Murine Spleen Cell Cultures Research Articles

Mast Cells Stimulated with Peptidoglycan from Staphylococcus aureus Augment the Development of Th1 Cells.

The skin of patients with atopic dermatitis (AD) is superficially colonized by Staphylococcus aureus. We have previously found that percutaneous permeation of peptidoglycan (PEG) from S. aureus increases the number of mast cells in the dermis, as seen in skin lesions of AD patients. The purpose of the present study was to clarify the influence of PEG on T helper type 1 (Th1)/ T helper type 2 (Th2) cell development mediated by mast cells. Mast cells were induced by long-term culture of murine spleen cells in medium supplemented with tumor necrosis factor (TNF)- a. Ovalbumin (OVA) peptide-pulsed mast cells were incubated with naïve Th cells in the presence or absence of PEG. Five days later, Th cells in the culture were stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin, and Th1/Th2 cytokine production was investigated by enzyme-linked immunosorbent assay. It was confirmed that the mast cells we obtained had surface expression of I-Ad, worked as antigen-presenting cells, and induced Th1 cell and Th2 cell development. The stimulation of mast cells with PEG enhanced the development of Th1 cells but not that of Th2 cells. The increase of Th1 cell development stimulated by PEG was associated with an increase in the expression of Notch ligand Delta 1 in the mast cells. Furthermore, treatment of mast cells with the macrolide antibiotic josamycin suppressed Th1 cell development and this was correlated with a reduction of both Delta 1 expression and interleukin (IL)-12 production in mast cells. Colonization of S. aureus on the lesioned skin of AD patients contributes to not only an increase in the number of mast cells but also Th1 cell development mediated by mast cells in the dermis and subsequent induction of chronic inflammation, which is characterized by up-regulation of the Th1 cytokine, interferon (IFN)- g. Therefore, application of josamycin to the lesional skin of AD patients may provide relief from chronic inflammation mediated by mast cells.

COMPARATIVE STUDY OF ECHINOCHROME A, OXYGENATED CAROTENOIDS, GINSENOSIDE Rh2, LUTEOLIN DISULFATE AND METFORMIN AS A MEAN TO POTENTIATE ANTITUMOR EFFECT OF DOXORUBICIN

This work concerned a comparative study of potentiating effects produced by some drugs of natural origin, as well as metformin, upon antitumor activity of doxorubicin. An increased therapeutic efficacy of doxorubicin used in combination with echinochrome A, oxycarotenoids, ginsenoside Rh2, and metformin was tested in the in vivo Ehrlich ascites carcinoma model. The negative effect of luteolin disulfate (10 mg/kg body weight) upon efficacy of antitumor therapy was revealed. When studying the effect of these substances on the level of intracellular ROS in primary cultures of murine spleen cells, using a selective fluorescent indicator (2’,7’-dichlorofluorescein-diacetate), we did not find a positive correlation between induction of excessive ROS synthesis and potentiation of doxorubicin action with the compounds studied. Some of the studied agents showed a positive correlation between interferon-inducing activity, as well as a negative correlation between increased IL-1 content, and higher antitumor effect of doxorubicin. The obtained results suggest expedience of further studies of antitumor effect potentiation using preparations of echinochrome, ginsenoside Rh2, a mixture of oxygenated carotenoids and metformin.

Mucosal co-application of lactic acid bacteria and allergen induces counter-regulatory immune responses in a murine model of birch pollen allergy

Recent epidemiological studies and clinical trials suggest a possible role of certain lactic acid bacteria (LAB) strains in the prevention of allergic diseases. In this study, we aimed at evaluating the immunomodulatory potential of two LAB strains, Lactococcus lactis and Lactobacillus plantarum, for prophylaxis and therapy of allergic immune responses. Both LAB strains-induced high levels of IL-12 and IFN-γ in naive murine spleen cell cultures. Intranasal co-application with recombinant Bet v 1, the major birch pollen allergen, prior or after allergic sensitization, led to increased levels of allergen-specific IgG2a antibodies and in vitro IFN-γ production, indicating a shift towards Th1 responses. Successful immunomodulation by the mucosal pre-treatment was further demonstrated by suppression of allergen-induced basophil degranulation. We conclude that these LAB strains in combination with an allergen could be promising candidates for mucosal vaccination against type I allergy.

CpG penta- and hexadeoxyribonucleotides as potent immunomodulatory agents

We demonstrate a new design for immunomodulatory CpG DNA containing two sequences each with as few as five or six-nucleotides joined together via 3 ′–3 ′ linkers. These do not require the –PuPu(Py)CGPyPy– hexameric motif generally found essential for CpG DNA immune stimulation. These novel, short-immunomers show potent immunostimulatory activity manifested by IL-12 and IL-6 secretion in murine spleen cell and PBMC cultures and splenomegaly in vivo. Short-immunomers show strong activation of NF-κB and stress-activated signaling pathways and induce cytokines in J774 cell cultures. The same sequences also induce cytokines in healthy human PBMC cultures whereas conventional CpG DNA requires different optimal sequences for murine and human immune cells. Additionally, short-immunomers inhibit IL-5 secretion and induce IFN-γ secretion in conalbumin-sensitized mouse spleen cell cultures, suggesting reversal of established Th2 responses to Th1 type responses. Short-immunomer also inhibits growth of MCF-7 human tumor xenograft in nude mice. This is the first report of activity with such short DNA sequences and also of sequences lacking hexameric motifs proposed in earlier studies.

Synergistic and inhibitory interactions in the in vitro control of murine megakaryocyte colony formation.

The formation of megakaryocyte colonies in agar cultures of murine bone marrow or spleen cells can be stimulated by the addition of interleukin-3 (IL-3), erythropoietin (EPO), thrombopoietin (TPO), or IL-6. However, greater numbers of colonies developed if combinations of two or more of these stimuli were used, particularly combinations including stem cell factor, with maximal numbers of colonies developing with the combination of stem cell factor plus IL-3 plus EPO. The data indicate that most committed progenitor cells in the megakaryocyte lineage were unusual in that they required stimulation by two or more hematopoietic growth factors. In tests using a range of growth factors, G-CSF was exceptional in that it consistently specifically inhibited megakaryocyte colony formation stimulated by EPO, TPO, or IL-6 but not that stimulated by IL-3. The mechanisms involved in this inhibitory action of G-CSF are unknown, but the inhibitory action could be of relevance for the dose-dependent lowering of platelet levels observed in some subjects injected with G-CSF.

Leptin inhibits osteoclast generation.

Originally, leptin was described as a product of adipocytes that acts on the hypothalamus to regulate appetite. However, subsequently, it has been shown that leptin receptors are distributed widely and that leptin has diverse functions, including promotion of hemopoietic and osteoblastic differentiation. It has been recognized for some time that both serum leptin and bone mass are correlated positively to body fat mass and, recently, we have shown a direct positive relationship between serum leptin and bone mass in nonobese women. We now report that leptin inhibits osteoclast generation in cultures of human peripheral blood mononuclear cells (PBMCs) and murine spleen cells incubated on bone in the presence of human macrophage colony-stimulating factor (hM-CSF) and human soluble receptor activator of NF-kappaB ligand (sRANKL). The half-maximal concentration inhibitory of leptin was approximately 20 nM in the presence of sRANKL at 40 ng/ml but decreased to approximately 2 nM when sRANKL was used at 5 ng/ml. The majority of the inhibitory effect occurred in the first week of the 3-week cultures. Inhibition did not occur when the PBMC cultures were washed vigorously to remove nonadherent cells or when purified CD14+ monocytes were used to generate osteoclasts, indicating an indirect or permissive effect via CD14- PBMC. Leptin increased osteoprotegerin (OPG) messenger RNA (mRNA) and protein expression in PBMC but not in CD14+ cells, suggesting that the inhibitory effect may be mediated by the RANKL/RANK/OPG system. Leptin may act locally to increase bone mass and may contribute to linkage of bone formation and resorption.

Pentoxifylline inhibits the synthesis and IFN-gamma-inducing activity of IL-18.

The effect of phosphodiesterase-inhibiting anti-inflammatory drug pentoxifylline (PTX) on LPS-induced IL-18 synthesis and IL-18-mediated IFN-gamma-induction were investigated. In a dose-dependent manner PTX inhibited production of IL-18 in LPS-treated cultures of murine spleen cells and bone marrow-derived macrophages. Similarly, PTX treatment significantly reduced blood IL-18 levels and expression of spleen IL-18 mRNA in LPS-challenged mice. The inhibitory effect of PTX was specific for IL-18, since LPS-induced IL-12 p40 release was not suppressed either in splenocyte cultures or blood of LPS-injected animals. Synergistic induction of IFN-gamma by combined IL-12/IL-18 treatment was also inhibited by PTX in vitro and in vivo. Experiments with IL-12 pretreatment of splenocytes, followed by IL-18 stimulation, revealed that PTX suppressed both IL-12 and IL-18 signals responsible for IFN-gamma induction. These results suggest that interference with IL-18 synthesis and IFN-gamma-inducing activity might contribute to anti-inflammatory actions of PTX.

Rat mast cell protease-I enhances immunoglobulin E production by mouse B cells stimulated with interleukin-4.

Mast cell chymase plays important roles in inflammation and tissue remodeling. Here we show that mast cell chymase also functions as an enhancer of immunoglobulin production. In the culture of murine spleen cells stimulated with lipopolysaccharide and interleukin-4, purified rat chymase (rat mast cell protease-I; RMCP-I), at physiological concentrations, enhanced immunoglobulin E (IgE) and IgG1 syntheses but not IgG3 synthesis. The enhancement was also evident when spleen cells depleted of T cells and macrophages were employed as responding cells. Enzymatic activity of RMCP-I was required to enhance IgE and IgG1, because two inhibitors for chymotryptic enzymes, chymostatin and Y-40613, a novel chymase inhibitor, suppressed the enhanced immunoglobulin production, and phenylmethylsulphonyl fluoride, an irreversible inhibitor for serine proteases, totally abolished the enhancing effect. Furthermore, a specific inhibitor for Zn2+-dependent metalloproteases, GI 129471, could also completely inhibit the production of IgE and IgG1 that was enhanced by RMCP-I, suggesting that a metalloprotease also played an essential role in the immunoglobulin production. Our results together with others show that proteases from mast cell granules have important function not only in the efferent phase but also in the afferent phase of immune responses.

T-Cell-Immunity-Based Inhibitory Effects of Orally Administered Herbal Medicine Juzen-taiho-to on the Growth of Primarily Developed Melanocytic Tumors in RET-Transgenic Mice

We examined the effect of oral administration of juzen-taiho-to, one of the most popular herbal medicines in Japan, on primary melanocytic tumor growth in RET-transgenic mice. There was virtually no difference between the lengths of tumor-free stages in the juzen-taiho-to-treated mice and the untreated littermate control mice. The rate of tumor growth in the juzen-taiho-to-treated mice, however, was greatly suppressed during the entire period after the initial tumor development. Correspondingly, the life span of juzen-taiho-to-treated transgenic mice was longer (over 6 mo in mean value) than that of control mice. We partially elucidated the mechanism of the antitumor effect of juzen-taiho-to. The addition of juzen-taiho-to at any of a wide range (50-1600 microg per ml) of concentrations to in vitro cultures of Mel-Ret cells, a malignant melanoma cell line derived from a RET-transgenic mouse, caused neither cell death nor cell cycle arrest directly. The addition of 50-400 microg per ml of juzen-taiho-to to cultures of murine spleen cells, however, promoted their DNA synthesis. More importantly, peritoneal exudate cells from the juzen-taiho-to-treated transgenic mice, in which the ratio and number of T cells were increased, displayed an antitumor immunity against Mel-Ret cells in vitro. Interestingly, the peritoneal-exudate-cell-associated antitumor immunity was further augmented by the addition of 200-400 microg per ml of juzen-taiho-to in vitro. This immunity, which was primarily conveyed by Thy-1+ T cells, was antigen (RET/melanoma) specific and cytotoxic. Amongst various chemical ingredients of juzen-taiho-to examined in this study, glycirrhizin displayed an action, partially replacing that of juzen-taiho-to, in promoting anti-Mel-Ret immunity when supplementarily added in vitro. These results suggest that juzen-taiho-to suppresses once-developed primary melanocytic tumors through potentiation of T-cell-mediated antitumor cytotoxic immunity in vivo.

Influence of backbone chemistry on immune activation by synthetic oligonucleotides

Depending on base sequence, DNA displays immunological activities relevant to the design of novel therapeutic agents. To determine the influence of backbone structure on these activities, we tested a series of synthetic phosphodiester and phosphorothioate oligonucleotides in in vitro cultures of murine spleen cells. These compounds were 30 bases long and consisted of either a single base or an immunostimulatory sequence (AACGTT) flanked on 5′ and 3′ ends by 12 nucleotides of each base. Cell activation was assessed by both thymidine incorporation and expression of cell surface CD69; production of interleukin-6 and interleukin-12 was used as a measure of cytokine stimulation. In these assays, phosphorothioate oligonucleotides induced much higher levels of proliferation, CD69 expression, and cytokine production than the comparable phosphodiester compounds and had activity at lower concentrations. The sequence for optimal stimulation by phosphorothioates varied among responses, however. For example, whereas compounds containing an immunostimulatory sequence all induced similar levels of proliferation and CD69 expression, cytokine production was greatest with compounds with dA and dT flanks. Furthermore, while single base dG oligonucleotides stimulated proliferation as both phosphodiesters and phosphorothioates, they failed to stimulate cytokine production. Together, these findings indicate that base sequence as well as backbone chemistry influence immune activation by synthetic oligonucleotides, with the effects varying among responses. While suggesting differences in the structure–function relationships of nucleic acids in their immune activities, these findings also raise the possibility of the design of agents with specific patterns of immune modulation.

Immunostimulatory properties of genomic DNA from different bacterial species

Bacterial DNA has potent immunological properties because of its content of immunostimulatory sequences centering on CpG motifs. To investigate whether DNA from various bacterial species differ in these properties, the activity of a panel of DNA was assessed in in vitro cultures of murine spleen cells. This panel varied in base composition and included DNA from Clostridium perfringens (CP), Escherichia coli (EC), Micrococcus lysodeikticus (MC), Staphylococcus aureus (SA), , as a mammalian DNA control, calf thymus (CT) DNA. In assays of IL-12 and IFN-γ production as well as B cell mitogenesis, these DNA showed marked differences in their immunostimulatory activity. For both cytokine and B cell responses, EC DNA demonstrated the highest activity while CP DNA had the lowest activity among the bacterial DNA. To determine whether differences in stimulatory capacity resulted from differences in cell uptake, the activity of DNA complexed with lipof ectin was tested. While the addition of lipof ectin to DNA increased stimulation by all DNA, it did not change the relative potency of the DNA tested. These results indicate that bacterial DNA differ in their immunostimulatory capacity, most likely reflecting their content of CpG motifs. These differences could affect the induction of innate immunity as well as the consequences of infection.

Study of an Argentine Mistletoe, the hemiparasite Ligaria cuneifolia (R. et P.) Tiegh. (Loranthaceae)

Ligaria cuneifolia (R. et P.) Tiegh. is an hemiparasite species used in Argentine folk medicine as a substitute for the European mistletoe ( Viscum album L.) based on its putative activity of decreasing high blood pressure. This paper analyzes flavonoid composition, protein constituents and the possible immunomodulatory and antitumoral effects of this species. Micromolecular study disclosed quercetin-free, quercetin-glycosylated and proanthocyanidins corresponding to cyanidin monomers, which implies a particular metabolic pathway. Proteins present in L. cuneifolia extracts analyzed by SDS-PAGE presented multiple bands with molecular weights ranging from 14 to 90 kD. These features contribute to the characterization of the native mistletoe. As V. album is being used in cancer treatment due to its immunomodulatory and antitumoral activity, the action of aqueous L. cuneifolia extracts on murine lymphocytes was investigated. Culture of murine spleen cells alone or stimulated with Concanavalin A or lipopolysaccharide in presence of L. cuneifolia extracts indicated a certain stimulation of splenocytes alone and an inhibition of splenocytes stimulated with Concanvalin A or lipopolysaccharide. An inhibitory effect was also observed on the proliferation of murine leukemia cells. In addition, aqueous extracts increased nitric oxide production by murine macrophages. These results suggest that L. cuneifolia extracts exert an immunomodulatory effect on the mouse immune system.

Immunostimulation of Murine Spleen Cells by Materials Associated with Bovine Milk Protein Fractions

Purified bovine milk proteins that were added to cultures of murine spleen cells significantly increased cell proliferation and production of immunoglobulin M. Casein and a whey mixture consisting of α-lactalbumin, bovine serum albumin, bovine gamma globulin, and β-lactoglobulin (β-LG) were stimulatory. Of the three β-LG preparations that are commercially available (β-LG containing variants A and B, purified variant A, and purified variant B), the unseparated mixture containing both the A and B variants showed the most immunomodulatory activity. Both alkaline treatment and trypsin digestion of the β-LG preparation markedly reduced its effectiveness. Polymyxin B, while greatly diminishing the stimulatory effect of lipopolysaccharide, had no significant effect on either the enhancement of cell proliferation or the enhancement of immunoglobulin production by β-LG. In the presence of S-(n-butyl)-homocysteine sulfoxamine, the stimulatory effect of β-LG on cell proliferation and IgM production in vitro was markedly reduced.

Interleukin‐12 acts as an adjuvant for humoral immunity through interferon‐γ‐dependent and ‐independent mechanisms

Interleukin-12 (IL-12) is a pivotal cytokine that has dramatic effects on cell-mediated immunity. It is now becoming increasingly recognized that IL-12 also strongly controls humoral immunity. We have investigated the mechanism by which IL-12 induces alterations in antibody isotype expression by determining the influence of IL-12 on in vitro immunoglobulin (Ig) production in polyclonally activated murine spleen cell cultures. Cells exposed to IL-12 plus lipopolysaccharide or anti-CD40 monoclonal antibody showed dramatically elevated IgG2a and suppressed IgG1 production compared to cells cultured in the absence of IL-12. IL-12 treatment of spleen cell cultures induced expression of gamma2a germ-line transcripts, consistent with initiation of switch recombination to IgG2a. In addition, exposure of limiting dilution cultures to IL-12 increased IgG2a+ cell precursor frequency. All of the above results were dependent on interferon-gamma (IFN-gamma). However, in the absence of IFN-gamma, IL-12 still had significant effects on Ig secretion. Specifically, IL-12 enhanced IgG1 and IgG2b anti-DNP antibody levels in mice containing specific disruptions in the IFN-gamma gene. Our results suggest that IL-12 induces T helper type 1 and natural killer cells to secrete large amounts of IFN-gamma which then causes B cells to switch to IgG2a and IgG3 production. In addition, IL-12 has direct or indirect effects on B cells that are independent of IFN-gamma. The IFN-gamma-independent effects may include enhancement of Ig expression by post-switched cells.

An IL-3-Induced Splenic NC-1.1+Mast Cell Line Mediates Natural Cytotoxicity Independent of TNF-α

Long-term culture of mouse spleen cells in IL-3-conditioned medium induced a stable mast cell line. This mast cell line (MCL) which could not be cloned expressed NC-1.1, a receptor on cells which mediate natural cytotoxicity (NC), and CD32/CD16 but no markers of T cells, B cells, macrophages, or NK cells. The MCL cells were large and granular, with abundant cytoplasm and >90% stained with Alcian blue, a mast cell-specific stain. Probing total RNA with cDNA encoding a mast cell-specific proteinase mMCP-5 identified the ∼1-kb mMCP-5 transcript which was confirmed at the protein level. MCL cells mediated very high NC against WEHI-164 which increased with time in culture and which was blocked by anti-NC-1.1 but not by anti-TNF-α. When incubated with WEHI-164 tumor cells, MCL cells showed transient induction of TNF-α mRNA, but no detectable surface protein. Thus long-term culture of murine spleen cells in IL-3 induces mast cells which express the NC-1.1 receptor of cells which mediate NC, and utilize a cytotoxic effector mechanism which is not TNF-α.

Ex vivo coating of islet cell allografts with murine CTLA4/Fc promotes graft tolerance.

To test the hypothesis that blockade of B7-triggered costimulation by donor cells could preclude allograft rejection, we coated crude islet allograft preparations in vitro for 1 h with a murine CTLA4/Fc fusion protein. Murine CTLA4/Fc blocks the proliferative response in primary mixed lymphocyte cultures (MLC) and Con A-stimulated murine spleen cell cultures by 85 to 95%. Responder cells from a primary MLC containing mCTLA4/Fc were hyporesponsive upon restimulation to the same stimulator cells in a secondary MLC lacking mCTLA4/Fc. Because of mutations in the Fc gamma RI and C'1q binding sites of the Fc portion of the murine CTLA4/Fc fusion protein, the molecule binds to, but does not target, cells for Ab-dependent cellular cytotoxicity or complement-directed cytolysis. Although systemic immunosuppression was not applied, 42% (10 of 24) of B6AF1 recipients of islet allografts pretreated with CTLA4/Fc were permanently engrafted. Further, 50% of hosts bearing functioning islet allografts more than 150 days post-transplant were formally proved to be tolerant to donor tissues. A persistent CD4+ and CD8+ T cell infiltrate surrounding, but not invading, islet grafts in tolerant hosts was discerned. In control experiments, 89% (8 of 9) of islet allografts coated with mIgG3, and 100% (n = 10) pretreated with media alone were rejected. Thus, we conclude that 1) B7-triggered costimulation by donor APCs is an important element of rejection, and 2) blockade of the B7 pathway by in vitro allograft manipulation is able to induce tolerance.

Rapid communication: effect of irradiation on lymphocyte proliferation and differentiation: potential of IL-6 in augmenting antibody responses in cultures of murine spleen cells.

Ionizing radiation induces both quantitative and qualitative changes in the lymphoid cells of both man and experimental animals, including inhibition of antibody responses. However, the cellular basis of this immunological lesion is not clear. In the present study, groups of mice were exposed to 2.0 Gy gamma-rays or sham irradiated, and 2 days later animals were killed and spleen cells were cultured with TNP-Ficoll and assayed for antibody responses. Results indicated a significant decrease in the number of anti-TNP, plaque-forming cells in cultures from the irradiated mice compared with cultures from the control. When lymphocytes were stimulated in vitro with anti-IgM or anti-CD3, there was a decreased proliferation in spleen cell cultures derived from the irradiated mice compared with those from the control. Since radiation treatment was found to deplete both T and B cells in equal proportions in spleen and an equal number of both control or treated cells were used in culture, the immunological abnormalities may have been due to intrinsic defects in irradiated cells. Addition of IL-6 to irradiated spleen cell cultures was able to augment anti-TNP, plaque-forming cell responses indicating the possibility that in the future this cytokine can be used in vivo to induce protection from infectious diseases in irradiated individuals.

Colon adenocarcinoma cells inhibit anti-CD3-activated killer cell induction.

Adoptive immunotherapy with lymphokine-activated killer (LAK) cells has shown some promise in the treatment of certain cancers that are unresponsive to conventional treatment approaches. However, colon adenocarcinomas tend to respond poorly to LAK therapy, possibly as a result of tumor-induced immunosuppression. Recently, in vivo administration of anti-CD3 antibody has been shown to induce mouse T lymphocytes to mediate major-histocompatibility-complex(MHC)-unrestricted tumoricidal activity which is distinct from natural-killer-cell-derived LAK activity. It has therefore been suggested that anti-CD3 therapy may find application in tumor immunotherapy in humans. However, the effectiveness of anti-CD3-activated killer cell induction within the environment found in the vicinity of colon adenocarcinoma cells has not been evaluated. The present report demonstrates that colon cancer cells of human (HT-29) and mouse (MCA-38) origin markedly inhibit the generation of activated killer cells in murine spleen cell cultures. DNA synthesis and interleukin-2 production by spleen cells following stimulation with anti-CD3 antibody are also profoundly depressed in the presence of MCA-38 and HT-29 adenocarcinoma cells. MCA-38- and HT-29-mediated inhibition of activated killer cell development is exerted through the production of a tumor-associated soluble factor that is distinct from transforming growth factor beta or prostaglandins. Local immunosuppression associated with sites of tumor growth may therefore represent a major obstacle to successful anti-CD3 immunotherapy of certain colon adenocarcinomas.

Suppression of endotoxin mitogenicity of spleen cells by lipoxygenase inhibitors and its reversal by 13-hydroxyoctadecadienoic acid

The effects of several inhibitors of lipoxygenases were investigated in murine spleen cell cultures activated with endotoxin (lipopolysaccharide). It was found that these inhibitors interfere with the proliferative response of the cultures. Indomethacin, a specific cyclooxygenase inhibitor, had no such effect. Endotoxin induced the synthesis of tumour necrosis factor alpha in spleen cells which was prevented by treatment with a lipoxygenase inhibitor. The inhibition of the mitogenic effect of endotoxin could be reversed by addition of 13-hydroxyoctadecadienoic acid. This was not the case with leukotriene B4 and C4 or 15-hydroxyeicosatetraenoic acid. In contrast, these substances had inhibitory effects on the mitogenicity of spleen cells. It is suggested that 13-hydroxyoctadecadienoic acid is involved in the development of the mitogenic reaction, possibly on the level of tumour necrosis factor alpha production of macrophages present in the cultures.

Yersinia enterocolitica produces superantigenic activity.

We have recently observed that antigenic preparations from Yersinia enterocolitica are capable of inducing strong proliferative responses in normal murine spleen cell cultures. As a consequence of this observation, we evaluated whether Yersinia-derived Ag possess superantigenic activity. Stimulatory activity can be found in culture supernatants, as well as membrane and cytoplasmic fractions of Y. enterocolitica. Cell depletion studies indicate that the primary responding cell is a CD4+ T cell, which requires the presence of APC for responsiveness to Y. enterocolitica Ag. Furthermore, these APC must express MHC class II Ag, as evidenced by the fact that either antibody depletion of class II+ APC or addition of anti-class II antibodies (that block class II Ag on the surface of APC) eliminates the proliferative response. Evaluation of TCR usage by BALB/c T cells responsive to Y. enterocolitica revealed that those T cells bearing V beta 3, 6, and 11 and possibly 7 and 9 were expanded after exposure to Y. enterocolitica Ag preparations. By using a panel of T cell hybridomas, we have shown that hybridomas bearing V beta 3, 7, 8.1, 9, and 11 but not 2, 8.2, 8.3, and 13 respond to Yersinia. When cytoplasmic fractions of Y. enterocolitica were subjected to column chromatography, proliferative activity was enriched approximately 27-fold, and the elution characteristics of the active material suggest that it possesses hydrophobic regions and is, therefore, probably membrane associated. These data indicate that Y. enterocolitica produces antigenic material that has properties consistent with those of T cell superantigens.