Immunomodulatory effect of Tylophora indica on Con A induced lymphoproliferation.

Our preliminary studies with tylophora alkaloids had shown that they inhibit cellular immune responses like contact sensitivity to dinitro-flurobenzene and delayed hypersensitivity to sheep red blood cells, in vivo. Investigations were hence carried out to determine the cellular targets of tylophora alkaloids in in vitro systems. Con A induced proliferation of splenocytes was used as a model system to study the effect of the alkaloids on cellular immune responses. The alkaloid mixture was found to inhibit proliferation of splenocytes at higher concentrations and augment the same at lower concentrations. Both macrophages and T cells were found to be vulnerable to tylophora alkaloids. The alkaloid mixture suppressed IL-2 production in Con A stimulated splenocytes at the inhibitory or higher concentrations and enhanced production at the lower concentrations. IL-1 production by activated macrophages on the contrary was doubled in the presence of inhibitory concentrations of tylophora. These studies indicate that tylophora alkaloids have a concentration dependent biphasic effect on Con A induced mitogenesis. At lower concentrations they augment Con A induced lymphoproliferation by enhancing IL-2 production. Inhibition of proliferation at higher concentrations of the alkaloid is due to inhibition of IL-2 production and activation of macrophages, which have a cytostatic effect.     

Immunomodulatory effect of Tylophora indica on Con A induced lymphoproliferation

Our preliminary studies with tylophora alkaloids had shown that they inhibit cellular immune responses like contact sensitivity to dinitro-flurobenzene and delayed hypersensitivity to sheep red blood cells, in vivo. Investigations were hence carried out to determine the cellular targets of tylophora alkaloids in in vitro systems. Con A induced proliferation of splenocytes was used as a model system to study the effect of the alkaloids on cellular immune responses. The alkaloid mixture was found to inhibit proliferation of splenocytes at higher concentrations and augment the same at lower concentrations. Both macrophages and T cells were found to be vulnerable to tylophora alkaloids. The alkaloid mixture suppressed IL-2 production in Con A stimulated splenocytes at the inhibitory or higher concentrations and enhanced production at the lower concentrations. IL-1 production by activated macrophages on the contrary was doubled in the presence of inhibitory concentrations of tylophora. These studies indicate that tylophora alkaloids have a concentration dependent biphasic effect on Con A induced mitogenesis. At lower concentrations they augment Con A induced lymphoproliferation by enhancing IL-2 production. Inhibition of proliferation at higher concentrations of the alkaloid is due to inhibition of IL-2 production and activation of macrophages, which have a cytostatic effect.     

Effects of lectins on cAMP production and steroidogenesis in cultured adrenal tumor cells

The plant lectins, concanavalin A (conA), wheat germ agglutinin (WGA), and phytohemagglutinin (PHA) stimulate steroidogenesis in cultured adrenal tumor cells. ConA maximally stimulated steroidogenesis at 100 μg/ml following an approximate 4 h lag phase. ConA stimulation was completely inhibited by α-methyl-d-mannopyranoside and the WGA effect was prevented by N-acetyl-d-glucosamine. It was also found that conA alone did not cause a measurable increase in either intra- or extracellular cyclic adenosine 3′5′-monophosphate (cAMP) production. In addition, conA when added simultaneously with adrenocorticotropin (ACTH) doubled the intra- and extracellular cAMP production over controls treated with ACTH alone. This enhancement effect was dose dependent. When Y-1 cells were preincubated with conA and then treated with either ACTH or cholera enterotoxin (CT) there was a dose- and time-dependent inhibition of induced cAMP production. In the case of CT, the inhibitory effect occurred even with simultaneous addition of conA and CT. This effect was reversed by addition of both α-methyl-d-mannopyranoside and washing with Eagle's minimal essential medium (MEM) 1 h after CT had bound to its receptor. This reversal was not apparent for the inhibitory effect of conA on ACTH-induced cAMP production which occurred after 2 h of preincubation with conA. These results demonstrate that conA, as well as the other plant lectins, interact with specific membrane receptors to reversibly stimulate steroid production as well as enhancing or inhibiting ligand-induced cAMP production in cultured adrenal tumor cells.     

Chloroquine's modulation of endothelial cell activity induced with basic fibroblast growth factor and human serum: effect on mitogenesis, protease production and cell migration

Chloroquine modulates the activity of cultured human microvascular endothelial (HOME) cells in a complex fashion. At concentrations of 5-25 microM, CQ inhibits basic fibroblast growth factor (bFGF-) and human serum-induced mitogenic activity in these cells, in a dose-dependent manner. The kinetics of CQ's inhibitory actions on serum-induced mitogenesis in HOME cells slowly develops with only 30% of maximum inhibition reached after 24 hours. In HOME cells grown in serum-free medium, CQ raised tissue-plasminogen activator antigen levels in cell extracts. There was also a potentiation of bFGF-induced t-PA production. The kinetics of CQ's stimulatory effect on t-PA production by HOME cells, suggest that this effect precedes its inhibitory actions on mitogenesis. This effect of CQ on t-PA generation in endothelial cells was susceptible to cycloheximide inhibition. In wound assays, HOME cell migration, induced with bFGF and HS, was potentiated by CQ.     

Interleukin-2 mRNA expression, lymphokine production and DNA synthesis in glutathione-depleted T cells

The stimulation of DNA synthesis in lymphocyte populations was previously shown to depend strongly on the intracellular glutathione (GSH) level. Since T cell growth is known to depend on interleukin 2 (IL-2), the experiments in this report were designed to determine whether intracellular GSH depletion may inhibit IL-2 production or the IL-2 dependent DNA synthesis. Our experiments revealed that IL-2 production and DNA synthesis of mitogenically stimulated splenic T cells have indeed different requirements for GSH. The addition of relatively high concentrations of GSH (5 mM) to cultures of concanavalin A (Con A)-stimulated splenic T cells was found to augment strongly the DNA synthesis but inhibited the production of IL-2. Moderate intracellular GSH levels, however, are apparently not inhibitory for IL-2 production, since intracellular GSH depletion by cysteine starvation or by graded concentrations of DL-buthionine sulfoximine (BSO) had virtually no effect on IL-2-specific mRNA expression and the production of T cell growth factor (TCGF). The DNA synthesis activity, in contrast, was strongly suppressed after GSH depletion with either method. As in cultures of splenic T cells, GSH depletion had no substantial effect on the induction of IL-2 mRNA and TCGF production in several mitogenically stimulated T cell clones. Taken together, our experiments suggest that complex immune response may operate best at intermediate GSH levels that are not too high to inhibit IL-2 production but sufficient to support DNA synthesis.     

The effect of concanavalin A on egestion of food vacuoles in Tetrahymena

The ability of concanavalin A (conA) to disrupt food vacuole elimination at the cytoproct of Tetrahymena pyriformis, strain GL-C, was investigated using fluorescence microscopy and thin section electron microscopy. ConA was found to induce "tails" in Tetrahymena. These tails were specifically stained by fluorescent conA. Thin section observations of conA-treated cells revealed that these tails were the result of abnormal egestion of food vacuole contents at the cytoproct. Tail formation appears to result from an inhibition of endocytosis of food vacuole membrane during egestion. Instead, the food vacuole membrane appears to be cast out of the cell, along with the contents of the vacuole. The mechanism of this inhibition may be related to an apparent absence of microtubules or microfilamentous mat in the cytoproct region of conA-treated cells. Although conA is ingested into food vacuoles in large amounts, conA appears to affect endocytosis only from outside the cell; ingested conA does not appear to be effective. ConA may exert its influence by binding to the cytoproct region. The ability of conA to induce tail formation is inhibited by sugars specific to it. Numerous membranous vesicles are found in association with the oral cilia and cytoproct region of conA-treated cells. These vesicles may be the conA-binding material reported to be secreted by Tetrahymena.     

Oxidatively stressed lymphocytes remain in G0/G1a on mitogenic stimulation

Thiol modifiers and oxidants inhibit lymphocyte activation. To investigate which of the many cell functions sensitive to oxidation are critical in this inhibition, mouse splenic lymphocytes were treated with oxidants prior to exposure to mitogen, and progression into the cell cycle was assayed. Different treatments were used to chemically dissect different potential targets within the cell: copper phenanthroline (CuP), to oxidize surface sulfhydryls; N-ethyl maleimide (NEM), to alkylate extra- and intracellular thiols; and hydrogen peroxide, which generates the highly reactive hydroxyl radical within the cell. Progression into the cell cycle was assayed with acridine orange (AO) and assays of interleukin-2 (IL-2) production and IL-2 receptor (IL-2R) expression. The contribution of ADP-ribosylation to inhibition of mitogenesis was assessed using 3-aminobenzamide (3AB) to inhibit adenosine 5'-diphosphate (ADP)-ribose transferases. The results indicate that the CuP and NEM treatments both produce two independent inhibitory effects, that is, a failure in the production of and response to IL-2. Cells treated with these compounds were able to progress only through G1a upon mitogenic stimulation. H2O2 had more complex effects. Both ADP-ribosylation and modulations of cytosolic Ca2+ were involved in the inhibitory effects. With lower inhibitory doses of H2O2, lymphocytes were completely unresponsive to mitogen and failed to exit Go upon mitogenic stimulation. If intra- and extracellular Ca2+ were buffered before treatment with H2O2, higher concentrations were required, and under these conditions cells were able to enter G1a but could not progress into G1b. Under neither of these conditions could cells produce IL-2 or express IL-2R.     

Inhibition of T-lymphocyte mitogenic responses and effects on cell functions by bovine herpesvirus 1.

The mitogenic response of bovine peripheral blood mononuclear cells stimulated by concanavalin A (ConA) was suppressed by infectious bovine herpesvirus 1 (BHV-1). Proliferation in response to interleukin-2 (IL-2) by IL-2-dependent lymphocyte cultures was also inhibited by BHV-1. Although inhibition of mitogenesis approached 100%, less than 1 cell in 1,000 was productively infected by BHV-1 in ConA-stimulated cultures. Neither conditioned medium from mitogen-stimulated peripheral blood mononuclear cell cultures nor human recombinant IL-2 reversed suppression by the virus. Infection by BHV-1 did not influence the expression of IL-2 or IL-2 receptor mRNA in ConA-stimulated cultures, nor did it affect the cytolytic capabilities of lymphocytes. The data suggest that the inhibition of T-lymphocyte proliferation is the result of a nonproductive BHV-1 infection.     

Potentiation of lymphokine-activated killer cell differentiation and lymphocyte proliferation by stimulation of protein kinase C or inhibition of adenylate cyclase

Interleukin 2 (IL-2) stimulated the differentiation of human peripheral blood leukocytes into lymphokine-activated killer cells, as well as DNA synthesis of human T lymphocytes. Both effects of IL-2 could be inhibited by prostaglandin E2, a potent stimulator of adenylate cyclase; however, the inhibitory effect of prostaglandin E2 could be overcome by increased concentrations of IL-2. The opposite effects of IL-2 and prostaglandin E2 were paralleled by their respective abilities to inhibit and stimulate cAMP production in intact cells. Other agents, which inhibit adenylate cyclase directly (somatostatin, beta-endorphin, UK 14.3041) or indirectly by activation of protein kinase C (phenylephrine), could stimulate both differentiation and proliferation. None of these agents alone or in combination were as effective as maximal concentrations of IL-2. However, all agents potentiated differentiation and proliferation induced by submaximal and maximal concentrations of IL-2. Additionally, combinations of agents which stimulated protein kinase C with those that inhibited adenylate cyclase were additive in the potentiation of IL-2-induced differentiation. Neither inhibition nor potentiation of IL-2-induced lymphokine-activated killer cell differentiation was accompanied by changes in Tac expression or gamma-interferon production. The data indicate that the stimulation of lymphokine-activated killer cell differentiation and lymphocyte proliferation in human cells share a common initial biochemical signal. Although the inhibition of adenylate cyclase is not sufficient to maximally stimulate either process and cannot bypass the requirement for IL-2, modulation of this enzyme complex, positively or negatively, can regulate the ultimate physiologic response to IL-2.     

Retinoic acid blocks potassium channels in human lymphocytes

Using the whole-cell variation of the patch-clamp technique, we have determined that retinoic acid, an active metabolite of natural vitamin A that possesses potent immunomodulating activity, reduces the K+ current in human T lymphocytes and natural killer cells in a dose-dependent manner: acute treatment with 5 X 10(-5) M caused over a 70% reduction while concentrations less than 1 X 10(-5) M caused less than 30% inhibition. Natural killer activity and T cell mitogenesis was inhibited by RA at concentrations that reduced the K+ conductance and correlated with the ability of a variety of classical ion-channel blockers to inhibit the functional activity of these cells. Thus, the reported inhibitory effects on natural killer activity and T cell mitogenesis by high concentrations of retinoic acid can be explained by its effect on the K channel.     

Oxidatively stressed lymphocytes remain in Go/Gla on mitogenic stimulation

Thiol modifiers and oxidants inhibit lymphocyte activation. To investigate which of the many cell functions sensitive to oxidation are critical in this inhibition, mouse splenic lymphocytes were treated with oxidants prior to exposure to mitogen, and progression into the cell cycle was assayed. Different treatments were used to chemically dissect different potential targets within the cell: copper phenanthroline (CuP), to oxidize surface sulfhydryls; N-ethyl maleimide (NEM), to alkylate extra- and intracellular thiols; and hydrogen peroxide, which generates the highly reactive hydroxyl radical within the cell. Progression into the cell cycle was assayed with acridine orange (AO) and assays of interleukin-2 (IL-2) production and IL-2 receptor (IL-2R) expression. The contribution of ADP-ribosylation to inhibition of mitogenesis was assessed using 3-aminobenzamide (3AB) to inhibit adenosine 5′-diphosphate (ADP)-ribose transferases. The results indicate that the CuP and NEM treatments both produce two independent inhibitory effects, that is, a failure in the production of and response to IL-2. Cells treated with these compounds were able to progress only through G1a upon mitogenic stimulation. H₂O₂ had more complex effects. Both ADP-ribosylation and modulations of cytosolic Ca²⁺ were involved in the inhibitory effects. With lower inhibitory doses of H₂O₂, lymphocytes were completely unresponsive to mitogen and failed to exit Go upon mitogenic stimulation. If intra- and extracellular Ca²⁺ were buffered before treatment with H²O², higher concentrations were required, and under these conditions cells were able to enter G1a but could not progress into G1b. Under neither of these conditions could cells produce IL-2 or express IL-2R.     

Toxic effects of interleukin-2-activated lymphocytes on vascular endothelial cells

IL-2-activated lymphocytes (LAK cells) show increased adherence to, and killing of, human vascular endothelial cells compared to resting lymphocytes. In the present work, we have found that supernatants from LAK cell cultures also are toxic to human umbilical vein endothelial cells (HUVEC) when tested for 48 h in a neutral red uptake assay. Recombinant TNF-alpha and IFN-gamma at high concentrations are also toxic under the same test conditions, and TNF-alpha was directly detected in LAK cell supernatants. An inconsistent inhibition of toxicity was found with anti-TNF-alpha whereas anti IFN-gamma antibodies had a partial inhibitory effect. The susceptibility of HUVEC to cellular killing by LAK cells could be up- and down-regulated with insulin-like growth factor I and IFN-gamma, respectively. It is concluded that damage to vascular endothelium during high dose IL-2 treatments may be partially related to an excessive production of lymphokines such as IFN-gamma and TNF-alpha. IFN-gamma may, in addition, be protective for HUVEC during cellular interactions with LAK cells.     

Pre-exposure effects of 1 and 3 MHz therapeutic ultrasound on ConA activated spleenocytes

This study was performed to evaluate the pre-exposure effects of ultrasound (1 MHz or 3 MHz) on ConA activated spleenocyte proliferation and cytokine production. Cells were treated for 10 min at various intensities, rested for 1h and stimulated with the T cell activator ConA. The cells were then analyzed for the effects of non-thermal ultrasound on cell growth and the presence of IL-2, IL-4 and IFN-g. The data show that pre-exposure of spleenocytes had no significant effects on the proliferation of ConA activated spleenocytes at either 1 or 3 MHz (10 min at 0.1 or 0.5 W/cm(2)). Significant increases in IL-2 were observed in both 1 and 3 MHz pre-treated and ConA activated spleenocytes. Cells pre-treated with 1 MHz and stimulated with ConA showed a significant increase in IL-4 and IFN-g. Conversely, cells pre-treated with 3 MHz and stimulated with ConA show a significant decrease in IL-4 and IFN-g. Interleuken-4 is known to increase the growth of mast cells, inhibit macrophage activation and increases the activity of the T cell subpopulation, T(H2). Interferon-gamma is known to stimulate production of collagen in fibroblasts, enhance debridement activity of macrophage and inhibit activity of the T cell subpopulation, T(H2).     

Inhibitory effect of physiological concentrations of cortisol but not estradiol on interleukin (IL)-6 production by human osteoblast-like cell lines with different constitutive IL-6 expression

Estrogen deficiency and glucocorticoid excess are two well-known conditions that account for osteoporosis. Interleukin (IL)-6 plays an important role in bone resorption; both estrogens and glucocorticoids are credited with an inhibitory effect on osteoblast production of IL-6. The aim of the study was to investigate whether endogenous hormones, which lead to opposite changes in bone mass, have a common inhibitory effect upon constitutive and inducible IL-6 production by human osteoblast-like cells. We used two human osteosarcoma cell lines (MG-63 and Saos-2) with a different degree of differentiation and constitutive production of IL-6 [2587+/-536 (mean+/-SE) and 3.65+/-0.06 pg/10(6) cells, respectively]. We examined the effects of physiological and supraphysiological concentrations of 17beta-estradiol (E2) and cortisol on basal and IL-1beta-induced IL-6 release in the medium. In all experimental conditions, cellular estrogen receptors (ERs) and glucocorticoid receptors (GRs) were measured by binding assay. Both MG-63 and Saos-2 cell lines had measurable GRs (106 300+/-24 996 and 18 100+/-3215 binding sites/cell, respectively) and ERs (2197+/-377 and 1261+/-66.5 binding sites/cell, respectively). In MG-63 cells, cortisol treatment for 20 h decreased both basal and IL-1beta-induced IL-6 release in a dose-dependent manner; in Saos-2 cells the same effect was apparent for IL-1beta-induced release. Mifepristone (RU-486) did function as partial agonist and antagonist of cortisol. At variance with cortisol, E2 did not exert any effect on IL-6 secretion. Treatment with 1,25(OH)2D3 increased by 100-200% ER concentrations, but did not change ineffectiveness of E2 in modifying IL-6 production; furthermore, when E2 was combined with cortisol, there was no additive effect on cortisol-induced inhibition. The dissociation between glucocorticoid and estrogen effects observed in these human cell lines is a sufficiently robust phenomenon to raise questions about the pathogenetic role of IL-6 in osteoporosis associated with estrogen deficiency. Conversely inhibition of osteoblast production of IL-6 may offer an explanation why bone resorption is not the dominant factor in the pathogenesis of glucocorticoid-induced osteoporosis.     

Eosinophil cationic protein inhibits immunoglobulin production and proliferation in vitro in human plasma cells.

The effect of eosinophil cationic protein (ECP) on immunoglobulin (Ig) production by and proliferation of human plasma cells was studied. ECP inhibited Ig production by and proliferation of the human plasma cell lines, IM-9 and AF-10, in a dose-dependent fashion. As little as 0.05 ng/ml ECP was found to be inhibitory, and the maximal inhibition was achieved at doses of 0.1-0.5 ng/ml ECP. This inhibition was not due to cytotoxicity, since viability was always greater than 98%. Kinetic experiments demonstrated that inhibition was observable after 24 hr of culture with ECP and that the inhibitory effect of ECP was reversible. The inhibitory effect of ECP could be blocked by anti-ECP serum, but not by control serum. Of the various cytokines tested, including interleukin (IL)-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6, interferon (IFN)-alpha, IFN-gamma, granulocyte-macrophage colony-stimulating factor (GM-CSF) and erythropoietin (Epo), IL-6 reversed the inhibition, while other cytokines failed to do so. ECP also inhibited Ig (IgG1, IgG2, IgG3, IgG4, IgM, and IgA) production by and proliferation of PCA-1+ plasma cells generated in vitro with a similar dose-response pattern. This inhibition also was blocked by anti-ECP serum but not by control serum, and was restored by IL-6. These results suggest that ECP may interact with IL-6 in controlling plasma cell responses.     

Lymphocyte mitogenesis induced by monoclonal antibodies to the T3 complex. Differential modulation by human IgG

Murine monoclonal antibodies OKT3 (IgG2), 64.1 (IgG2), and Leu 4 (IgG1) react with a common membrane antigen on human T cells and induce potent mitogenesis at concentrations of 1 ng/ml, 10 ng/ml, and 100 ng/ml, respectively. Human serum inhibits the mitogenic effect of antibodies OKT3 and 64.1, but not that of Leu 4. The inhibitor in serum has been identified as immunoglobulin G (IgG) as evidenced by the ability of anti-human IgG-Sepharose affinity columns to retain the inhibitory activity. Various immunoglobulin classes and subclasses obtained from human myelomas differ in their ability to inhibit the OKT3-induced activation. The best inhibition is obtained with the IgG subclasses IgG1 and IgG3, followed by IgG2; IgG4, IgM, and IgA have little if any effect. None of the IgG subclasses inhibit the Leu 4-induced mitogenesis. Indomethacin as well as supernatants containing interleukin 2 (IL-2) can reverse the inhibitory effects of IgG. Prostaglandins (PGE1 and PGE2) inhibit both the OKT3- and Leu 4-induced mitogenesis, thus lacking the selectivity seen with IgG. Since stimulation by the monoclonal antibodies requires the participation of monocytes, an interpretation consistent with the present data is that IgG stimulates monocytes via its Fc portion to release prostaglandins and/or other suppressor factors via an indomethacin-sensitive pathway. The inability of IgG to inhibit Leu 4-induced mitogenesis may therefore relate to an inability of the monocyte subpopulation, which mediates the Leu 4 response, to secrete suppressor factors. These data suggest a potential value of the mitogenic monoclonal antibodies as probes in studying monocyte heterogeneity and T-cell-monocyte interactions.     

LC－CW的抗肿瘤作用及其机理的研究

提取干酪乳酸杆菌细胞壁成分（ＬＣ－ＣＷ）,研究其抗肿瘤作用及其机理。结果表明：１００μｇＬＣ－ＣＷ,ｉｐ,连续４天,可明显抑制小鼠Ｓ１８腹水瘤移植物的生长,抑瘤率为５４％。增强ＩＬ－２诱导的ＬＡＫ杀伤活性,可明显促进小鼠ＮＫ杀伤活性,明显促进小鼠Ｔ细胞转化,促进ＣｏｎＡ和ＰＨＡ－Ｐ诱导的ＩＬ－２产生,促进ＳＩＬ－２Ｒ的减少。研究结果表明ＬＣ－ＣＷ是一种重要的抗肿瘤免疫调节因子。     

Interleukin 1 stimulates proliferation of a nontransformed T lymphocyte line in the absence of a co-mitogen

Although interleukin (IL) 2-responsive T cell lines provide an opportunity to study the cellular effects of this lymphokine on homogeneous T lymphocyte populations, T cell clones which proliferate in response to IL-1 alone have not been available. We have isolated from cultures of the nontransformed murine T helper cell line, D10 . G4 . 1, a variant (MD10 cells) which proliferates (no lectin or antigen needed) in response to IL-1 alone. The MD10 cells are markedly sensitive to either murine or human recombinant IL-alpha (HrIL-1 alpha) with half-maximal responses observed at monokine concentrations as low as 0.4 X 10(-12) M or 0.8 U/ml, respectively. MD10 cells show the maximal IL-1 effect at 72 hr where the response exceeds the base line by 100-fold (approximately 3,000----300,000 cpm of [3H]thymidine). Whereas both HrIL-2 and purified murine B cell-stimulatory factor 1 (MpBSF-1) induce MD10 proliferation, the maximal response to either is much lower (HrIL-2: 50X baseline; MpBSF-1: less than 20X base line) than to IL-1. Conditioned media from control, concanavalin A-, or IL-1-treated MD10 cells fail to stimulate CTLL or HT-2 cell proliferation alone or inhibit CTLL mitogenesis in the presence of added HrIL-2. Furthermore, monoclonal antibodies to BSF-1 fail to inhibit IL-1-stimulated MD10 replication, and neither HT-2 nor CTLL cells proliferate despite direct cell-to-cell contact with IL-1-treated MD10 cells. When combined, IL-1 (10(-13), 10(-12) M) and IL-2 (10(-13) to 10(-10) M) act synergistically in their MD10 cell growth-promoting effects. MD10 proliferation induced by either IL-1 or IL-2 is relatively resistant to cyclosporine A, with the ID50 of cyclosporine for both IL-1- and IL-2-exposed MD10 cells (ID50 5000 ng/ml) exceeding that for concanavalin A-activated splenocytes (ID50 20 ng/ml) by 2 to 3 orders of magnitude. Finally, MD10 cells bear the L3T4 antigen, IL-2 receptors, and the same clonotypic antigen receptor as the parent clone as recognized by monoclonal antibody 3D3. These data suggest that, in respect to this particular T cell line, IL-1 is directly growth-promoting or, alternatively, induces the production of undetectable, intermediate growth factor(s) resistant to inhibition by cyclosporine A.     

The Ba fragment of complement factor B inhibits human B lymphocyte proliferation

Normal human B lymphocyte function is finely regulated by both positive and negative signals at each stage of activation, proliferation, and differentiation. Activation signals include antigen and surface Ig cross-linking agents such as anti-mu or anti-delta. Signals inducing proliferation include IL-2, high m.w.-B cell growth factor (BCGF), and low m.w.-BCGF. IL-2 as well as IL-6 and other partially characterized B cell differentiation factors can induce terminal differentiation of proliferating B cells into Ig-secreting plasma cells. Various C components have been described to regulate B cell function including Bb that enhances proliferation, C5a that enhances Ig production, and C3a that inhibits Ig production. In our study, we examined the ability of the factor B cleavage fragment Ba to influence human B cell function. Ba did not affect the activation of resting B cells but inhibited the proliferation of activated B cells stimulated with either high m.w.-BCGF or low m.w.-BCGF. The inhibition occurred with doses of Ba as low as 1 microgram/ml (29 nM). Ba was found to bind to activated human B lymphocytes in a saturable manner with an apparent K of approximately 25 nM and an apparent Bmax of 56,000 sites/cell. A peptide made of the carboxy terminal 10 amino acids of Ba (GHGPGEQQKR), was also found to inhibit growth factor induced proliferation of activated B cells but at an ID50 of approximately 5 microM. Finally, Ba was found to inhibit the terminal differentiation of Staphylococcus aweus Cowan-activated B cells stimulated with B cell differentiation factors but not Ig secretion by the partially differentiated EBV-transformed cell line SKW.6. Thus, concentrations of Ba achievable in vivo at sites of active inflammation were found to act on human B lymphocytes by inhibiting their proliferation. This may act to limit the immune response to a specific antigenic challenge.     

Zinc ions suppress mitogen-activated interleukin-2 production in Jurkat cells

Calcineurin (CN) is thought to play an important role in the immune system by regulating cytokine production, for example, interleukin-2 (IL-2) in T-lymphocytes. We have previously shown that physiological concentrations of Zn2+ inhibit CN activity in vitro [K. Takahashi, E. Akaishi, Y. Abe, R. Ishikawa, S. Tanaka, K. Hosaka, Y. Kubohara, Zinc inhibits calcineurin activity in vitro by competing with nickel, Biochem. Biophys. Res. Commun. 307 (2003) 64-68], in spite of the fact that Zn2+ is an essential element of the CN catalytic domain. In this study, in order to assess whether Zn2+ regulates (suppresses) CN activity in vivo and whether Zn2+ can be used as an anti-inflammatory and/or immunosuppressive drug, we examined the effects of Zn2+ on IL-2 production induced by the mitogen, concanavalin A (ConA), in Jurkat T-cells. Zn2+ at 0.2 mM suppressed ConA-induced IL-2 accumulation in the medium of an in vitro culture of Jurkat cells. Zn2+ at 0.03-0.3 mM dose-dependently suppressed ConA-induced IL-2 mRNA expression in Jurkat cells. Zn2+ also suppressed IL-2 mRNA expression induced by phorbol ester (PMA) and ionomycin. Furthermore, Zn2+ and the immunosuppressant FK506 showed an additive inhibitory effect on ConA-induced IL-2 mRNA expression. These results suggest that exogenously added Zn2+ may disturb (increase) the intracellular Zn2+ concentration and inhibit CN activity, thereby suppressing IL-2 production in Jurkat cells. The present study further indicates that Zn2+ may have therapeutic potential in the treatment of T-cell related inflammation and also that Zn2+ may be utilized as a supplemental drug with FK506.