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PubMed1996

Phorbol ester and calcium ionophore can replace TCR signals that induce positive selection of CD4 T cells.

Positive selection of immature thymocytes is a developmental process in which TCR ligation by low avidity interaction induces the generation of mature T cells. However, biochemical signals that can induce positive selection have been unclear. By using TCR-alpha beta- mutant thymus cultures, the present study shows that direct stimulation of intracellular signals by PMA and calcium ionophore ionomycin can induce the generation of mature CD4+8- T cells, bypassing TCR-induced positive selection signals. Interestingly, the concentrations of phorbol ester that induced positive selection were more restricted than those that induced mature T cell activation. Moreover, the combination of phorbol ester and ionomycin restored the generation of CD4+8- T cells in class II MHC- thymus cultures, but did not induce the generation of CD4-8+ T cells in class I MHC- thymus cultures. These results identify that the combination of protein kinase C activation and calcium elevation is the biochemical signal that can induce positive selection of CD4+ T cells.

Animals↗

PubMed1997

Thrombopoietin and thrombin induce tyrosine phosphorylation of Vav in human blood platelets.

Thrombopoietin has an essential role in megakaryopoiesis and thrombopoiesis. To investigate the signaling processes induced by thrombopoietin, we have employed human platelets and recently demonstrated that thrombopoietin induces rapid tyrosine phosphorylation of Jak-2, Tyk2, Shc, Stat3, Stat5, p120(c-cbl) and other proteins in human platelets. Because the apparent molecular weight of a major tyrosine phosphorylated protein in platelets stimulated by thrombopoietin is approximately 85 to 95 kD, we examined the possibility that this could be Vav, a 95-kD proto-oncogene product. Specific antisera against Vav recognized the same 95 kD protein in lysates of Jurkat cells, which are known to express Vav, and platelets, indicating that platelets have Vav. Thrombopoietin induced rapid tyrosine phosphorylation of Vav in platelets without an elevation in cytosolic free calcium concentration or activation of protein kinase C. Vav was also tyrosine phosphorylated upon treatment of platelets with thrombin, collagen, or U46619, which activate phospholipase C, leading to an increased ionized calcium concentration and activation of protein kinase C. Ionomycin or phorbol 12-myristate 13-acetate (PMA) also induces tyrosine phosphorylation of Vav, suggesting that an increase in ionized calcium concentration or activation of protein kinase C may lead to phosphorylation of Vav. Thrombopoietin also induced tyrosine phosphorylation of Vav in FDCP-2 cells, genetically engineered to express human c-Mpl (FDCP-hMpl5). However, neither ionomycin nor PMA induced an increase in tyrosine phosphorylation of Vav in FDCP-hMpl5 cells, suggesting that the calcium and protein kinase C pathways of Vav phosphorylation may be unique to platelets. Further, Vav became incorporated into the Triton X-100 insoluble 10,000 g sedimentable residue in an aggregation-dependent manner, suggesting that it may have a regulatory role in platelet cytoskeletal processes. Vav was constitutively associated with a 28-kD adapter protein, Grb2, which is also incorporated into the cytoskeleton in an aggregation-dependent fashion. Lastly, we found that Vav is cleaved when there is activation of calpain, a protease that may have a role in postaggregation signaling processes. Our data suggest that thrombopoietin and other agonists may induce tyrosine phosphorylation of Vav by different mechanisms and Vav may also be involved in signaling during platelet aggregation by its redistribution to the cytoskeleton.

Animals↗

PubMedDate not supplied

[Cryopreservation of female gametes after the second meiotic division].

During the past few years, experimentation in the field of assisted fertilisation has been focused on the freezing of the female gamete. This technique would allow the ethical problems relating to embryo conservation to be resolved. Unfortunately, in spite of the continuous progress made in embryology, conventional freezing methods have proved lethal to ovocytes owing to the profound morphological and functional changes that take place caused by the low temperature and the toxic action of cryoprotective agents. These alterations include: the thickening of the pellucid zone, thus preventing fertilisation; the confluence of cytoplasmatic organules into vesicles and masses; the depolymerisation of the microtubular apparatus of the meiotic spindle and loss of its orientation towards the egg cell, leading to lack of fertilisation or in the event that fertilisation takes place, a high incidence of chromosomal alterations, such as polyploidy, which are incompatible with subsequent embryonal development. This study aimed to evaluate the possibility of eliminating part of these alterations, in particular those affecting the meiotic spindle, an element of central importance in the formation of the zygote. For this purpose, ovocytes matured in vitro were treated with ionomycin, a substance capable of activating the second meiotic division, and then frozen. The mechanism of action of ionomycin can be attributed to the depolarisation of the plasmatic membrane of the ovocyte, with an increased flow of calcium ions and inhibition of a protein that blocks the second meiotic division. The activation of the second meiotic division has enabled the authors to avoid damage to the meiotic spindle resulting from freezing and to obtain encouraging results in terms of the percentage of fertilisation and embryonal development after freezing, and the subsequent insemination of the ovocyte. Embryonal development ceased during the subsequent phases for reasons that are not yet clear. However, this study showed that severe cellular damage induced by freezing is attributable to alterations to the meiotic spindle that condition the later phases of fertilisation and embryonal development.

Cryopreservation↗

PubMed1998

Autocrine and paracrine apoptosis are mediated by differential regulation of Fas ligand activity in two distinct Jurkat T cell populations.

Fas ligand (FasL) produced by activated T cells mediates autocrine-induced apoptosis to limit T cell expansion. To investigate the regulation of FasL activity, Jurkat cells were stably transfected with a 2.3-kb fragment of human FasL promoter that controlled the expression of a GFP reporter gene. Two populations of Jurkat cells with different levels of GFP expression were obtained. One population constitutively expressed high levels of GFP (GFP+), while the other population expressed low levels of GFP (GFP-). The level of GFP expression in the two populations correlated with their levels of FasL transcription and its functional activity. Autocrine regulation of apoptosis was demonstrated by increased FasL activity after stimulation of GFP- cells with anti-CD3, phorbyl myristyl acetate plus ionomycin, or Con A. Paracrine regulation of apoptosis was suggested by the induction of apoptosis of GFP- cells after coculture with unstimulated GFP+ cells. GFP+ cells exhibited a decreased sensitivity to FasL-mediated apoptosis compared with GFP- cells. Furthermore, the cell surface expression of Fas and CD4 was lower on GFP+ cells than GFP- cells, whereas the expression of CD45RO was higher. A decreased level of IL-2 was produced by GFP+ cells after phorbyl myristyl acetate and ionomycin stimulation. Our results indicate that a subpopulation of T cells that express low levels of FasL and IL-2, which are responsive to up-regulation of these molecules after activation, can undergo apoptosis either by suicide after activation or by a paracrine pathway mediated by T cells that constitutively express higher levels of FasL.

Apoptosis↗

PubMed1998

Expression of protein kinase C isozymes in human basophils: regulation by physiological and nonphysiological stimuli.

The expression of protein kinase C (PKC) isozymes in human basophils and the regulation of PKC isozymes during basophil activation by phorbol 12-myristate 13-acetate (PMA) +/- ionomycin, f-met-leu-phe (FMLP), and anti-IgE antibody were examined. In human basophils (> 98% purity), PKCbetaI, betaII, delta, and were expressed, PKCalpha was difficult to detect, and PKCgamma and eta were undetectable. In unstimulated basophils, PKCbetaI and betaII were found primarily in the cytosol fraction (95% +/- 3% of total and 98% +/- 1%, respectively). Within 5 minutes of stimulation with PMA (100 ng/mL), both PKCbetaI and betaII were translocated to the membrane fraction (85% +/- 4% and 83% +/- 6%, respectively). In resting cells, 48% +/- 3% and 61% +/- 10% of PKCdelta and , respectively, existed in the membrane fraction. Within 1 minute of stimulation with PMA, 90% +/- 6% of PKC was found in the membrane fraction, however, no translocation of PKCdelta was apparent. Stimulation with FMLP caused modest translocation ( approximately 20%) of all PKC isozymes by 1 minute, whereas stimulation with anti-IgE antibody led to no detectable changes in PKC location throughout a 15-minute period of measurement. However, concentrations of PMA and ionomycin that alone caused no PKC translocation and little histamine release, together caused significant histamine release but no apparent PKC translocation. Studies with bis-indolylmaleimide analogs showed inhibition of PMA-induced, but not anti-IgE-induced, histamine release. These pharmacological studies suggest that PKC does not play a prodegranulatory role in human basophil IgE-mediated secretion.

Antibodies, Anti-Idiotypic↗

PubMed1998

Sterol-independent, sterol response element-dependent, regulation of low density lipoprotein receptor gene expression.

Stimulation with phorbol 12-myristate 13-acetate (PMA) and the calcium ionophore ionomycin increased native low density lipoprotein (LDL) receptor gene expression in the human leukemic T cell line Jurkat when cells were cultured in the absence of sterols and also increased nuclear accumulation of sterol regulatory element binding protein (SREBP)-1. PMA and ionomycin likewise increased LDL receptor mRNA levels when cells were cultured in the presence of suppressive concentrations of sterols, when neither SREBP-1 nor SREBP-2 was detectable in the nucleus. These findings indicated that mitogen-induced up-regulation of the LDL receptor gene could be independent of sterol-regulated transcription factors. The involvement of sterol regulatory element (SRE)-1 was analyzed by transfection of LDL receptor promoter constructs. Promoter fragments of either the 5' 1472 or 142 base pairs induced reporter gene expression after mitogenic stimulation when cells were cultured in the absence or presence of sterols. Mutation of the SRE-1 sequence in either construct abolished sterol-mediated regulation of transcription. However, mutation of the SRE-1 sequence in the 1472 base pair promoter fragment did not alter mitogenic induction of transcription, whereas mutation of SRE-1 in the 142 base pair promoter fragment completely prevented up-regulation of transcription. Taken together, these results demonstrate that the LDL receptor promoter contains at least one 5' SRE-independent as well as an SRE-dependent response element. Furthermore, the data suggest that the SRE-dependent response may not involve the action of either SREBP-1 or -2. Thus, mitogen-induced transcription of the LDL receptor promoter is regulated by diverse sterol-independent mechanisms.

Base Sequence↗

PubMed1998

Mechanism of tissue factor activation on cells.

The activation of tissue factor (TF) procoagulant activity (PCA) on the surface of cells may be the first step in the extrinsic pathway of blood coagulation. The latent TF PCA on intact cells is expressed following mechanical disruption or exposure to ionomycin, a Ca2+ ionophore. Within seconds of ionophore addition, an increase in TF PCA of more than 100-fold is observed. The ionophore effect is blocked by pretreating the cells with calmodulin inhibitors. Complexes of TF and activated factor VII (FVIIa) form on both untreated and ionophore-treated cells. However, pseudosubstrate inhibitors bind only to TF-FVIIa on ionophore-treated cells. When proteins on unperturbed cells are crosslinked with 3-3'-dithiobis(sulphosuccinimidylpropionate), crosslinked homodimeric TF is produced. This TF crosslinking is prevented by first treating the cells with ionomycin. Thus, there is an apparent change in TF quaternary structure that is coincident with the ionophore-induced change in TF PCA. These results suggest a simple mechanism for the activation of TF PCA on cells which is triggered by Ca2+ influx into the cytosol. A Ca2+-binding protein, possibly calmodulin, appears to be an essential link in the signal transduction pathway going from increased cytosolic Ca2+ to increased expression of TF PCA on the cell surface. This activation of TF PCA may result from exposure of an essential macromolecular-substrate-binding site on the TF-FVIIa complex that is the direct result of a change in TF quaternary structure, i.e. the conversion of cryptic TF dimers to procoagulant TF monomers.

Calcium↗

PubMed1998

Functional responses of neonatal chicken and turkey heterophils following stimulation by inflammatory agonists.

OBJECTIVE: To determine functional responses of neonatal chicken and turkey heterophils to various inflammatory agonists. ANIMALS: 100 one-day-old chickens and turkeys. PROCEDURE: Blood heterophils were isolated and stimulated for 30 minutes at 39 C with ionomycin, phorbol myristate acetate (PMA), opsonized zymosan (OZ), or formyl-methionyl-leucyl-phenylalanine (FMLP). Functional responses (shape change, adherence, phagocytosis, influx of intracellular calcium, and oxidative burst) of stimulated heterophils were measured and compared with responses of unstimulated (control) heterophils. RESULTS: Turkey and chicken heterophils did not respond to FMLP stimulation. Stimulation of chicken and turkey heterophils with ionomycin resulted in significant increases in adherence, percentage of cells with a shape change, phagocytosis, intracellular calcium concentration, and oxidative burst. Turkey heterophils did not respond to PMA stimulation, whereas stimulation of chicken heterophils with PMA resulted in significant increases in adherence, percentage of cells with a shape change, phagocytosis, and oxidative burst but not intracellular calcium concentration. Stimulation of chicken and turkey heterophils with OZ resulted in significant increases in oxidative burst. CONCLUSIONS: Mechanisms regulating initiation of heterophil activation in neonatal chicken and turkey heterophils are consistent with those described for heterophils isolated from mature birds. The biochemical and cytoskeletal systems of neonatal avian heterophils undergo functional alterations following stimulation with inflammatory agonists. CLINICAL RELEVANCE: Understanding heterophil activation and regulation should eventually lead to methods for controlling bacterial diseases in poultry.

Animals↗

PubMed1999

Proteolysis in human lens epithelium determined by a cell-permeable substrate.

PURPOSE: To develop a system for continuous evaluation of proteolytic activity in human lens epithelium and to characterize factors of importance for the regulation of proteolytic activity in lens epithelial cells. METHODS: Human lens epithelial cells were obtained during cataract surgery. Capsule epithelium specimens consisted of the central parts of the anterior capsule and the underlying lens epithelium. The sample, with the cell-permeable substrate Suc-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin, was placed in a chamber, which was placed in a thermostat-controlled aluminum block. Fluorescence changes were continuously measured by the fiber optics of the luminometer, which was placed 5 mm above the buffer surface. RESULTS: After administration of substrate to the medium overlying the cells, the substrate was degraded at a relatively slow rate. Approximately 10 picomoles of amino-4-methylcoumarin were formed per minute. A significant increase of proteolytic activity could be observed after application of 1 microM ionomycin or 2 microM thapsigargin. No leakage of lactate dehydrogenase from the cells was observed during these procedures. Basal proteolytic activity was totally inhibited by the proteasome inhibitor lactacystin. Lactacystin also attenuated the response to ionomycin and thapsigargin. CONCLUSIONS: Human lens epithelium responds to increased Ca levels from external or internal stores with an increased proteolytic activity that may be mediated by calpain, by the proteasome, or by both. This calcium-dependent change in proteolytic activity may be of importance in the development of cataract.

Acetylcysteine↗

PubMed2000

Flow cytometric visualisation of cytokine production by CD3-CD56+ NK cells and CD3+CD56+ NK-T cells in whole blood.

BACKGROUND: Natural killer (NK) cells produce multiple cytokines with potential immune regulatory roles. We standardised a whole-blood flow cytometry method to visualise intracellular cytokine production by NK cells for the study of NK cell biology and for clinical monitoring. METHODS: With a three-colour fluorescent labelling technique, specific cytokine production by NK or T cells was visualised directly in whole blood in the same sample after stimulation by phorbol 12-myristate 13-acetate (PMA) and ionomycin and by electronically gating on the CD3-ve/CD56+ve NK population or on the CD3+/CD56+ NK-T-cell population. RESULTS: Detectable levels of tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) but not of interleukin-2 (IL-2) or IL-4 were easily observed in NK cells. The visualisation of the cytokine production by NK cells was dependent on the addition of a Golgi transport inhibitor, Brefeldin A. Other known stimuli for NK cells (IL-2 and CD16 monoclonal antibody and incubation with K562, the NK-sensitive cell line) promoted IFN-gamma and TNF-alpha production in NK cells to a lesser extent than did PMA and ionomycin stimulation. CONCLUSIONS: This whole-blood flow cytometric assay appears to be an useful and easy method to examine cytokine production by NK cells and/or by CD3+CD56+ NK-T lymphocytes in patients with relevant diseases.

Brefeldin A↗

PubMed1996

Calcium-calmodulin plays a major role in bradykinin-induced arachidonic acid release by bovine aortic endothelial cells.

We provided evidence that calcium-calmodulin plays a major role in bradykinin-induced arachidonic acid release by bovine aortic endothelial cells. In cells labeled for 16 hr with 3H-arachidonic acid, ionomycin and Ca2(+)-mobilizing hormones such as bradykinin, thrombin and platelet activating factor induced arachidonic acid release. However, arachidonic acid release was not induced by agents known to increase cyclic AMP (forskolin, isoproterenol) or cyclic GMP (sodium nitroprusside). Bradykinin induced the release of arachidonic acid in a dose-dependent manner (EC50 = 1.6 +/- 0.7 nM). This increase was rapid, reaching a maximal value of fourfold above basal level in 15 min. In a Ca2(+)-free medium, bradykinin was still able to release arachidonic acid but with a lower efficiency. Quinacrine (300 microM), a blocker of PLA2, completely inhibited bradykinin-induced arachidonic acid release. The B2 bradykinin receptor antagonist HOE-140 completely inhibited bradykinin-induced arachidonic acid release. The B1-selective agonist DesArg9-bradykinin was inactive and the B1-selective antagonist [Leu8] DesArg9-bradykinin had no significant effect on bradykinin-induced arachidonic acid release. The phospholipase C inhibitor U-73122 (100 microM) decreased bradykinin-induced arachidonic acid release. The calmodulin inhibitor W-7 (50 microM) drastically reduced the bradykinin- and ionomycin-induced arachidonic acid release. Also, forskolin decreased bradykinin-induced arachidonic acid release. These results suggest that the activation of PLA2 by bradykinin in BAEC is a direct consequence of phospholipase C activation. Ca2(+)-calmodulin appears to be the prominent activator of PLA2 in this system.

Animals↗

PubMed1996

Role of anion exchange and thiol groups in the regulation of potassium efflux by lead in human erythrocytes.

Pb2+ is thought to enter erythrocytes through anion exchange (AE) and to remain in the cell by binding to thiol groups. To define the role of AE mechanisms and thiol groups in Pb2+ toxicity, we studied the effects of drugs and conditions that modify AE and that modify thiol groups on the ability of Pb2+ to stimulate potassium efflux as measured with 86Rb. The most potent stimulation of 86Rb efflux by Pb2+ occurred when conditions were optimal for the AE mechanism--that is, when bicarbonate was included in the buffer or a buffer made with Nal or NaCl rather than NaClO4 or NaNO3 was used. Furthermore, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid and 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfuonic acid, potent inhibitors of the AE mechanism, completely inhibited stimulation of the 86Rb efflux by Pb2+. These conditions or inhibitors did not affect stimulation of the 86Rb efflux by ionomycin plus Ca2+. A role for Ca2+ channels was dismissed because the inorganic Ca2+ channel blockers, Cd2+ or Mn2+, did not prevent stimulation of 86Rb efflux by Pb2+ but did inhibit stimulation by ionomycin plus Ca2+. 86Rb efflux was more sensitive to Pb2+ if erythrocytes were treated for 15 min with thiol-modifying reagents that enter cells, such as iodoacetamide, N-ethylmaleimide, or dithiothreitol, than to reduced glutathione, a thiol-modifying reagent that is not permeable to the cell. Thus, in erythrocytes the AE mechanism and internal thiol groups are critical factors that affect the stimulation of a Ca(2+)-dependent process by Pb2+.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1996

Potentiation of bradykinin-induced inositol phosphates production by cyclic AMP elevating agents and endothelin-1 in cultured astrocytes.

Cultured astrocytes express bradykinin (BK) receptors, which are coupled to phospholipase C (PLC) through G-protein to mediate phosphoinositide (PI) hydrolysis. The regulation of this BK receptor-G protein-PLC pathway by cAMP and endothelin-1 (ET-1) was explored by short-term (20 min) and long-term (24 h) treatment with 100 mu M dibutyryl cyclic AMP (dBcAMP) or 10 nM ET-1. Short-term treatment of cells with dBcAMP had no effect on BK-induced PI hydrolysis; however, long-term treatment resulted in potentiation of the BK response. Similar effects were seen after 10 mu M forskolin pretreatment of the cells. We further explored the site of action of 24 h dBcAMP pretreatment and found that AlF(4)-, ionomycin- or A3187-induced PI hydrolysis was not affected but (3H)BK binding was increased. These results indicate that the site of action of dBcAMP is the BK receptor and Scatchard plot analysis showed that the Bmax was increased but the Kd decreased. Cycloheximide (0.5 mu M) blocked the increase in (3H)BK binding, indicating that new synthesis of receptor protein might occur during 24 h pretreatment with dBcAMP. Twenty minutes pretreatment of cells with ET-1 resulted in desensitization of the ET-1 induced P1 response, while the BK response was unaffected. After 24 h pretreatment with ET-1, desensitization to ET-1 still occurred, while BK-induced PI hydrolysis was markedly potentiated. (3H)BK binding and AlF(4)--induced but not A23187- or ionomycin-induced PI hydrolysis were increased, indicating that the site of action of long-term ET-1 treatment was the BK receptor and G protein; Scatchard analysis showed an increase in Bmax but no effect on Kd. These effects were blocked by cycloheximide, indicating that new synthesis of both receptor protein and G protein might occur during 24 h pretreatment with ET-1. (3H)Thymidine uptake was inhibited or potentiated by dBcAMP and ET-1, respectively. Possible dBcAMP-induced differentiation and ET-1-induced proliferation may contribute to the increased expression of receptor proteins.

Aluminum Compounds↗

PubMed1996

Studies related to progesterone-induced hamster sperm acrosome reaction.

Experiments were designed to characterize the effect of progesterone on the hamster sperm acrosome reaction (AR). Progesterone stimulated exocytosis of previously capacitated spermatozoa in a dose-dependent manner. Progesterone-3-(O-carboxymethyl)oxime:BSA conjugate also induced AR when added to capacitated sperm suspensions. EGTA and La3+, added 10 min before progesterone, completely abolished the steroid-stimulatory effect. Benzamidine, a trypsin inhibitor, also inhibited AR when added to sperm cells 10 min before progesterone. This effect was avoided when spermatozoa were treated with the Ca2+ ionophore ionomycin. Conversely, the H+ ionophore FCCP, or the Na+/K+ ionophore nigericin, did not prevent the effect of the inhibitor. Results suggest that progesterone acts on the hamster sperm plasma membrane to stimulate exocytosis, which requires external Ca2+ and presumably Ca2+ influx. In addition, a sperm trypsin-like protease may be part of the mechanism by which progesterone stimulates AR. Since the ionomycin-induced AR does not require this proteolytic activity, the possible involvement of such an enzyme in the progesterone-stimulated Ca2+ influx necessary for the occurrence of AR is discussed.

Acrosome↗

PubMed1998

Shedding of the soluble IL-6 receptor is triggered by Ca2+ mobilization, while basal release is predominantly the product of differential mRNA splicing in THP-1 cells.

The soluble IL-6 receptor (sIL-6R) is generated through either proteolytic shedding of the cognate receptor (PC-sIL-6R), or released as the product of differential mRNA splicing (DS-sIL-6R). Using monocytic THP-1 cells, we demonstrate that both mechanisms are independently regulated, and that each process contributes to sIL-6R production. Shedding of the IL-6R was activated by the Ca2+ ionophore, ionomycin, and inhibited by the TNF-alpha protease inhibitor (TAPI). In contrast, basal sIL-6R release was unaffected by Ca2+ depletion and largely insensitive to TAPI. Moreover, although IL-6R shedding was inactivated by serum starvation, non-stimulated production remained intact. Basal sIL-6R production via differential mRNA splicing was shown through the inhibitory action of brefeldin A and an enzyme-linked immunosorbent assay specific for DS-sIL-6R. Release of this isoform was unaffected by ionomycin or TAPI, indicating that Ca2+ mobilization activates PC-sIL-6R generation, but not DS-sIL-6R. The divergent control of these sIL-6R isoforms indicates that they may independently influence the inflammatory response.

Animals↗

PubMed1988

Subpopulations of CD4- CD8- murine thymocytes: differences in proliferation rate in vivo and proliferative responses in vitro.

Cell sorting and cytotoxic depletion procedures were used to subdivide the population of CD4- CD8- ("double-negative") thymocytes from adult CBA mice on the basis of expression of Ly-1, HSA (the "heat-stable antigen" M1/69 or B2A2), Pgp-1 glycoprotein, Thy-1, MEL-14 and the PC61 antigenic determinant on the IL2 receptor (IL2R). The level of dividing cells within these subsets was assessed by brief in vivo administration of [3H]-thymidine, followed by radioautography, or by flow cytometric cell cycle analysis after DNA staining. The capacity of the subsets to proliferate in culture, in response to stimulation with concanavalin A (Con A), or with phorbol myristate acetate (PMA) and the calcium ionophore ionomycin, was assessed in high cloning efficiency single-cell culture systems. In general, the proliferative response in culture was inversely related to the rate of cell division in vivo. Response of the double-negative subsets to Con A correlated with expression of the T cell antigen receptor complex; although a high cloning efficiency was obtained from the receptor-positive fractions, very few of the clones were cytotoxic. In particular, a major Ly-1+ HSA- Pgp-1+ double-negative subset, as well as minor Ly-1- HSA- Pgp-1+ subsets, contained very few cells in cycle in vivo, but showed a high cloning efficiency in both culture systems. Conversely, the other major double-negative subset, Ly-1- HSA+ Pgp-1-, included most of the cells in cycle, but showed a reduced cloning efficiency in response to PMA and ionomycin and failed to respond to Con A. The dividing cells within the Ly-1- HSA+ Pgp-1- group were strongly enriched in the IL2R- rather than in the IL2R+ subset, suggesting IL2 was not the growth factor maintaining their proliferation in vivo.

Animals↗

PubMed1988

Development of autoreactive L3T4+ T cells from double-negative (L3T4-/Ly-2-) Thy-1+ spleen cells of normal mice.

Thy-1+/L3T4-/Ly-2- spleen cells were purified from normal C57BL/6 (B6) and C,B-17 mice. Cells within this subset expressed the T cell receptor (TcR) for antigen: the majority of cells in this subset were CD3+; a fraction of the cells was stained with the monoclonal antibody (mAb) F23.1; and the TcR molecule was immunoprecipitable with mAb F23.1 from cells within this subset. In limiting dilution analyses, about 1/30 cells within this subset were growth inducible in vitro by stimulation with phorbol myristate acetate (PMA) plus ionomycin; conditioned media containing interleukin (IL) 1, IL2, IL3 or IL4 activity neither triggered nor promoted in vitro growth of these cells. The in vitro generated T cells displayed the Thy-1+/L3T4+/Ly-2- surface phenotype and were self-reactive, i.e., proliferated preferentially in response to syngeneic stimulator cells, and secreted IL2 and IL3 only in response to syngeneic but not allogeneic stimulator cells. The proliferative response of these cells to syngeneic stimulator cells was blocked by anti-self Ia mAb. This autoreactive helper T cell subset was not inducible in purified Thy-1+ spleen cell subsets from athymic nude mice or scid mice. Autoreactive helper T cells did not express detectable levels of the IL2 receptor (IL2R), and their proliferative response was not blocked by anti-IL2R mAb. From PMA plus ionomycin-stimulated double-negative Thy-1+ spleen cells, 14 T cell clones were established in long-term culture which displayed the CD3+CD4+CD8- surface phenotype and were self-reactive.

Animals↗

PubMed1992

Inhibitory and stimulatory signaling via immunoglobulin receptors: dichotomous responses elicited in clonal B cell populations.

The paradox that cross-linking of IgM antigen receptors on B cells by native or surrogate antigen can inhibit, as well as stimulate, B cell functions has previously been attributed to changes during maturation and activation, programming either a negative or a positive response at defined developmental stages. In contrast to this concept, we show here that some B cells possess the potential for both types of response at the same stage. In three clonal malignant human B cell populations, bivalent soluble monoclonal antibodies to IgM or idiotype, but not IgD completely inhibited spontaneous DNA synthesis, but significantly induced [3H]thymidine uptake when coupled to insoluble compounds. In co-incubation experiments mitogenic stimuli were dominant over inhibitory ones and were still effective after prolonged pretreatment of the B cells with inhibitory reagents. Ionomycin, known to increase intracellular calcium levels, and low doses of phorbol ester, described to activate protein kinase C, also suppressed DNA synthesis. High doses of phorbol ester alone or in combination with ionomycin, however, induced DNA synthesis in two of the lymphomas. We conclude that some B cells may respond to cross-linking of surface IgM in a dose-dependent manner so that all signals increase DNA synthesis once a threshold has been reached. These dose-dependent effects may in part involve signaling via breakdown of membrane inositol phosphates.

Antibodies, Monoclonal↗