in vitro, T cells primed to nitrosobenzene reacted in anamnestic fashion to white bone marrow cells (WBMCs) pulsed with aniline. Hence, we propose that aniline is a prohapten that can be metabolized by WBMCs, which form neoantigens that are recognized by T cells. The possible significance of these findings for the pathogenesis of TOS is discussed.

Increased production of interferon-gamma, but not IL-4 mRNA, by streptozotocin in the popliteal lymph node assay.

The popliteal lymph node (PLN) assay has been proposed as a tool to predict systemic autoimmune reactions induced by medicinal products and chemicals, the mechanisms of which are poorly understood. To determine whether PLN responses involved Th1 or Th2 cell control, or both, the effects of streptozotocin (STZ), a prototypic immunotoxic compound, were analysed on the production of interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) mRNA by lymph node cells after injection into the hind footpad of C57 BL/6 mice. Streptozotocin induced a dramatic increase in IFN-gamma mRNA production, which correlated with PLN responses as evidenced by augmented weight and cellularity indices. No effect on IL-4 mRNA synthesis was noted. These results suggest that a Th1 response is involved in the PLN response to STZ.

The popliteal lymph node response to streptozotocin is under type 1, MHC class-I restricted, CD8 + T-cell control

The popliteal lymph node (PLN) assay has been proposed to predict the ‘autoimmunogenic’ potential of xenobiotics. A better understanding of the processes involved in PLN responses is needed to establish the value of this assay for preclinical safety evaluation. In order to determine whether PLN responses involve CD4 + or CD8 + T-cells, the effects of streptozotocin (STZ), a prototypic immunotoxic compound, were analyzed after injection into the hind footpad of C57 BL/6 mice and major histocompatibility complex (MHC) class I or II deficient mice. The involvement of type 1 or type 2 cell control on the production of cytokine mRNAs was analyzed in lymph node cells by quantitative RT-PCR, together with the analysis of a wide range of cytokine mRNAs after STZ injection (IL-1α, IL-1β, TNF-α, IFN-γ, IL-2, IL-2 receptor, IL-4, IL-5, IL-6, IL-10 and IL-12). We have found that mice depleted in CD8 + T-cells did not respond to STZ, whereas mice depleted in CD4 + T-cells exhibited the expected positive PLN responses, with increased weight and cellularity indices. STZ induced a low production of interleukin (IL)-2 mRNAs, a mild increase in IL-1α and IL-6 mRNAs production, and a dramatic increase in IFN-γ, IL-1β, TNF-α, IL-12 and IL-2 receptor mRNAs, which correlated with positive PLN responses. No effects on IL-4, IL-5 and IL-10 mRNAs synthesis were noted. In CD8 + T-cell deficient mice, there was no production of IFN-γ or IL-6 mRNAs. These results suggest that PLN responses to STZ are under the control of type 1, MHC class-I-restricted, CD8 + T-cells. This is in accordance to the known physiopathology of STZ-induced diabetes. Additional studies are necessary to establish the mechanism of CD8+ T-cell activation.

Mouse popliteal lymph node assay for assessment of allergic and autoimmunity-inducing potentials of low-molecular-weight drugs.

In the present collaborative study, popliteal lymph node (PLN) responses to penicillin G (an allergenic chemical), D-penicillamine (an autoimmunity-inducing chemical), and barbital (a negative reference chemical) were investigated in three different mouse strains by ten pharmaceutical companies. Two inbred mouse strains (BALB/c and A/J) and one outbred strain (ICR) were subcutaneously injected with saline solutions containing penicillin G (1.25, 2.5 and 5 mg/mouse), D-penicillamine (0.5, 1 and 2 mg/mouse), or barbital (2 mg/mouse) into one hind footpad and saline only was injected into the contralateral footpad. PLN cellularity indices were determined on day 7. In the three strains tested, the penicillin G and D-penicillamine injections resulted in approximately dose-dependent responses. In contrast, barbital failed to generate a significant PLN reaction. In the typical data from one of the participating laboratories, the PLN responses of A/J, BALB/c, and ICR to penicillin G were high, intermediate and low, respectively, while their PLN responses to D-penicillamine were all high. Some variation in PLN cellularity indices was observed among the participating laboratories, but reproducibility of the popliteal lymph node assay (PLNA) evaluation was partly confirmed. Although the appropriate selection of mouse strains and drug dosage levels has to be considered, these results suggest that the PLNA may be an appropriate screening system for prediction of the allergic or autoimmunity-inducing potentials of low-molecular-weight drugs.

Airborne house dust elicits a local lymph node reaction and has an adjuvant effect on specific IgE production in the mouse

Indoor suspended particulate matter (SPM) consists of many different types of particles, the vast majority of which are less than 2.5 μm in diameter. An important question is how these particles, being inhalable, contribute to asthma and respiratory symptoms. One possibility is that these particles have an adjuvant effect on the immune response and increase the IgE production, or cause a non-specific irritation in the airways, contributing to bronchial hyper-responsiveness. In this study, the adjuvant activity of indoor SPM on the response to the model allergen ovalbumin (OA) in BALB/c mice was investigated, using the popliteal lymph node (PLN) assay. The adjuvant activity on the local lymph node response was determined by measuring the PLN weight, cell numbers and cell proliferation, and the adjuvant activity on the IgE production by measuring the levels of serum IgE specific to OA. SPM was found to give a significant PLN response, both when injected alone and together with OA. SPM was also found to enhance the production of specific IgE to OA when injected together with OA, after reinjection with OA, compared with immunisation with OA alone.

The popliteal lymph node assay in 1996

The popliteal lymph node (PLN) assay is based on the assumption that a mechanism similar to a graft-versus-host (GvH) reaction is involved in ‘GvH-like’ drug-induced side-effects, including generalized lymphadenopathy, serum sickness-like disease, scleroderma-like reaction and the lupus syndrome. An increased PLN weight 7–10 days after injection of the test article into the footpad is generally held as a positive response. Most, if not all compounds reported to induce pseudo-GvH side-effects in man (namely positive model compounds) have been shown to induce positive PLN responses in mice and/or rats. Reproducible results have been obtained in several laboratories, in some instances blindly. However, positive responses have also been obtained with the negative model compounds acetone and imipramine. Flow cytometry analysis and conventional histology failed to help differentiate between a true GvH response and a primary irritative effect. In order to confirm the potential value of the PLN assay to predict the risk for drug-induced GvH-like reactions, mechanistic studies are urgently needed.

Immunostimulatory effects of platinum compounds: correlation between sensitizing properties in vivo and modulation of receptor-mediated endocytosis in vitro.

The sensitizing properties of different complex salts of platinum were defined in vivo by means of the popliteal lymph node (PLN) assay in mice. Hexa- and tetrachloroplatinates were confirmed to be highly immunogenic, inducing vigorous primary immune responses in the draining PLN following single subcutaneous injections. Flow-cytometric analysis revealed a dramatic increase in the total number of cells expressing proliferating cell nuclear antigen. The majority of these cells were of the T helper phenotype (CD4+) reflecting the T-cell dependence of the PLN response induced by Pt salts such as Na2[PtCl6] or Na2[PtCl4]. In contrast, [Pt(NH3)4]Cl2 failed to elicit a significant increase in PLN cell proliferation when compared with saline-treated controls. The differential immunogenicity of the Pt compounds found in vivo directly correlated with their capacity to modulate mechanisms of receptor-mediated endocytosis in murine Langerhans cells in vitro. The reactivity of Na2[PtCl6] or Na2[PtCl4] resembled that of potent contact sensitizers in this endocytosis assay whereas [Pt(NH3)4]Cl2 proved to be mert. These results suggest that [Pt(NH3)4]Cl2 might be less harmful to humans than hexa- or tetrachloroplatinates. As demonstrated with Pt compounds, monitoring of direct effects of low-molecular-weight chemicals on antigen-presenting dendritic cells in vitro is able to predict their sensitizing potential in vivo.

Lymphocyte activation by liposome-trapped streptozotocin in murine popliteal lymph node (PLN) test

The lymphoproliferative potential of liposome-trapped streptozotocin (STZ) was compared to the effect of saline-dissolved STZ injected locally into the foot pad of CD-1 mice. Popliteal lymph node (PLN) enlargement and early cell activation of lymphocyte subsets were monitored during the onset of STZ-induced autoimmune-like reaction. Injection of the optimal STZ dose, 0.5 mg/foot pad, markedly increased the absolute PLN cell number as well as specific T-helper (CD4 +), T-suppressor/cytotoxic (CD8 +), and B-(Ig +) cell subsets stained with fluorescent monoclonal antibodies. Furthermore, there was a marked increase in the number of large/activated CD4 + and CD8 + cells and subsets bearing specific markers of early activation. These included cells stained with fluorescein-conjugated monoclonal antibodies against interleukin-2 receptor (CD25 +) and early activation marker (EAM +) (CD69 +), and with fluorescein-conjugated peanut agglutinin (PNS +). Surprisingly, the injection of liposome-trapped STZ, at a 1 10 of the optimal dose only, induced a marked PLN enlargement comparable to the effect of optimal STZ dose. The effect of liposome-STZ could be dissociated from the non-drug-containing MLV-related lymphocyte activation. The data suggest several possible advantages from the introduction of chemicals by the liposome route and the subsequent PLN test for chemical-induced autoimmunity. Toxicological advantages could involve better control of chemical exposure, controlled exposure to the water-insoluble substances, drastic reduction of xenobiotic dose, a stronger, clear PLN response and possible elimination or at least restriction of false-negative results, due to the liposome adjuvancity. Overall, application of liposomes as an exposure route potentialized the STZ-induced early lymphocyte activation.

Comparison of popliteal lymph node responses in various strains of rats.

Outbred (namely Wistar and Sprague-Dawley) and inbred (Wistar-Furth, Lewis, Fisher 344 and Brown-Norway) strains of rats were screened for their responses to reference compounds in the popliteal lymph node (PLN) assay. Streptozotocin and diphenylhydantoin gave positive responses as evidenced by increased weight and cellularity indices in all strains used whereas procainamide, isoniazid and barbital consistently gave negative responses. Although these findings overall are in agreement with previous investigations involving these compounds, the lack of marked interstrain differences in PLN responses argues against a strong immunogenetically controlled mechanism as could be assumed in presumably auto-immune reactions. The question is raised whether drug-induced side-effects predicted by the PLN assay are basically non-autoimmune as suggested by clinical and immunological findings in man.

Morphology of popliteal lymph node responses in brown‐norway rats

The popliteal lymph node (PLN) assay has been proposed as a tool to predict in rodents those xenobiotics likely to induce autoimmune reactions in humans. To further validate this assay and to study the mechanisms involved, histologic changes in PLNs from rats injected with streptozotocin, diphenylhydantoin, pure acetone, or 50% ethanol were compared to a local graft-versus-host (GvH) reaction. This study suggests that routine histology of PLNs is instrumental to discard primary irritants. In addition, the hypothesis of a GvH-like mechanism in positive PLN responses is supported by the finding that the reference compounds streptozotocin and diphenylhydantoin produced histologic changes similar to a "true" local GvH reaction.

Popliteal lymph node response to procainamide and isoniazid. Role of β-naphthoflavone, phenobarbitone and S9-mix pretreatment

The popliteal lymph node assay (PLNA) was proposed for the preclinical prediction of xenobiotics-induced autoimmune reactions in humans. Among the substances so far tested in this model, procainamide and isoniazid gave negative PLNA responses despite reports of lupus syndromes in man. To confirm the hypothesis that a metabolite instead of the parent molecule is involved, rats were pretreated with phenobarbital or β-naphthoflavone, then injected with procainamide or isoniazid. In additional groups of animals, procainamide or isoniazid were injected together with S9-mix following various incubation times. Pretreated rats had a positive PLNA response when injected with procainamide, whereas preincubation with S9-mix resulted in a positive response to isoniazid. These results further support the validity of the PLNA.

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.