Effects of extracellular ATP and adenosine on different thymocyte subsets: possible role of ATP-gated channels and G protein-coupled purinergic receptor.

To explore the possible role of purinergic receptors in thymocyte development and in pathogenesis of adenosine deaminase SCID, we studied effects of extracellular adenosine triphosphate (ATP(ext)) and adenosine on TCR- and steroid hormone-triggered processes in mouse thymocytes. Reverse transcriptase-PCR analysis confirms the mRNA expression of several types of purinergic receptors, while the functioning of ATP receptors in thymocytes is reflected by ATP(ext)-induced intracellular calcium increases and by thymocyte subset-specific sensitivity to the effects of ATP(ext) and adenosine. Only ATP(ext), but not the ATP catabolites, adenosine, dexamethasone, or TCR cross-linking, was efficient in triggering rapid protein synthesis independent lysis of CD4+8- thymocytes and peripheral CD4+ T cells. In contrast, extracellular adenosine specifically induced the apoptosis of CD4+8+ thymocytes. ATP(ext) also induced a slower process of DNA fragmentation and protein synthesis-dependent apoptosis in all thymocyte subsets. ATP(ext) had an additive effect with TCR cross-linking in the induction of thymocyte death, but, unexpectedly, the effects of ATP(ext) at high concentration were antagonistic to steroid-induced apoptosis. Described here, the properties of ATP(ext) and adenosine are consistent with their involvement in the regulation of T cell development due to differential expression and signaling through purinergic receptors in different thymocyte subsets. The possible role of purinergic receptor signaling in T cell differentiation and adenosine deaminase SCID is discussed.

Murine T lymphocytes modulate activity of an ATP-activated P2Z-type purinoceptor during differentiation.

Murine T, but not B, lymphocytes constitutively express a membrane receptor for adenosine nucleotides that opens a nonspecific pore that admits Ca2+ and ethidium (314 Da), but not propidium (415 Da) ions. ATP, ADP, and AMP show decreasing potency; UTP and adenosine are inactive. Nonhydrolyzable ATP analogues are completely ineffective. Oxidized ATP inhibits the response. Activity is detectable at ATP concentrations of 125 microM and peaks at 1 mM. The intracellular free Ca2+ ([Ca2+]i) rise is not reversed by removing ATP by centrifugation or apyrase. The kinetics, agonist and antagonist profiles, and the passage of ions as large as ethidium are the characteristics of a P2z-type purinoceptor. No expression of classical P2x-, P2u-, or P2Y-type purinoceptors can be detected. The [Ca2+]i elevating activity of the ATP receptor is modulated during T cell differentiation. CD4+8+ double-positive thymocytes are the least responsive. CD4-8+ single-positive thymocytes, CD8+ splenic T cells, CD4+8- single-positive thymocytes, and CD4+ splenic T cells show increasing reactivity. Measurement of P2Z expression by the rate of ethidium ion uptake correlates with the [Ca2+]i. The trimodal expression of P2Z by splenic CD4+ T cells correlates with the subsets defined by CD44 and CD45RB, differentiation Ags that distinguish memory cells: P2Zlow cells are CD44brightCD45RBbright; P2Zint are CD44dullCD45RBint; P2Zhigh are CD44brightCD45RBdull. It is suggested that P2Z receptor-mediated signaling could be involved in the regulation of differentiation and cell death in the thymus and peripheral T lymphocytes.

Antigen-specific cell conjugate formation and long-lasting calcium responses in recognition of Mls cellular superantigen by cloned murine T lymphocytes.

T lymphocyte recognition of cell-associated minor lymphocyte stimulation (Mls) superantigen was studied by simultaneous flow cytometric measurement of T cell free ionized intracellular calcium ([Ca2+]i) with the fluorescent probe indo-1 and T cell binding to antigen-presenting cells stained with a long-chained, membrane-fixed cyanine dye. Cloned T cell-B lymphocyte antigen-presenting cell conjugate formation and increased T cell [Ca2+]i were antigen specific and tightly linked in four Mls-reactive T cell clones. The T cell-antigen-presenting cell conjugates were extremely stable and, like T cell [Ca2+]i elevation, were maintained for more than 2 hr. Three ligand-receptor pairs, (i) T cell receptor/antigen+class II, (ii) CD4/class II, and (iii) LFA-1/ICAM-1, were obligatory participants in T cell recognition of cell-bound superantigen since monoclonal antibodies to any of them blocked formation of cell conjugates. Despite previous reports, Mls recognition occurred by the conventional T cell recognition pathway with easily detected phosphoinositide hydrolysis. B cell activation greatly enhances their recognition by Mls-specific T cells.

Regulation of IgD-receptor expression on murine T cells. II. Upregulation of IgD receptors is obtained after activation of various intracellular second-messenger systems; tyrosine kinase activity is required for the effect of IgD.

The presence of IgD receptors (IgD-R) on T cells during a primary response to antigen causes augmented antibody production and facilitates priming for a secondary response. Cross-linked, but not monomeric IgD leads to a rapid upregulation of these receptors on T cells. As shown in the present study, the rapid upregulation of IgD-specific receptors is also induced by cross-linking of T cell surface molecules known to mediate triggering of T cell activation, such as CD3, CD2, and Thy 1. Furthermore, IgD-R are also upregulated by pharmacologically active compounds that increase intracellular cAMP and by PMA/DiOG plus ionomycin, but not by either PMA or ionomycin alone. The upregulation of IgD-R by anti-CD3 is inhibited by both calphostin C and herbimycin A, while that due to DiOG plus ionomycin is only inhibited by calphostin C. Upregulation of IgD-R by increased cAMP is blocked by HA1004, but not by low concentrations of staurosporine or herbimycin A. IgD itself does not cause an increase in intracellular cAMP, protein kinase C translocation, influx of extracellular Ca2+, or a change in membrane potential. Relatively specific inhibitors of these activation pathways, HA1004, calphostin C, and neomycin, also fail to interfere with IgD-receptor upregulation by IgD itself. However, tyrosine kinase inhibitors, including herbimycin A, tyrphostin C11, and genistein, completely prevent the effect of IgD on IgD-R expression. Although an influx of Ca2+ is apparently not involved, a role for intracellular Ca2+ in the upregulation of IgD-R by IgD on T cells is indicated by the susceptibility to inhibition by BAPTA, W7, and FK520. We conclude that activation of at least three different second-messenger systems can cause IgD-R upregulation, but that the effect of IgD itself requires tyrosine kinase activity, perhaps in an intracellular Ca(2+)-dependent manner.

Two separate mechanisms of T cell clonal anergy to Mls-1.

T cell tolerance to superantigen can be mediated by clonal anergy in which Ag-specific mature T cells are physically present but are not able to mount an immune response. We induced T cell unresponsiveness to minor lymphocyte stimulations locus antigen (Mls)-1a in mice transgenic for TCR V beta 8.1 in three different systems: 1) injection of Mls-1a spleen cells, 2) mating with Mls-1a mice, and 3) bone marrow (BM) chimeras in which Mls-1a is present only on nonhematopoietic cells. CD4+8-V beta 8.1+ cells from all these groups did not proliferate in response to irradiated spleen cells from Mls-1a mice. We compared the response of these cells by T cell/stimulator cell conjugate formation, Ca2+ mobilization, and proliferation assays. The mechanisms underlying the unresponsiveness of these T cells appear to differ. CD4+8-V beta 8.1+ cells from Mls-1a spleen cell-injected mice mobilized cytoplasmic Ca2+ but proliferated at a reduced level in response to cross-linking with anti-TCR mAb. However, these cells formed conjugates, mobilized Ca2+, and proliferated in response to Mls-1a when activated B cells were used as stimulators, although they produced reduced levels of IL-2. In Mls-1a/b V beta 8.1 transgenic mice, a subset in CD4+8-V beta 8.1+ cells did not mobilize cytoplasmic Ca2+ after TCR cross-linking. Their conjugate formation, Ca2+ mobilization, or proliferation in response to Mls-1a on activated B cells was undetectable. Finally, CD4+8-V beta 8.1+ cells from the BM chimeras proliferated to TCR cross-linking at a partially reduced level and formed conjugates, mobilized Ca2+, and proliferated in response to Mls-1a on activated B cells. These features suggest that the mechanisms underlying the maintenance of anergy in Mls-1a spleen cell-injected mice are distinct from those in Mls-1a mice.

Lack of voltage sensitive potassium channels and generation of membrane potential by sodium potassium ATPase in murine T lymphocytes.

Voltage sensitive K+ channels, which are responsible for generation of membrane potential in most cells, are functionally absent in about one-third of peripheral murine T cells and greatly reduced in the rest as shown by resistance of their membrane potential to changes in extracellular potassium concentration and failure of K+ channel dependent volume regulation. Despite the absence of voltage- sensitive K+ channels, the membrane potential of peripheral T cells is between -60 and -70 mV, the same as thymocytes. A total of 40 to 70 mV of the membrane potential of peripheral T cells is produced by the direct electrogenic action of the asymmetric Na+K+ ATPase pump because the cells are depolarized by ouabain, an inhibitor of the pump, removal of extracellular potassium or reduction of temperature. The residual, ouabain-resistant membrane potential, is sensitive to the K+ channel blocker, quinine, and thus due to electrodiffusion through K+ channels. Na+ and K+ turnover, and sensitivity to ouabain, are the same in peripheral T cells and thymocytes. The predominant mechanism of membrane potential generation changes during T lymphocyte differentiation from electrodiffusion in the thymus to electrogenic in peripheral T cells and back to electrodiffusion upon peripheral cell activation.

T cell receptor-mediated recognition of self-ligand induces signaling in immature thymocytes before negative selection.

Shaping of the T cell repertoire by selection during intrathymic maturation involves T cell receptor (TCR) recognition of major histocompatibility complex/self-antigen complexes. In this communication, we studied the ability of minor lymphocyte stimulating (Mls) determinants to act as self-tolerogens in the selection of the T cell repertoire. We demonstrate that unprimed T cells from normal as well as TCR transgenic mice form Mls-specific conjugates with antigen-presenting cells, and that this TCR-ligand interaction leads to elevation of intercellular Ca2+ ([Ca2+]i). Peripheral T cells from TCR transgenic mice expressing receptors specific for self-Mls antigen show no reactivities to Mlsa. However, a proportion of immature thymocytes from these mice show specific binding and strong [Ca2+]i elevation in response to self-antigen-presenting cells, although these thymocytes do not proliferate. This self-reactivity of thymocytes is inhibited by antibodies specific for TCR, CD4, CD8, class II molecules, lymphocyte function-associated antigen 1, and intercellular adhesion molecule 1. These results demonstrate for the first time that before thymic negative selection, immature T cells can specifically interact with cells bearing self-antigen, and suggest that the resulting TCR-dependent signal transduction events provide a basis for negative selection of self-reactive T cells.

Murine splenic null cell compartment contains distinct haemopoietic subpopulations: enlargement of a myeloid and an undifferentiated subset with the development of splenomegaly in New Zealand black mice.

We have previously reported that non-T, non-B 'null' cells increase with age in New Zealand Black (NZB) mice resulting in splenomegaly. Using a panel of monoclonal antibodies recognizing lineage-specific cell surface antigens we demonstrate four distinct subsets within this null cell compartment: (1) undifferentiated; (2) T lineage with undetectable Thy-1.2; (3) myeloid/erythroid; and (4) a pre-B/plasma cell type. All four subsets also occur in non-autoimmune mice. The frequency of these populations are similar in the young mice of all the strains examined, although the total number of null cells is higher in NZB. The elevation of null cells in young NZB mice is controlled by a single dominant gene in the genetic cross with New Zealand White (NZW) mice and does not appear closely related to the subsequent development of autoimmune disease. The proportion of myeloid/erythroid null cells increases with age in NZB as splenomegaly develops.

Induction of proliferation by high molecular weight B cell growth factor or low molecular weight B cell growth factor is associated with increases in intracellular calcium in different subpopulations of human B lymphocytes.

The activation of resting B cells with anti-surface Ig is associated with transient increases in intracellular calcium. In the present study, we demonstrate that stimulation of B cells which have already been activated by Staphylococcus aureus Cowan I (Sac), with high molecular weight B cell growth factor (HMW-BCGF) or low molecular weight B cell growth factor (LMW-BCGF), but not IL-2, IL-4, or interferon-gamma, is associated with an increase in intracellular calcium, which is modest compared to that seen with anti-Ig (approximately 100 nM vs approximately 400 nM). The increases in intracellular calcium induced by HMW-BCGF or LMW-BCGF occur in distinct but overlapping subpopulations of B cells. Thus, increases in intracellular calcium in human B cells occur not only upon activation but also upon the induction of proliferation by certain (but not all) B cell growth factors. Presumably, the effect of increasing intracellular calcium during the induction of proliferation is to modify a different group of intracellular molecules than those induced during activation.

Effects of induced anemia in normal and autoimmune mice.

Normal and autoimmune mice were studied with regard to signals eliciting differentiation and division of bone marrow stem cells. The erythropoiesis induced by anemia following serial bleedings was analyzed in young autoimmune New Zealand Black (NZB) mice and non-autoimmune strains. No difference in the response to the stimulus created by anemia was noted between the strains. After serial bleedings as a stimulus to stem cell proliferation, a five-fold increase in numbers of proliferating spleen cells occurred in both NZB and DBA/2 strains; the increased proliferating spleen cells in both strains were non-lymphoid. The bled animals had decreased percentages of B cells. The production of autoantibodies was not significantly altered by the experimentally induced anemia. In contrast, anti-immunoglobulin activation of resting B cells was increased in response to anemia. Young mice which had experimentally induced anemia had several characteristics in common with old autoimmune NZB mice. Both old NZB mice and young anemic animals had splenomegaly, increased numbers of proliferating spleen cells, decrease in splenic Ly 5+ cells and an increase in splenic colony forming units (CFUs). The anemic normal strains of animals lacked other characteristics of old NZB mice such as hyperimmunoglobulinemia or autoantibody production or elevated CD5+B cell numbers. This work supports the concept that the increase in spleen cell number, proliferating spleen cells, CFUs and the increased percentages of non-Ly-5 cells (which include erythroid precursors) found in the spleens of old NZB mice may in part result from their autoimmune hemolytic anemia.

Heterogeneity of lymphocyte calcium metabolism is caused by T cell-specific calcium-sensitive potassium channel and sensitivity of the calcium ATPase pump to membrane potential.

Calcium management differs in T and B lymphocytes. [Ca2+]i elevation in response to calcium ionophores is up to 10 times greater in T cells than B cells. There is no difference between them in ionophore uptake. T cells, but not B cells, possess a calcium-sensitive potassium channel which produces membrane hyperpolarization at [Ca2+]i above 200 nM. This alters T cell density providing a rapid and easy method of cell separation. In contrast, B cells depolarize when [Ca2+]i is increased. Isolated B cell membrane vesicle ATP-dependent calcium pump activity is higher than T cell vesicles. Membrane depolarization reduces the [Ca2+]i response to ionomycin, most dramatically in T cells because they are hyperpolarized by increased [Ca2+]i. The most likely basis of this behavior is an effect of membrane potential on lymphocyte membrane calcium pump activity. This mechanism provides an explanation of the inhibitory effect of membrane depolarization on T lymphocyte responses.

Fixation and long-term storage of human lymphocytes for surface marker analysis by flow cytometry.

A method to preserve stained human lymphocytes for subsequent cell surface analysis by flow cytometry (FCM) is described. Cells stained with fluorescein isothiocyanate (FITC) and phycoerythrin (PE)-conjugated monoclonal antibodies and then fixed in 1% paraformaldehyde, followed by extensive washing and resuspension in 1% BSA medium, could be stored at 4 degrees C for at least 2 weeks prior to FCM analysis without significant alteration in the light scatter or fluorescence properties of the cells. Furthermore, the method was also suitable for analyzing lymphocytes that express T-cell activation markers in certain disease conditions. In addition, we have identified monoclonal antibody combinations that discriminate different lymphocyte subsets that are satisfactory for multiparameter analysis after 2 weeks of storage. This method should prove useful for enumerating lymphocyte subsets in field study sites remote from flow cytometry laboratories.

Aluminum fluoride induces phosphatidylinositol turnover, elevation of cytoplasmic free calcium, and phosphorylation of the T cell antigen receptor in murine T cells.

Antigen activation of murine T lymphocytes leads to phosphorylation of three subunits of the murine T cell antigen receptor (L.E. Samelson, M.D. Patel, A.M. Weissman, J.B. Harford, and R.D. Klausner. 1986. Cell 46:1083). Two kinases are activated in this process: protein kinase C which leads to phosphorylation of the gamma and, to a lesser extent, the epsilon subunits on serine residues and a tyrosine kinase which phosphorylates the p21 subunit (M.D. Patel, L.E. Samelson, and R.D. Klausner. 1987. J. Biol Chem. 262:5831). We sought to determine whether treatment of these cells with NaF could simulate any of these antigen-induced events. Indeed NaF treatment resulted in breakdown of polyphosphoinositides and production of phosphoinositols. This treatment also resulted in a rise in cytosolic free Ca2+. EGTA failed to block this rise suggesting that NaF liberated intracellular stores of Ca2+. Finally NaF treatment resulted in phosphorylation of the gamma and epsilon chains of the T cell receptor indistinguishable from the effects of phorbol esters. The NaF effect was potentiated by addition of A1Cl3 consistent with the view that the active moiety is A1F4-. The A1F4--induced phosphorylations were abolished in cells in which protein kinase C was depleted by prior treatment with phorbol myristate acetate. All of these observations are compatible with the interpretation that the A1F4- phosphorylation is mediated by protein kinase C. Antigen and anti-receptor antibody-induced receptor serine phosphorylation and phophatidylinositol turnover are blocked by raising intracellular levels of cyclic adenosine monophosphate. In contrast, A1F4--induced effects were insensitive to cyclic adenosine monophosphate.

Molecular events in the induction of a nonresponsive state in interleukin 2-producing helper T-lymphocyte clones.

Exposure of normal interleukin 2 (IL-2)-producing helper T-cell clones to antigen and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide-treated antigen-presenting cells results in proliferative unresponsiveness to subsequent stimulation with antigen and normal antigen-presenting cells. In the present study, we have examined the molecular events that accompany the induction of this unresponsive state. T cells stimulated in this manner failed to produce IL-2, but interleukin 3, interferon-gamma, and IL-2 receptors were partially induced and T-cell receptor beta mRNA was fully induced. Although T-cell unresponsiveness correlated with an IL-2 production defect, addition of IL-2 during the induction phase failed to prevent development of the unresponsive state. The critical biochemical event appeared to be an increase in intracellular calcium. Removal of calcium from the medium prevented induction of the unresponsive state, whereas addition of the calcium ionophore ionomycin induced unresponsiveness as well as all of the related partial activation events. Thus, an increase in intracellular calcium under nonmitogenic conditions appears to initiate an alternative activation program that prevents the T cell from producing IL-2 in response to subsequent normal activation signals. The significance of this in vitro model for tolerance induction in vivo is discussed.

Flow cytometric analysis of murine splenic B lymphocyte cytosolic free calcium response to anti-IgM and anti-IgD.

The accurate measurement of ionized intracellular calcium [Ca++]i in single cells by flow cytometry with the use of a new fluorescent calcium chelator, indo-1, is described. We have developed a dependable in situ calibration technique that indicates a resting [Ca++]i in lymphocytes of 100 nM. The enhanced fluorescence of this probe permits its use at sufficiently low cytoplasmic concentrations that buffering of [Ca++]i transients does not occur. The [Ca++]i response of small resting B lymphocytes to crosslinking of surface antigen receptors by anti-immunoglobulin is heterogeneous. With maximal stimulus, the peak [Ca++]i response occurs in 10 to 20 seconds with most cells reaching levels greater than/1 microM. Mean [Ca++]i falls to between 300 and 800 nM by 100 seconds where it remains for more than 10 min. Anti-delta is a more potent stimulus of increased [Ca++]i than anti-mu in terms of both [Ca++]i level and fraction of B cells responding. Whether this is due to the greater density of surface IgD than IgM, a difference in signal transduction efficiency, or both, is not yet known. Surface immunoglobulin receptors are present in great excess. Less than 3% of surface immunoglobulin is crosslinked at the peak of the [Ca++]i response.

HLA-DR antigens in systemic lupus erythematosus: association with specificity of autoantibody responses to nuclear antigens.

HLA-DR antigens and autoantibodies to the nuclear or cytoplasmic antigens Ro/SSA, La/SSB, Sm, and RNP were determined in North American and Austrian patients with systemic lupus erythematosus (SLE). Analysis of the association of antibodies to these ribonucleic acid (RNA)-protein antigens with HLA-DR antigens showed that HLA-DR3 was related to the presence of anti-Ro/SSA or anti-La/SSB, or both. In contrast, anti-Sm or anti-RNP, or both were associated with HLA-DR4. HLA-DR5 was associated with absence of these autoantibodies. The data extend evidence for the complexity and heterogeneity of SLE. Moreover, they indicate that, in SLE, genes linked to those coding for HLA-DR antigens, are related to the specificity of autoantibody responses rather than to the primary immunological abnormalities of this disorder.

Proteins associated with B lymphocyte hyperactivity in New Zealand black mice.

New Zealand Black (NZB) mice exhibit polyclonal B cell activation and elevated immunoglobulin production, an abnormality associated with the spontaneous autoimmune disease that affects this strain. To further our understanding of this abnormality of B cell differentiation and maturation, we have employed two-dimensional polyacrylamide gel electrophoresis to analyze the proteins synthesized by lymphocytes of several strains. Two proteins were produced by lymphocytes from NZB mice but not those from normal strains. One was a 16 kd protein with a pI of 5.1, and the other was a 27.5 kd protein with a pI of 4.5. The presence of the xid gene on the NZB background suppressed production of both proteins. They were synthesized by spleen cells but not by bone marrow or lymph node cells, and production was restricted to enlarged B lymphocytes. p16 was synthesized by normal mouse strain B cells upon stimulation with LPS. The 27.5 kd protein was shown to be secreted. On the basis of partial amino acid sequence determination of proteins eluted from gels, p27.5 was identified as J chain and p16 as the C terminal fragment of mu-chain. The synthesis of two other proteins, 13 kd and 18 kd in size, was elevated in NZB spleen lymphocytes. The 18 kd protein was identified as translation initiation factor eIf-4D. The increased level of this protein may be related to the upregulation of immunoglobulin synthesis.

The B lymphocyte calcium response to anti-Ig is diminished by membrane immunoglobulin cross-linkage to the Fc gamma receptor.

We report the cytosolic free calcium, [Ca2+]i, responses of single murine B lymphocytes to whole and F(ab')2 fragments of anti-Ig measured in the flow cytometer with indo-1, a new fluorescent chelator of calcium. The principle advantages of this recording system are these: Indo-1 is highly fluorescent; hence, loading concentrations that introduce artifacts in the reported [Ca2+]i signal may be avoided. The measurement of [Ca2+]i by fluorescence ratio corrects for nonuniform dye uptake, making possible quantitative estimates of [Ca2+]i in single cells and an assessment of the variability of population responses. Baseline recordings of unstimulated lymphocytes indicated a narrow, stable range of [Ca2+]i (75 to 125 nM). The [Ca2+]i rise induced by various anti-Ig preparations exhibited considerable heterogeneity. The initial mean value for F(ab')2 anti-Ig-stimulated cells peaked above 1 microM and was due only to the release of Ca2+ from intracellular stores. A steady state elevation of [Ca2+]i was reached by 5 min and persisted for hours. Cells stimulated with intact anti-Ig reached similar initial peak [Ca2+]i values, but then declined toward baseline. This difference was due to membrane Ig-IgG Fc receptor (mIg-Fc gamma R) cross-linkage, because blocking the Fc gamma R with a monoclonal antibody made the [Ca2+]i responses to F(ab')2 and intact anti-Ig identical. The attenuation of the [Ca2+]i signal by mIg-Fc gamma R cross-linkage is proceeded by a corresponding Fc gamma-mediated reduction in anti-Ig-induced inositol trisphosphate elevation. These findings outline a biochemical basis for mIg- and Fc gamma R-mediated activation and regulation intrinsic to the B cell, and demonstrate the advantages of indo-1 over quin2 for fluorescent measurement of [Ca2+]i in small cells.