Gillian Grafton

Calcium channels in lymphocytes

On B and T lymphocytes, ligation of the antigen receptor (AgR) induces a biphasic Ca2+ response. In the initial phase there is a large elevation in the intracellular Ca2+ concentration as a consequence of Ca2+ release from intracellular stores. This is followed by a lower, but prolonged elevation that is dependent on extracellular Ca2+.1,2 This simple description belies the complexity of the response. The initial phase may involve as many as three different intracellular Ca2+ channels, while the second phase depends not only on plasma membrane Ca2+ channels, but also on at least two different intracellular channels. The complexity of the signal, and the many opportunities for regulation of individual components of the signalling mechanism, lead to a tremendous flexibility in outcome, ranging from single, brief elevated Ca2+ transients, through a range of oscillatory responses, each of which can be decoded by the cell into a differing outcome.2 In this review we concentrate on the Ca2+ channels involved in the AgR-mediated Ca2+ signal, but we briefly discuss other Ca2+ channels present in lymphocytes. Figure 1 shows two possible schemes for the involvement of Ca2+ channels in TCR signalling, and Fig. 2 shows possible roles for Ca2+ channels in B cells. Figure 1 A possible scheme for the involvement of Ca2+ channels in TCR signalling (a) depicts the simplest possible scheme for the role of Ca2+ channels in TCR-induced Ca2+ signalling. TCR-induced Ins(1,4,5)P3 production causes Ca2+ release from intracellular ... Figure 2 Possible roles for Ca2+ channels in B cells. The BCR-induced Ca2+ signal involves the production of Ins(1,4,5)P3 and the release of Ca2+ from intracellular stores gated by InsP3Rs and RyR1. This is followed by an influx of Ca2+ through an unidentified ... INTRACELLULAR Ca2+ CHANNELS A plethora of studies of AgR signalling have highlighted the role of inositol trisphosphate [Ins(1,4,5)P3]-mediated release of Ca2+ from internal stores (reviewed in refs 1–3). However, it is becoming apparent that there is more to the regulated release of intracellular Ca2+ in lymphocytes than inositol trisphosphate receptors (InsP3Rs). Recent studies are beginning to unravel roles for ryanodine receptors (RyRs) and the newly described and little understood NAADP receptor. Inositol trisphosphate receptors Three types of InsP3R are known, and they vary in their sensitivities to Ins(1,4,5)P3 and in the properties of their activation by Ca2+. InsP3Rs must bind Ins(1,4,5)P3 for Ca2+ release to occur. The response of the InsP3R can be regulated by phosphorylation, by various accessory proteins and by ATP, but by far the most important regulator is Ca2+. The exact mechanism is disputed4–6 but it is apparent that the differing sensitivities of the InsP3R isoforms to regulation by Ca2+ allow cells to fine-tune the temporal and spatial aspects of the Ca2+ signal.5 Much recent work has been directed towards determining the roles of the various isoforms. B and T cells express all three types of InsP3R to varying degrees depending on their stage of differentiation.7–11 It is not clear why lymphocytes simultaneously express all three isoforms, particularly since Sugawara et al.8 clearly demonstrated the redundancy of InsP3R expression. In a series of knockouts in DT40 B lymphocytes the BCR-induced Ca2+ signal could not be ablated until all three InsP3R isoforms were simultaneously knocked out.8 InsP3R3 was the first isoform to have a specific role ascribed to it. In both T and B cells, InsP3R3 is up-regulated in cells undergoing apoptosis.10 Inhibition of InsP3R3 expression using antisense RNA prevents TCR-induced apoptosis.10 However, Sugawara et al.8 provided convincing evidence against a specific role for InsP3R3 in apoptosis when they showed that knockout of any two isoforms in DT40 cells inhibited BCR-induced apoptosis. InsP3R3 can be expressed on the external surface of the plasma membrane of T and B cells and in T cells it can cocap with the TCR.9,10 The validity of this observation has been questioned, but support was provided by Tanimura et al.12 who demonstrated InsP3R3 expression on the external surface of Jurkat T cells. They showed that this pattern of expression was not limited to InsP3R3, and in fact the predominant isoforms in the plasma membrane were InsP3R1 and InsP3R2.12 The role of plasma membrane InsP3Rs is unknown, but Putney13 has suggested that, in some cell types, InsP3R3 may be expressed as an integral plasma membrane protein and function as all or part of a store-operated Ca2+ channel. However, the properties of InsP3R, in particular its Ca2+ selectivity profile, do not accord with those of known store-operated Ca2+ channels and the prevailing evidence strongly indicates that these channels are formed by distinct molecules from InsP3Rs (discussed below). In an attempt to explore the role of InsP3R1, Jayaraman et al.11 used antisense RNA to show that TCR-induced Ca2+ signals are exclusively transduced through this subtype. However, this is a controversial finding since the construct they used could cross-react with the type 2 and type 3 receptors. Hirota et al.14 subsequently demonstrated that T lymphocytes could develop normally in InsP3R1 knockout mice and showed normal Ca2+ fluxes in response to anti-CD3 stimulation, contradicting the claim that InsP3R1 was absolutely required for TCR signalling. In the most convincing study to ascribe roles for the various isotypes, Miyakawa et al.15 showed that InsP3R3 was involved in the generation of monophasic single Ca2+ transients following BCR ligation, whereas InsP3R1 and InsP3R2 were involved in the generation of Ca2+ oscillations with differing frequencies. The knockouts studied by Sugawara et al.8 suggest that inhibition of downstream events may be achieved simply by reducing the overall levels of InsP3Rs, rather than the specific levels of one particular isotype. This raises the possibility that cells do not express homotetramers of InsP3Rs; rather they may express heterotetramers of varying amounts of each isoform, allowing the cell to express a graded array of hybrid receptors with a wide variety of subtly different properties made up of combinations of the properties of the ‘pure’ homotetramers. Clearly much remains to be done to unravel the role of InsP3Rs in AgR signalling.

Effect of Mg2+ on Na+-Dependent Inositol Transport: Role for Mg2+ in Etiology of Diabetic Complications

Diabetes mellitus is associated with a significant reduction in the serum concentration of Mg2+. Several studies have suggested that hypomagnesemia may be implicated in the etiology of diabetic complications; however, no mechanism has been proposed. This study demonstrates that Mg2+ is a positive effector of inositol transport and is capable of promoting a 2.5-fold increase in the affinity of the transporter for inositol. Analysis of the kinetics of inositol transport shows that, at physiological concentrations of inositol, the reductions in Mg2+ concentrations that occur in diabetic patients would result in a significant decline in the rate of inositol transport (1.5- to 2-fold). We suggest that hypomagnesemia may be linked to the development of diabetic complications via reduction in the rate of inositol transport and subsequent intracellular inositol depletion. This assertion allows hypomagnesemia and the polyol theory to be unified into one mechanistic model for the development of diabetic complications.

Type I cytokine profiles of human naïve and memory B lymphocytes: a potential for memory cells to impact polarization

B cells bifurcating along ‘type 1’ or ‘type 2’ pathways under the influence of polarizing cytokines can, in turn, influence the direction of an immune response. Here, we compare the capacity of human B cells residing within naïve and memory compartments to participate in type 1 polarizing responses. B‐cell receptor (BCR) engagement provided the main signal for interleukin (IL)‐12Rβ1 expression in the two subsets: this was potentiated by CD154 together with interferon‐γ (IFN‐γ) but inhibited by IL‐12. IL‐12Rβ2 could be induced on a minority of B cells by the same signals, and also by IFN‐γ alone. WSX‐1, a receptor for IL‐27, was expressed in both subsets with no evidence for its regulation by the signals studied. While neither subset was capable of secreting much IL‐12 p70, memory B cells could produce a small amount of IL‐12 p40 on CD40 ligation. Memory B cells also, exclusively, expressed IL‐23 p19 mRNA on BCR triggering. Importantly, products of appropriately stimulated memory – but not naive – B cells were shown to promote the synthesis of IFN‐γ in uncommitted T‐helper cells. The data indicate an equal capacity for naïve and memory B cells to respond within a type 1 polarizing environment. Although poorly equipped for initiating type 1 responses, B cells – by virtue of the memory subset – reveal a capacity for their maintenance and amplification following T‐dependent signalling.

Naive and memory B cells respond differentially to T-dependent signaling but display an equal potential for differentiation toward the centroblast-restricted CD77/globotriaosylceramide phenotype.

Resting (CD38low) tonsillar B cells differentiate to express the centroblast‐restricted CD77/globotriaosylceramide antigen on high‐level engagement of CD154. As the CD38low population comprises both naive and memory subsets, we wished to compare the propensity of each to develop this germinal center phenotype; particularly as the capacity of memory B cells to re‐enter afollicular reaction remains unclear. Resting B lymphocytes were therefore separated into CD27–IgA–IgG– and IgD– fractions to generate subsets enriched for naive and memory cells, respectively. Following stimulation via BCR and/or CD40 – surrogate signals for B cells engaged in T‐dependent signaling – differences between the two subsets were seen in the kinetics and/or magnitude of responses such as entry into DNA synthesis, induction of the costimulatory molecules CD80 and CD86; up‐regulation of CD23, and changes in BCL‐6 mRNA expression. Nevertheless, naive and memory cells revealed a nigh identical capacity for acquiring CD77: both appeared equally sensitive in this regard, with high‐level CD40 engagement via cell‐bound CD154 being required for both subsets to achieve the hallmark centroblast phenotype. These findings suggest that, provided with the opportunity to encounter cell membrane CD154 in abundance, both naive and memory B cells display the potential to be diverted towards a germinal center pathway of differentiation.

Changes in the kinetics of inositol transport during TPA-induced differentiation of HL60 cells towards monocytes

When exposed to the phorbol ester TPA, HL60 cells undergo growth arrest and differentiate towards monocytes. During TPA‐induced differentiation there was a 2.6‐fold increase in the rate of inositol transport (V max), a 2.1‐fold increase in intracellular inositol and a 1.5‐fold increase in inositol lipid. An increase in the V max of inositol transport did not occur when the variant cell line HL60Ast3 was exposed to TPA, which has been shown in this cell line to induce growth arrest but not differentiation. This observation suggests that the change in inositol transport during HL60 monocyte differentiation is specifically associated with the process of cell differentiation as opposed to growth arrest.

Novel vitamin D analogues; cytotoxic and anti-proliferative activity against a diffuse large B-cell lymphoma cell line and B-cells from healthy donors

Calcitriol (1,25-dihydroxyvitamin D3, 1,25D3) and vitamin D side-chain modified analogs (VDAs) have gained considerable attention as potential drugs in the treatment of acute myeloid leukemia (AML), yet studies of the impact of 1,25D3 and VDAs upon other haematological malignancies are more limited. To address this gap in knowledge, we have examined the action of 1,25D3 and VDAs on a human cell line (DOHH2, K422) typifying diffuse large B-cell lymphoma (DLBCL) and also peripheral blood B-cells isolated from healthy donors. 1,25D3 and certain VDAs displayed moderate cytotoxic and pro-apoptotic actions upon DLBCL cells. 1,25D3 and VDAs (100nM) caused the death of approximately 40% DOHH2 cells after 24h stimulation, similar to their impact on HL-60 cells (acute myeloid leukaemia cell line). In addition, 1,25D3 and VDAs displayed concentration and time-dependent anti-proliferative actions upon stimulated B-cells from healthy donors. The VDAs inhibited proliferation by approximately 30%. Hence VDAs may offer therapeutic potential for the treatment of DLBCL or conditions benefitted by B-cell depletion.

Hippocampal 5-HT7 receptors signal phosphorylation of the GluA1 subunit to facilitate AMPA receptor mediated-neurotransmission in vitro and in vivo.

The 5‐HT7 receptor is a GPCR that is the target of a broad range of antidepressant and antipsychotic drugs. Various studies have demonstrated an ability of the 5‐HT7 receptor to modulate glutamatergic neurotransmission and cognitive processes although the potential impact upon AMPA receptors has not been investigated directly. The purposes of the present study were to investigate a direct modulation of the GluA1 AMPA receptor subunit and determine how this might influence AMPA receptor function.

Signals for Survival and Apoptosis in Normal and Neoplastic B Lymphocytes

B lymphocytes are subject to selection within germinal centers following somatic hyper-mutation on immunoglobulin variable region genes based on their ability to bind antigen with high affinity. Non-selected cells die by apoptosis. Tumors with features of germinal center B cells include follicular center cell lymphoma and Burkitts lymphoma. We have used the latter extensively as a neoplastic model of germinal center cells and have compared directly the behaviour of cell lines derived from biopsy material with that of the normal counterparts. Here we describe some of our findings in the two systems with regard to signals regulating survival and apoptosis.

5‐Chloroindole: a potent allosteric modulator of the 5‐HT3 receptor

The 5‐HT3 receptor is a ligand‐gated ion channel that is modulated allosterically by various compounds including colchicine, alcohols and volatile anaesthetics. However the positive allosteric modulators (PAMs) identified to date have low affinity, which hinders investigation because of non‐selective effects at pharmacologically active concentrations. The present study identifies 5‐chloroindole (Cl‐indole) as a potent PAM of the 5‐HT3 receptor.

Sodium dependent inositol transport in HL60 cells is not related to Na+/K+, ATPase activity

In HL60 cells, inositol transport is sodium‐dependent but functionally independent of Na+/K+ ATPase activity. This observation has implications for the currently proposed theory for the development of diabetic complications.