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Serotonin (5-HT), a well-known neurotransmitter of the central nervous system, has been implicated in diverse aspects of immune regulation. Here we show that 5-HT can efficiently drive programmed cell death in established Burkitt lymphoma (BL) lines that remain faithful to the original biopsy phenotype (group 1). Group 1 BL cells cultured in the presence of 5-HT exhibited marked suppression of DNA synthesis that was accompanied by extensive apoptosis-serotonindriven apoptosis was complete within 24 hours, was preceded by early caspase activation, and was accompanied by a decline in mitochondrial membrane potential. BL cells that had drifted to a lymphoblastic group 3 phenotype were relatively resistant to these actions of serotonin, and the forced ectopic expression of either bcl-2 or bcl-x L provided substantial protection from 5-HT-induced apoptosis. 5-HT receptor antagonists (SDZ205-557 IntroductionOutside the central nervous system, serotonin (5-HT) is produced mainly by enterochromaffin cells of the gut and is taken up by an active transport mechanism to a number of cell types, with platelets providing the richest reservoir of 5-HT in the periphery. 1 Release of platelet-stored 5-HT can be rapid and triggered, for example, by platelet-activating factor, thrombin, C3a, C5a, and immunoglobulin (Ig)E-containing immune complexes. Thus, at sites of inflammation and platelet activation, local concentrations of 5-HT could be expected to greatly exceed the relatively low amounts normally found in serum. [2][3][4] Moreover, primary and secondary lymphoid organs are innervated with noradrenergic nerve fibers that have the potential to accumulate serotonin and to release it on stimulation. Because the nerve terminals are in close contact with, for example, lymphocytes at these sites, this indicates that immune cells can be exposed directly to 5-HT flow. 5 There are numerous reports of 5-HT modulating natural killer cell, macrophage, and T-cell function [reviewed in 5 ]. Of the 14 known receptor subtypes for 5-HT, mRNA for 8 of these was recently demonstrated in rat immune tissues. 6,7 Particularly for the promotion of T-cell proliferation, a major target for serotonin action appears to be the 5-HT 1A receptor, a property conserved between mammals and fish. 8,9 B lymphocytes also carry this receptor subtype (among others) and, as with T cells, display NF-B-dependent up-regulation of mRNA and protein for the 5-HT 1A receptor. 10 At least for rodent splenic B cells, this receptor subtype seems to be involved in the 5-HT-promoted augmentation of mitogenic responses to lipopolysaccharide and dextran sulfate. 11 B cells additionally express the serotonin transporter (SERT) with Epstein-Barr virus (EBV)-transformed B-cell lines providing an important resource for genotyping polymorphisms in the promoter region of the transporter. 1,12 However, no function has as yet been ascribed to B-cell SERT.Monoamines, including 5-HT, have been reported to induce apoptosis in cultured neuronal cells, and cerebellar granule neurons are p...

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Population size is governed through cells reacting to a variety of intrinsic and extrinsic cues. Tumors, while liberated from many of the homeostatic constraints placed on physiologic counterparts, can nonetheless remain subject to both social and environmental control. Burkitt lymphoma cells faithful to the biopsy phenotype were used to model the reliance of the colony, if any, on an inbuilt population sensor. Below a normally suicidal threshold number of cells, low picomolar quantities of exogenous CD40 ligand (CD40L/CD154) were found to sustain the clone without the discernible shift in phenotype that accompanies high CD40L encounter. Although CD154 was undetectable in populous cultures, message was induced as numbers became limiting. Correspondingly, attempts to neutralize endogenous CD40L activity failed to perturb cells at optimal densities but resulted in their marked decline as the critical threshold was approached. These data reveal an auto-inducible survival mechanism seemingly regulated through the monitor- IntroductionAlthough progressive liberation from homeostatic control is a feature of tumor development, malignant cells are rarely emancipated completely from the multitude of environmental constraints that are placed on normal counterparts. 1 Indeed, before any progression toward metastasis, a dependency on specific tissue microenvironment is often observed in malignancy. For example, follicular lymphoma, one of the most common nonleukemic lymphoid tumors, develops within and is primarily limited to the lymphoid follicles that harbor their normal equivalents, the germinal center (GC) B cells. [2][3][4][5][6] Another tumor bearing a GC B-cell phenotype is Burkitt lymphoma (BL), although here development is not constrained to conventional follicles. [7][8][9][10] Nevertheless, BL presents with a classical "starry sky" histology reflecting the presence of large infiltrating macrophages that are there to scavenge tumor cells entering apoptosis. 9,11,12 This feature of spontaneous apoptosis is a vestige of the tumor's GC origin in which the constitutive B cells need to be selected by antigen during the development of a memory response. 6,13,14 Unlike their normal equivalents, however, the maintenance of the BL population proceeds independently of discernible antigen input and, despite their residual propensity for apoptosis, the balance is clearly in favor of survival.BL cells established in culture and kept at early passage remain remarkably faithful to their GC origins. 7,12,15,16 The L3055 cell line, derived from an Epstein-Barr virus (EBV)-negative case of sporadic BL, has been used extensively to model both GC B cells and BL itself. [17][18][19][20][21][22][23][24] These "biopsylike" cultures display a low background of apoptosis that can be accelerated by a number of factors, including B-cell receptor engagement, exposure to transforming growth factor ␤, cold shock, and serum deprivation. [17][18][19][20]24 With regards to survival factors, roles for both autocrine and paracrine factors have bee...

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In T lymphocytes, engagement of the antigen receptor leads to a biphasic Ca 2؉ flux consisting of a mobilization of Ca 2؉ from intracellular stores followed by a lower but sustained elevation that is dependent on extracellular Ca 2؉ . The prolonged Ca 2؉ flux is required for activation of transcription factors and for subsequent activation of the T cell. Ca 2؉ influx requires as yet unidentified Ca 2؉ channels, which potentially play a role in T cell activation. Here we present evidence that human T cells express a non-voltage-gated Ca 2؉ channel related to L-type voltage-gated Ca 2؉ channels. Drugs that block classical L-type channels inhibited the initial phase of the antigen receptor-induced Ca 2؉ flux and could also inhibit the sustained phase of the Ca 2؉ signal suggesting a role for the L-type Ca 2؉ channel in antigen receptor signaling. T cells expressed transcripts for the ␣ 1 1.2 and ␣ 1 1.3 pore-forming subunits of L-type voltage-gated Ca 2؉ channels and transcripts for all four known ␤-subunits including several potential new splice variants. Jurkat T leukemia cells expressed a small amount of full-length ␣ 1 1.2 protein but the dominant form was a truncated protein identical in size to a truncated ␣ 1 1.2 protein known to be expressed in B lymphocytes. They further expressed a truncated form of the ␣ 1 1.3 subunit and auxiliary ␤1-and ␤3-subunit proteins. Our data strongly suggest that functional but non-voltage-gated L-type Ca 2؉ channels are expressed at the plasma membrane in T cells and play a role in the antigen receptor-mediated Ca 2؉ flux in these cells.

SUMMARYWhether CD5 on B cells marks a subset functionally distinct from the conventional CD5 negative (CD5 neg ) adult population or is more an indicator of activation, remains contentious. Here we have investigated whether CD5 positive (CD5 pos ) and CD5 neg B cells can be distinguished in terms of their response to surrogate signals aimed to model, in vitro, T-cell dependent (TD) and Tindependent (TI) encounters with antigen in vivo: the predominantly CD5 pos B-cell population found in cord blood, CD5 B cells positively selected from tonsils and their CD5 neg counterparts, were compared. Neonatal B cells displayed a near-identical phenotype to that of adult CD5 pos B cells, being characterized by uniform immunoglobulin M (IgM), immunoglobulin D (IgD), CD23 and CD44 coexpression. When cultured with anti-IgM maintained at high density on CD32-tranfected mouse L cells to model TI responses or on CD40 ligand (CD40L)-bearing L cells (with or without captured anti-IgM) to model TD encounters, DNA synthesis was stimulated to a similar extent in all three populations. Focusing on CD5 and CD23, we found that ± although the signals delivered promoted distinct pro®les of expression ± under each condition of activation, the phenotypes that emerged for adult CD5 pos and CD5 neg B cells were remarkably similar. Neonatal B cells displayed a greater diminution in CD5 expression than adult CD5 pos B cells following CD40 signals but otherwise the two populations again behaved similarly. The inclusion of interleukin-4 (IL-4) to cultures where cells were costimulated via surface (s)IgM and CD40 resulted in a complete loss of CD5 expression and a corresponding hyperexpression of CD23, irrespective of the population studied. The near-identical response of CD5 pos and CD5 neg B cells to surrogate TD or TI signals in vitro and their convergence to indistinguishable phenotypes is wholly supportive of CD5 being a¯uctuating marker of activation rather than it delineating functionally distinct subsets.

Resting (CD38 low ) tonsillar B cells differentiate to express the centroblast-restricted CD77/ globotriaosylceramide antigen on high-level engagement of CD154. As the CD38 low 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 a follicular 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.