Cadmium-induced synthesis of metallothioneins in human lymphocytes and monocytes

Cd2+-binding proteins of peripheral blood lymphocytes and monocytes have not well been characterized so far, although they are expected to be a clue for understanding Cd2+ toxicity in those immune competent cells. We separated a family of Cd2+-binding proteins from Cd2+-exposed human peripheral blood lymphocytes by gel filtration chromatography, and characterized them by SDS-gel electrophoresis. The proteins showed electrophoretic behaviours closely similar to metallothioneins (MTs) of HeLa cells derived from human cervical carcinoma. The proteins were also found in Cd2+-exposed monocytes, and were inducible by Cd2+ in both lymphocytes and monocytes. Anti-MT serum specifically precipitated these proteins, which were thus identified as MTs. These results suggest that the two classes of the cells involved in the immune system possess a protective mechanism against Cd2+ through MTs. A variety of human lymphoid cell lines derived from both T and B cells were also found to have capacity to synthesize MTs in response to Cd2+.     

Protective effect of metallothioneins against oxidative stress evaluated on wild type and MT-null cell lines by means of flow cytometry

It is generally accepted that metallothioneins (MTs) are devoted to the regulation of the metabolism of essential trace metals and to chelation of toxic metals. Nowadays, there is increasing evidence that MTs also act as free radical scavengers. We employed wild type mouse embryo fibroblast cell line, GKA1, and its MT-null variant, GKA2, in order to correlate the presence of MTs to the sensitivity of cells to reactive oxygen species (ROS), spontaneously generated by the aerobic cellular metabolism, or chemically induced by hydrogen peroxide. The absence of MTs in GKA2 cells was unambiguously correlated to higher sensitivity to ROS attack, as evaluated by detection and quantification of 8-oxo-2'-deoxyguanosine (8-oxo-G), the first product of oxidative attack to DNA, using Fluorescence-Activated Cell Sorter (FACS). When compared to MT-null cell line, the wild type cells (GKA1) were less sensitive to ROS attack. In GKA1 cells, MT biosynthesis is readily induced by Cd2+ treatment, and such an induction caused a further decrease in sensitivity to ROS injury. On the contrary, the MT-null cells (GKA2) expressed no detectable metallothioneins either constitutively, or after heavy metal pretreatment. Indeed, in GKA2 cell line, pretreatment with Cd2+ did not reduce but even enhanced the oxidative stress.     

Two phenotypically distinct suppressor T cell subpopulations inhibit the induction of B cell differentiation by phytohemagglutinin

Human B lymphocytes can be induced to differentiate into antibody-secreting plasma cells by Leu-3+ T lymphocytes stimulated with pokeweed mitogen (PWM), a polyclonal T cell activator. In contrast, other polyclonal T cell mitogens, such as phytohemagglutinin (PHA), also activate Leu-3+ T cells but are relatively ineffective inducers of B cell differentiation. We have performed a series of experiments to investigate the mechanism underlying this apparent paradox. When human B cells were cultured with unfractionated T cells and PWM or PHA, only PWM was able to induce plasma cell formation and immunoglobulin (Ig) secretion. However, when the T cells were treated with mitomycin C (MMC) before culture, both PWM and PHA were able to induce significant B cell differentiation. These data indicated that both mitogens were able to activate the helper T cells required for B lymphocyte differentiation and suggested that MMC-sensitive suppressor T cells were responsible for inhibiting the induction of antibody-secreting cells by MMC-untreated T cells stimulated with PHA. Phenotypic analysis of the T cells capable of suppressing PHA-induced B cell differentiation revealed that small numbers of either Leu-2+ or Leu-3+ T cells could profoundly suppress the B cell differentiation induced by PHA. In contrast, significant suppression of PWM-stimulated B cell differentiation was observed only with relatively large numbers of Leu-2+ T cells. These data confirm previous reports that OKT4+/Leu-3+ T cells can suppress human B cell differentiation and indicate that the difference in B cell differentiation induced by PWM and PHA with MMC-untreated T cells is largely a reflection of the relative potency of these mitogens to activate these phenotypically distinct suppressor T cell subpopulations.     

Evidence for more than two metallothionein isoforms in primates

Two isoforms of metallothionein (MT) have in general been identified in mammalian cells. We have analyzed Cd2+-induced MTs of primate origin and demonstrated the presence of more than two isoforms. Four low molecular weight Cd2+-binding proteins were separated from Cd2+-exposed HeLa cells by gel filtration and ion-exchange chromatography and identified as MTs by amino acid analysis. These were carboxymethylated and analyzed by electrophoresis under denaturing conditions. Three of these proteins were found to be distinct molecules. We also analyzed hepatic MTs from Cd2+-exposed rhesus monkeys, which have previously been partially separated. In this case, five distinct isomers were detected.     

Effect of metallothionein on cell viability and its interactions with cadmium and zinc in HEK293 cells

Metallothioneins (MTs) are thought to participate in a wide variety of physiological roles, but the mechanisms involved are still unclear. The study was designed to examine the possible factors related to these mechanisms. Methods, including transfection, MTT assay and flow cytometry, were used to investigate the effect of MTs on cell viability and their interactions with cadmium and zinc in HEK293 cells. The results showed that transient overexpression of human MT1A, MT2 and MT3 genes dynamically affected cell viability, and the effect was influenced by zinc and cadmium ions. Overexpressed MTs with added zinc showed a greater inhibitory effect on cell viability. Overexpressed MTs protected cells against low concentrations of cadmium ions (10 microM), but increased cell death in response to high concentrations (20-50 microM). Out of the three MTs, MT1A was more efficient than MT2 and MT3 in its resistance to cadmium (10 microM), and MT3 together with zinc showed more cell growth inhibition than MT1 and MT2. These results indicate that both of the divalent metal ions that could bind MTs, as well as the individual MT isoforms, affect the role of MTs on cell viability, which may explain in part why the comprehensive effect of MTs on the cells was elusive.     

T8 cell regulation of human B cell responsiveness: regulatory influences of CD45RA+ and CD45RA- T8 cell subsets

The immunoregulatory functions of human T8 cell subpopulations defined by mAb to the CD45RA molecule (2H4) were examined. Both CD45RA+ and CD45RA- T8 cells that had been treated with mitomycin C provided help for the production of immunoglobulins by B cells in cultures stimulated with immobilized mAb to CD3 (64.1). In contrast, both CD45RA+ and CD45RA- T8 cells that had not been treated with mitomycin C suppressed B cell responses in anti-CD3-stimulated cultures, although CD45RA+ T8 cells were more effective in this regard. Interleukin 2 (IL2) enhanced suppression by anti-CD3-activated CD45RA- T8 cells, whereas suppression by CD45RA+ T8 cells was almost maximal and not as much increased by IL2. The differentiation into suppressor-effector cells in this system appeared to involve the production of IL2, but not the production of interferon (INF)-gamma. Thus, CD45RA+ T8 cells produced higher amounts of IL2 but lower amounts of IFN-gamma than CD45RA- T8 cells in anti-CD3-stimulated cultures. Moreover, addition of mAb to the p55 component of IL2 receptor (anti-Tac) inhibited the generation of suppressor activity from CD45RA+ and CD45RA- T8 cells. The pattern and magnitude of suppression of B cell responses by CD45RA+ and CD45RA- T4 cells were similar to that by CD45RA+ and CD45RA- T8 cells in this system. Finally, preactivated CD45RA+ T8 cells that had lost CD45RA expression suppressed the B cell responses as effectively as fresh CD45RA+ T8 cells. The results indicate that both CD45RA+ and CD45RA- T8 cells can help or suppress B cell responses. More importantly, the data suggest that the suppressor-effector function of human T cells may rather be related with the stages of the post-thymic differentiation as evidenced by the expression of the CD45RA molecule than represent the fully differentiated T cell subsets, such as T4 and T8 cells. In addition, the CD45RA molecule appeared not to be involved in the suppressor-effector function, but to determine the stage of post-thymic differentiation.     

T cell tolerization in vitro: modulation of helper and suppressor cell activities

This paper analyzes the conditions for in vitro tolerization of purified whole T cell populations and the consequences on helper and suppressor T cell functions. Highly purified splenic T cells from adult DBA/2 mice were incubated in vitro for 24 hr with high doses of trinitrophenyl coupled to human gamma-globulins (TNP-HGG). A profound inhibition of the TNP-specific helper function of these T lymphocytes was observed in a cooperative culture with normal purified splenic B cells and TNP-SRBC as antigen. This state of specific unresponsiveness was maintained after trypsin treatment of the cells, at the end of the 24-hr incubation with the tolerogen. We checked that this procedure removed the vast majority of F23.1 T cell receptor determinants from the cells. This result indicates that T cell receptors for antigen were not merely blocked by the tolerogen. In addition, B cells preincubated with tolerized T cells for 24 hr remained as responsive to TNP as B cells mixed with normal T cells in similar conditions. This demonstrates that the decreased response is not the result of secondary B cell tolerization. In addition, anti-Ia monoclonal antibodies were shown to block the induction of tolerance. We also showed that tolerized T cells significantly decreased the anti-TNP response of normal T and B cells in vitro, whereas the anti-SRBC response in the same cultures was unaffected. When tolerized T cells were separated into Lyt-2- and Lyt-2+ cells, it was found that tolerized Lyt-2- cells had lost about 75% of their helper activity and that Lyt-2+ cells suppressed 70% of the response of a normal T and B cell culture. Thus, in vitro induction of T cell tolerance results in a specific T cell unresponsiveness which is due to both helper T cell inactivation and induction of specific suppressor T cells.     

T cell regulation of human B cell proliferation and differentiation. Regulatory influences of CD45R+ and CD45R- T4 cell subsets

The immunoregulatory functions of human T4 cell subpopulations defined by mAb to the CD45R molecule (2H4) were examined. Both CD45R- and CD45R+ T4 cells that had been treated with mitomycin C (CD45R- and CD45R+ T4-mito) provided help for the generation of Ig-secreting cells (ISC) in cultures stimulated by PWM or by immobilized mAb to CD3 (64.1). IL-2 enhanced the generation of ISC in PWM-stimulated cultures and in anti-CD3-stimulated cultures containing CD45R+ T4-mito. The generation of ISC was maximal in cultures containing anti-CD3-activated CD45R- T4-mito and was not increased by IL-2. By contrast, CD45R+ T4 cells that had not been treated with mitomycin C suppressed B cell responses in cultures stimulated with PWM or anti-CD3, whereas CD45R- T4 cells suppressed the generation of ISC only in cultures stimulated with anti-CD3. IL-2 enhanced suppression by anti-CD3, but not PWM, activated CD45R- T4 cells. Suppression by CD45R+ T4 cells was maximal and not increased by IL-2. CD45R+ T4-mito were more effective suppressor-inducers in PWM-stimulated cultures, promoting the differentiation of suppressor-effector cells from CD8+ T cells. However, both CD45R+ and CD45R- T4-mito exerted comparable suppressor-inducer function in anti-CD3-stimulated cultures. Moreover, in anti-CD3-stimulated cultures, T8 cells could function as both suppressor-effector cells and suppressor-inducer cells. One of the functions of suppressor-inducer cells in this system appeared to involve the production of IL-2. Thus, the addition of IL-2 facilitated the induction of suppressor-effector T8 cells by CD45R- T4-mito in PWM-stimulated cultures. Although IL-2 production by the T cell subsets varied widely depending on the nature of the stimulus, these differences could not entirely explain their capacity to function as helper cells, suppressor-effector cells or suppressor-inducer cells. These results indicate that both CD45R+ and CD45R- T4 cells can help or suppress B cell responses, as well as induce suppressor-effector T8 cells. Moreover, suppressor-inducer function of T cells is not limited to the T4 cell population, but rather can also be accomplished by T8 cells. The results indicate that both T4 cell subsets and T8 cells exert multiple regulatory effects on human B cell function, with the nature of the activating stimulus playing a major role in determining the functional capacity of various T cell subsets.     

Colitis immunoregulation by CD8+ T cell requires T cell cytotoxicity and B cell peptide antigen presentation

Deficient immunoregulation by CD4+ T cells is an important susceptibility trait for inflammatory bowel disease, but the role of other regulatory cell types is less understood. This study addresses the role and mechanistic interaction of B cells and CD8+ T cells in controlling immune-mediated colitis. The genetic requirements for B cells and CD8+ T cells to confer protective immunoregulation were assessed by cotransfer with colitogenic Galphai2-/- T cells into immune-deficient mice. Disease activity in Galphai2-/- T cell recipients was evaluated by CD4+ T intestinal lymphocyte abundance, cytokine production levels, and large intestine histology. B cells deficient in B7.1/B7.2, CD40, major histocompatibility complex (MHC) II (Abb), or native B cell antigen receptor (MD4) were competent for colitis protection. However, transporter-1-deficient B cells failed to protect, indicating a requirement for peptide MHC I presentation to CD8+ T cells. CD8+ T cells deficient in native T cell receptor repertoire (OT-1) or cytolysis (perforin-/-) also were nonprotective. These finding reveal an integrated role for antigen-specific perforin-dependent CD8+ T cell cytotoxicity in colitis immunoregulatory and its efficient induction by a subset of mesenteric B lymphocytes.     

异常汉逊酵母BD102金属硫蛋白的分离纯化和鉴定

从异常汉逊酵母中分离出拮抗Cu²⁺、Cd²⁺等重金属、并经铜、镉诱导产生金属硫蛋白的异常汉逊酵母 (Hansenulaanomala)BD1 0 2。无细胞抽提液经SephadexG 50、DEAESepharoseCL 6B、SephadexG 2 5三次凝胶及阴离子交换柱层析分离纯化 ,Cu²⁺诱导得到Cu MTs两个亚型 ,Cd²⁺诱导得到Cd MT一个亚型。Mr分别约为 7kD和 7 5kD ,由 60和 61个氨基酸组成 ,其中半胱氨酸含量各为 6.8%和 1.0 %。每分子金属硫蛋白 (Cu MTs或Cd MT)可结合 4个铜或镉原子     

Antigen-induced T suppressor cells regulate the autoreactive T helper-B cell interaction

The T suppressor (Ts) cell population that functions to regulate antigen-specific MHC-restricted T helper (Th)-B cell interactions also regulates the activation of B cells by cloned autoreactive Th cells. Activated Ts cells were generated by in vivo priming and restimulation in vitro with high concentrations of the specific priming antigen. Once generated, this Ts population inhibits the Th-dependent activation of primed B cells by both antigen-specific and autoreactive T cells in an antigen-nonspecific manner. This suppression requires the participation of both Lyt-1+2- and Lyt-1-2+ T cells. It was also demonstrated that accessory cells were required for the induction of Ts cells. Moreover, the generation of suppression was MHC-restricted and required the recognition by T cells of Ia antigens on accessory cells. These studies demonstrate that the same or a very similar Ts cell population can function to inhibit the activation of B cells by antigen-specific as well as autoreactive T cells.     

Morphologic and phenotypic features of the subpopulation of Leu-2+ cells that suppresses B cell differentiation

The Leu-2 antigen is expressed on a subpopulation of human T cells that perform suppressor and cytotoxic functions. In addition, this antigen is also present on a portion of cells with morphologic characteristics of granular lymphocytes. Although both Leu-2+ cells and granular lymphocytes have been shown to suppress B cell differentiation, the interrelationship of these two suppressor populations has not previously been fully characterized. We recently produced a monoclonal antibody, termed D12 (anti-Leu-15), which reacts with a variety of cell types, including a subpopulation of Leu-2+ cells. Previous studies have indicated that the Leu-2+ cells that suppress T cell proliferative responses express the Leu-2+15+ phenotype, whereas the precursor and effector cytotoxic T cells that recognize class I major histocompatibility antigens are Leu-2+15- lymphocytes. For this report, we used the anti-Leu-2 and anti-Leu-15 monoclonal antibodies and fluorescence-activated cell sorter techniques to characterize the E+ cells that suppress PWM-induced B cell differentiation. These studies indicate that the vast majority of Leu-2+ cells that suppress this T cell-dependent B cell response have the Leu-2+15+ phenotype. Furthermore, when the morphologic and cytochemical characteristics of these Leu-2+15+ cells were studied, virtually all of these cells were granular lymphocytes. Most of the Leu-2+15+ suppressor cells co-expressed the HNK-1 (Leu-7) antigen, which is detected only on granular lymphocytes. In contrast, virtually none of the Leu-2+15+ granular lymphocytes expressed Fc receptors for IgG molecules. These data indicate that the Leu-2+ cells that suppress PWM-induced B cell differentiation are Leu-2+15+ (and predominantly Leu-7+) granular lymphocytes that do not express Fc receptors. The implications of these observations concerning the relationship of human Leu-2+ suppressor cells to murine Ly-2+ cells and the lineage of granular lymphocytes are discussed.     

Cadmium and calcium uptake in isolated mitochondria-rich cell populations from the gills of the freshwater rainbow trout

A novel cell isolation technique was used to characterize cadmium and calcium uptake in distinct populations of gill cells from the adult rainbow trout (Oncorhynchus mykiss). A specific population of mitochondria-rich (MR) cell, termed the PNA+ MR cell (PNA is peanut lectin agglutinin), was found to accumulate over threefold more 109Cd than did PNA- MR cells, pavement cells (PV cells), and mucous cells during a 1-h in vivo exposure at 2.4 microg/l 109Cd. In vitro 109Cd exposures, performed in standard PBS and Cl- -free PBS, at concentrations from 1 to 16 microg/l 109Cd, were also carried out to further characterize Cd2+ uptake kinetics. As observed during in vivo experiments, PNA+ MR cells accumulated significantly more 109Cd than did other cell types when exposures were performed by an in vitro procedure in PBS. Under such conditions, Cd2+ accumulation kinetics in all cell types could be described with Michaelis-Menten relationships, with Km values of approximately 3.0 microg/l Cd (27 nM) for both MR cell subtypes and 8.6 microg/l Cd (77 nM) for PV cells. In similar experiments performed in Cl- -free conditions, a significant reduction in 109Cd accumulation in PNA+ MR cells was seen but not in PNA- MR or in PV cells. In vitro 45Ca fluxes were also performed to determine the cellular localization of Ca2+ transport in these functionally distinct populations of gill cells. 45Ca uptake was most pronounced in PNA+ MR cells, with levels over threefold higher than those found in either PNA(-) MR or in PV cells. Results from the present study suggest that the PNA+ MR cell type is a high-affinity and high-capacity site for apical entry of Cd2+ and Ca2+ in the gill epithelium of rainbow trout.     

Immunolocalization of metallothioneins in different tissues of turbot (Scophthalmus maximus) exposed to Cd

Metallothioneins (MT) were localized by immunochemistry in different organs and cell compartments of turbot exposed to sublethal concentrations (100 ppb) of Cd for 7 days. The polyclonal rabbit anti-cod MT antibody (NIVA, Norway) applied herein exhibited positive cross-reactivity with turbot MTs. Immunoreactive MTs were localized in the branchial epithelium, in the liver and in the kidney of turbot. In Cd exposed fishes MTs were demonstrated mainly in branchial chloride cells (CC) and to a lesser extend in the area where progenitor cells are located and in the cells of the respiratory epithelium (secondary lamellae). A higher staining intensity for MTs was observed in CC of the interlamellar space of the main branchial epithelium in comparison with control CC. MT-staining was also observed in the chondroblasts of the cartilage and in the erythrocytes within blood vessels both in control and Cd-exposed specimens. MT immunoreaction was high in the liver hepatocytes and weak in the epithelium of the proximal portion of the kidney in exposed turbot. The tegument, spleen and muscle were devoid of any immunolabelling in both treatments. Ultrastructural studies at the transmission electron microscope revealed that Cd-induced MTs were mainly located in the cytoplasm of gill CC, the lysosomes and the cytoplasm of hepatocytes and in the basal labyrinth of kidney proximal nephrocytes. The differential localization/induction of MTs in different cell types described hereby suggests that the quantification of the specific expression of MT may be used in biomonitoring programs as a biomarker of Cd exposure in aquatic environments.     

The cell biology of T-dependent B cell activation

The requirement that CD4+ helper T cells recognize antigen in association with class II Major Histocompatibility Complex (MHC) encoded molecules constrains T cells to activation through intercellular interaction. The cell biology of the interactions between CD4+ T cells and antigen-presenting cells includes multipoint intermolecular interactions that probably involve aggregation of both polymorphic and monomorphic T cell surface molecules. Such aggregations have been shown in vitro to markedly enhance and, in some cases, induce T cell activation. The production of T-derived lymphokines that have been implicated in B cell activation is dependent on the T cell receptor for antigen and its associated CD3 signalling complex. T-dependent help for B cell activation is therefore similarly MHC-restricted and involves T-B intercellular interaction. Recent reports that describe antigen-independent B cell activation through coculture with T cells activated by anti-T-cell receptor or anti-CD3 antibodies suggest that cellular interaction with T cells, independent of antigen presentation or lymphokine secretion, induces or triggers B cells to become responsive to T-derived lymphokines, and that this may be an integral component of the physiological, antigen- and MHC-restricted T-dependent B cell activation that leads to antibody production.     

Isolation and functional analysis of human B cell populations. I. Characterization of the B1+B2+ and B1+B2- subsets

Distinct populations of human B lymphocytes can be identified by their expression and/or co-expression of the B cell-restricted antigens B1 and B2. Dual fluorochrome staining and flow cytometric cell sorting permitted the isolation of the B1+B2+ and B1+B2- cells to homogeneity. In contrast, very few B1-B2+ cells were obtainable from normal lymphoid organs. Virtually all B1+B2+ cells expressed IgM and IgD, but lacked IgG and the plasma cell antigens PCA-1 and PC-1, whereas the B1+B2- cells more frequently expressed IgG, PCA-1 and PC-1. Both populations were noncycling and were composed of similar percentages of small and large cells. The B1+B2+ cells proliferate to anti-mu or to anti-mu + PHA-LCM, but not to PHA-LCM alone. They require both T cells and PWM to produce Ig. In contrast, B1+B2-cells do not significantly proliferate to anti-mu, PHA-LCM, or anti-mu and PHA-LCM. They produce Ig in response to T cells alone without PWM. These phenotypic and functional observations provide preliminary evidence that these populations are distinct and that the B1+B2+ cell may be a "resting" B cell, whereas the B1+B2- cell appears to be more "differentiated." The present studies further suggest that they will also be helpful in characterizing B cells in some human disease states. We believe that the identification and isolation of these and similar subsets of B cells defined by differing cell surface phenotype should aid our understanding both of normal B cell differentiation and of B cell disease states.     

Role of the major histocompatibility complex in T cell activation of B cell subpopulations: antigen-specific and H-2-restricted monoclonal TH cells activate Lyb-5+ B cells through an antigen-nonspecific and H-2-unrestricted effector pathway

Monoclonal T helper (TH) cell populations were employed to study the mechanism of activation of the Lyb-5+ B cell subpopulation in T cell-dependent antibody responses in vitro. It was demonstrated that monoclonal T cell populations were sufficient to help rigorously T-depleted unprimed (B + accessory) cells for direct plaque-forming cell responses to trinitrophenyl- (TNP) conjugated keyhole limpet hemocyanin (KLH). The activation of several lines of cloned (H-2b X H-2k)F1 TH cells was antigen (KLH) specific and H-2 restricted. Individual clones were restricted to H -2b, H-2k, or unique (H-2b X H-2k)F1 encoded determinants. Under the experimental conditions employed, responses mediated by cloned TH cells were found to result in the activation of the Lyb-5+ B cell subpopulation. The activation of Lyb-5+ B cells by cloned TH cells did not require covalent linkage of carrier and hapten, and responses could be stimulated in the presence of free KLH plus TNP conjugated to an irrelevant carrier. The H-2 restriction of TH cell function was shown to reflect a requirement for T cell recognition of determinants expressed by accessory cells, whereas no requirement existed for restricted T cell recognition of B cells. These findings suggest that the help provided by monoclonal TH cells, once activated, was both antigen nonspecific and H-2 unrestricted. Consistent with this interpretation, it was found that the supernatant of antigen-stimulated TH cells provided antigen-nonspecific help to T-depleted spleen cells. Thus, these results demonstrate that the activation of Lyb-5+ B cells by antigen-specific and H-2-restricted monoclonal TH cell populations is itself antigen nonspecific and H-2 unrestricted.     

Characterization of a T4+/Leu-8+ T cell clone that directly helps B cell Ig production by secreting B cell differentiation factor

A human T4+/Leu-8+ T cell clone (YA2) was established by phytohemagglutinin activation and interleukin 2 (IL 2) propagation. Functional characterization of this clone demonstrated that it provided potent help towards Ig production by pokeweed mitogen-stimulated B cells in the presence of small numbers of autologous T cells or by Staphylococcus aureus Cowan I (SAC)-activated B cells in the presence of B cell growth factor (BCGF). YA2 provided no help to resting B cells and minimal help to either unactivated B cells cultured with BCGF or SAC-activated B cells. Supernatant generated from clone YA2 by IL 2 stimulation had significant B cell differentiation activity but no BCGF or IL 2 activity. Thus, YA2 is a T4+/Leu-8+ potent direct helper only to B cells that are activated and proliferating due to its selective secretion of a differentiation factor, and not an activation and growth factor. The availability of phenotypically defined cloned populations of T cells with restricted functional helper activity related to the secretion of selected B cell tropic factors should prove useful in the dissection of the role of individual T cell subsets in the regulation of the human B cell cycle.     

Suppressor cell function of human granular lymphocytes identified by the HNK-1 (Leu 7) monoclonal antibody

The HNK-1 (Leu 7) differentiation antigen defines a subpopulation of human granular lymphocytes with natural killer (NK) and K cell function. In this study, we investigated whether HNK-1+ cells, identified with the monoclonal antibody and purified with a fluorescence-activated cell sorter (FACS), could function as suppressor cells. The results demonstrated that purified HNK-1+ cells efficiently suppressed both PWM-induced IgG production by B cells and T cell proliferation in mixed lymphocyte reactions (MLR). Manifestation of this suppressor cell activity required immune complex activation and was partially sensitive to 2000 rad irradiation. This suppressor cell activity was predominantly mediated by a subset of HNK-1+ cells that have previously been shown to have maximum NK function and lack expression of the E rosette (ER) receptor and T cell antigens (e.g., T3 and T8). Thus, HNK-1+ER- cells suppressed a MLR by an average 52%; HNK-1+ER+ were one-half as efficient, causing an average 23% suppression. For comparison, we also examined the characteristics of Leu 2a+ suppressor T lymphocytes. In contrast to HNK-1+ cells, unactivated Leu 2a+ cells suppressed both B and T cell responses. This suppressor activity was not augmented by immune complex activation and was absolutely radio-sensitive in PWM assays. HNK-1+ cells, especially the HNK+ER- subset, can therefore mediate suppressor cell function in addition to their spontaneous cytotoxic function. Furthermore, some of their suppressor cell properties are distinct from those attributed to other types of suppressor lymphocytes.     

Metal binding of metallothioneins in human astrocytomas (U87 MG,IPDDC-2A)

Astroglia cells structurally and nutritionally support neurons in the central nervous system. They play an important role in guiding the construction of the nervous system and controlling the chemical and ionic environment of neurons. They also represent the major sites for accumulation and immobilisation of toxic metal ions most probably connected with metallothioneins. For this reason astroglia cells possess high cytosolic levels of metallothioneins I, II and III (MT-I,II,III). Our aim was to establish the inducibility and metal binding of MTs in two human astrocytoma cell lines, U87 MG (astrocytoma–glioblastoma, grade IV) and IPDDC-2A (astrocytoma, grade II), on exposure to cadmium chloride (1 μM). MTs were identified by molecular weight (size exclusion chromatography) and their metal content (Cd, Zn and Cu) to follow the interactions between metals. We showed that MTs are constitutively expressed in both human astrocytoma cell lines. In accordance with the higher malignancy grade of U87 MG, the amount of MTs was higher in U87 MG than in IPDDC-2A cells. After 24 hours of exposure to Cd their expression greatly increased in both cell lines and they were capable of immobilising almost all water soluble Cd. Induction of MTs in U87 MG cells was additionally followed up to 48 hours with exposure to different concentrations of CdCl₂ (1, 10 μM). Induction was a time dependent process throughout the period. Isoform III (identified by chromatographic separation of isoform III from I/II) was present at all exposure times, but only in traces with respect to the prevailing amounts of MT-I/II isoforms. So induction can be attributed to isoform I/II only.