Michelle J. Holder

Close encounters of the monoamine kind: immune cells betray their nervous disposition

Here we review the evidence for immune cells expressing multiple components of the serotonergic and dopaminergic systems that are more commonly associated with the central nervous system (CNS). We discuss where and how peripheral encounters with these biogenic monoamines occur and posit reasons as to why the immune system would wish to deploy these pathways. A full taxonomy of serotonergic and dopaminergic constituents and their workings in component cells of the immune system should facilitate the formulation of novel therapeutic approaches in diseases characterized by immune dysfunction and potentially provide a range of surrogate peripheral markers for registering and monitoring disturbances within the CNS.

The serotonin transporter (SLC6A4) is present in B-cell clones of diverse malignant origin: probing a potential antitumor target for psychotropics

Following our previous description of the serotonin transporter (SERT) acting as a conduit to 5‐hydroxytryptamine (5‐HT)‐mediated apoptosis, specifically in Burkitts lymphoma, we now detail its expression among a broad spectrum of B cell malignancy, while exploring additional SERT substrates for potential therapeutic activity. SERT was readily detected in derived B cell lines with origins as diverse as B cell precursor acute lymphoblastic leukemia, mantle cell lymphoma, diffuse large B cell lymphoma, and multiple myeloma. Concentration and timecourse kinetics for the antiproliferative and proapoptotic activities of the amphetamine derivatives fenfluramine (an appetite suppressant) and 3,4‐methylenedioxymethamphetamine (MDMA; “Ecstasy”) revealed them as being similar to the endogenous indoleamine. A tricyclic antidepressant, clomipramine, instead mirrored the behavior of the selective serotonin reuptake inhibitor fluoxetine, both being effective in the low micromolar range. A majority of neoplastic clones were sensitive to one or more of the serotonergic compounds. Dysregulated bcl‐2 expression, either by t(14;18)(q32;q21) translocation or its introduction as a constitutively active transgene, provided protection from proapoptotic but not antiproliferative outcomes. These data indicate a potential for SERT as a novel anti‐tumor target for amphetamine analogs, while evidence is presented that the seemingly more promising antidepressants are likely impacting malignant B cells independently of the transporter itself.

Inflammation and Regeneration in the Dentin-Pulp Complex: A Double-edged Sword

Dental tissue infection and disease result in acute and chronic activation of the innate immune response, which is mediated by molecular and cellular signaling. Different cell types within the dentin-pulp complex are able to detect invading bacteria at all stages of the infection. Indeed, at relatively early disease stages, odontoblasts will respond to bacterial components, and as the disease progresses, core pulpal cells including fibroblasts, stems cells, endothelial cells, and immune cells will become involved. Pattern recognition receptors, such as Toll-like receptors expressed on these cell types, are responsible for detecting bacterial components, and their ligand binding leads to the activation of the nuclear factor-kappa B and p38 mitogen-activated protein (MAP) kinase intracellular signaling cascades. Subsequent nuclear translocation of the transcription factor subunits from these pathways will lead to proinflammatory mediator expression, including increases in cytokines and chemokines, which trigger host cellular defense mechanisms. The complex molecular signaling will result in the recruitment of immune system cells targeted at combating the invading microbes; however, the trafficking and antibacterial activity of these cells can lead to collateral tissue damage. Recent evidence suggests that if inflammation is resolved relatively low levels of proinflammatory mediators may promote tissue repair, whereas if chronic inflammation ensues repair mechanisms become inhibited. Thus, the effects of mediators are temporal context dependent. Although containment and removal of the infection are keys to enable dental tissue repair, it is feasible that the development of anti-inflammatory and immunomodulatory approaches, based on molecular, epigenetic, and photobiomodulatory technologies, may also be beneficial for future endodontic treatments.

The extrafollicular-to-follicular transition of human B lymphocytes: induction of functional globotriaosylceramide (CD77) on high threshold occupancy of CD40

Amongst lymphocytes, expression of CD77 (globotriaosylceramide, Gb3) is exclusive to B cells of the germinal center (GC). Its acquisition by extrafollicular B cells may thus herald their commitment to a follicular response. Here we show that high threshold occupancy of CD40 by its cognate ligand (CD40L) promotes rapid induction of CD77 expression in non‐GC (CD38lo) B cells. The kinetics of CD77 acquisition mirrored those of GC‐related markers CD95 and CD86 but contrasted with the more delayed increase in CD38 expression. Induction of CD77 was not a simple consequence of cell cycle entry: other conditions of stimulation equally capable of driving proliferation failed to promote CD77 expression. CD77 was functional in that cells were now sensitive to Verotoxin‐1, an Escherichia coli‐derived ligand of Gb3. These data indicate that acquisition by extrafollicular B cells of CD77 results from high threshold occupancy of CD40, a situation that should be reached physiologically only once a critical level of T cell priming has been achieved.

Effects of Red Light-emitting Diode Irradiation on Dental Pulp Cells:

Light irradiation activates a range of cellular processes in a variety of cell types, including stem cells, and can promote tissue repair. This study investigated the effects of light-emitting diode (LED) exposure on dental pulp cells (DPCs). Dose response analysis at 20-second intervals up to 120 seconds demonstrated that a LED array emitting 653-nm red light stimulated significantly increased cell growth at 3 and 7 days post-irradiation with 40 (149 mJ/cm2) and 60 (224 mJ/cm2) seconds of radiant exposure. Double-dosing cells at days 1 and 4 of a 7-day culture period with 60-second (224 mJ/cm2) LED exposure significantly increased cell growth compared with a single dosing regime. BrdU analysis demonstrated significantly increased proliferation rates associated with significantly increased ATP, nitric oxide (NO), and mitochondrial metabolic activity. LED-stimulated NO levels were not reduced by inhibition of NO-synthase activity. Light exposure also rescued the inhibition of mitochondrial dysfunction and increased levels of in vitro mineralization compared with control. Media exchange experiments indicated that autocrine signaling was not likely responsible for red-light-induced DPC activity. In conclusion, data analysis indicated that 653-nm LED irradiation promoted DPC responses relevant to tissue repair, and this is likely mediated by increased mitochondrial activity.

Dopamine targets cycling B cells independent of receptors/transporter for oxidative attack: Implications for non-Hodgkin’s lymphoma

Human B lymphocytes and derived lines from a spectrum of B cell malignancy were studied for expression of dopaminergic pathway components and for their cytostatic response to the catecholamine and related, potentially therapeutic compounds. Proliferating normal lymphocytes and dividing malignant clones rapidly arrested on exposure to dopamine in the low (≤10 μM) micromolar range. The antiparkinsonian drugs l-DOPA and apomorphine (particularly) were similarly antiproliferative. With the exception of D4, dopamine receptors D1–D5 were variably expressed among normal and neoplastic B cell populations, as was the dopamine transporter. Transcripts for D1 and D2 were frequently found, whereas D3 and D5 revealed restricted expression; dopamine transporter was detected in most cases. Nevertheless, pharmacological analysis disclosed that dopamine targeted cycling B cells independent of these structures. Rather, oxidative stress constituted the primary mechanism: the catecholamine’s actions being mimicked by hydrogen peroxide and reversed by exogenous catalase, and evidence for the intracellular redox protein thioredoxin contributing protection. Among proliferating clones, growth arrest was accompanied by cell death in populations deplete in antiapoptotic Bcl-2: resting lymphocytes escaping low micromolar dopamine toxicity. Dysregulated bcl-2 expression, although preventing oxidative-induced caspase-dependent apoptosis, by itself conferred only minor protection against dopamine cytostasis. The selective impact of dopamine on lymphocytes that are in active cycle indicates an axis for therapeutic intervention not only in B cell neoplasia but also in lymphoproliferative disturbances generally. Rational tailoring of drug delivery systems already in development for Parkinson’s disease could provide ideal vehicles for carrying the oxidative hit directly to the target populations.

Characterisation of the endogenous human peripheral serotonin transporter SLC6A4 reveals surface expression without N-glycosylation

RT-PCR confirmed that human cell lines of diverse peripheral origins express transcripts for the serotonin transporter (sert/slc6a4). Molecular weights reported for the translated protein appear to be quite variable however. Here we compared directly immunoreactive protein generated from cloned sert transfected into HEK293 with that carried endogenously among the cell lines. The dominant glycosylated 85-95 kDa immunoreactive species contained in HEK-sert transfectants was poorly represented in any native cell: instead, discrete 70 and 60 kDa bands were universally detected. Biotinylation of lymphoid cells revealed that the endogenous non-glycosylated 60 kDa but not 70 kDa protein was available at the surface to access exogenous ligands.

SLC6A4 expression and anti-proliferative responses to serotonin transporter ligands chlomipramine and fluoxetine in primary B-cell malignancies

B-cell lines of diverse neoplastic origin express the serotonin transporter (SERT/SLC6A4) and growth arrest in response to SERT-ligands, including the antidepressants chlomipramine and fluoxetine. Here we detail SLC6A4 transcript (Q-PCR) and protein (FACS) expression in primary cells from patients with: chronic lymphocytic leukaemia; mantle cell lymphoma; follicular lymphoma; Burkitts lymphoma; and diffuse large B-cell lymphoma. The ability of the SERT-binding antidepressants to impact the growth of these cells when sustained on CD154-transfected fibroblasts was also determined. The results reveal a broad spectrum of primary B-cell malignancies expressing SLC6A4 with a proportion additionally displaying growth arrest on SERT-ligand exposure.

Enhancing the anti-lymphoma potential of 3,4-methylenedioxymethamphetamine (‘ecstasy’) through iterative chemical redesign: mechanisms and pathways to cell death

SummaryWhile 3,4-methylenedioxymethamphetamine (MDMA/‘ecstasy’) is cytostatic towards lymphoma cells in vitro, the concentrations required militate against its translation directly to a therapeutic in vivo. The possibility of ‘redesigning the designer drug’, separating desired anti-lymphoma activity from unwanted psychoactivity and neurotoxicity, was therefore mooted. From an initial analysis of MDMA analogues synthesized with a modified α-substituent, it was found that incorporating a phenyl group increased potency against sensitive, Bcl-2-deplete, Burkitt’s lymphoma (BL) cells 10-fold relative to MDMA. From this lead, related analogs were synthesized with the ‘best’ compounds (containing 1- and 2-naphthyl and para-biphenyl substituents) some 100-fold more potent than MDMA versus the BL target. When assessed against derived lines from a diversity of B-cell tumors MDMA analogues were seen to impact the broad spectrum of malignancy. Expressing a BCL2 transgene in BL cells afforded only scant protection against the analogues and across the malignancies no significant correlation between constitutive Bcl-2 levels and sensitivity to compounds was observed. Bcl-2-deplete cells displayed hallmarks of apoptotic death in response to the analogues while BCL2 overexpressing equivalents died in a caspase-3-independent manner. Despite lymphoma cells expressing monoamine transporters, their pharmacological blockade failed to reverse the anti-lymphoma actions of the analogues studied. Neither did reactive oxygen species account for ensuing cell death. Enhanced cytotoxic performance did however track with predicted lipophilicity amongst the designed compounds. In conclusion, MDMA analogues have been discovered with enhanced cytotoxic efficacy against lymphoma subtypes amongst which high-level Bcl-2—often a barrier to drug performance for this indication—fails to protect.

Stimulation of tyrosine phosphorylation without inositol lipid hydrolysis in human B lymphocytes on engaging CD72.

Occupancy of CD72 on resting tonsillar B cells by monoclonal antibodies (mAb) promotes entry into the G1 phase of the cell cycle with an accompanying increase in MHC Class II expression and provides a co‐stimulus to immobilized anti‐μ for driving DNA synthesis. We now report that engagement of CD72 by mAb stimulates tyrosine phosphorylation in B cells with a peak of activity seen at 5–10 min. Two major substrates of 29 and 57 kDa showed a basal level of phosphorylation which increased with time, while a 40 kDa protein and several other minor components were phosphorylated de novo on the addition of mAb to CD72. Inositol lipid hydrolysis was found to be unperturbed, although a shallow rise in the basal level of intracellular free Ca2+ was provoked on engaging CD72. Receptor cross‐linking was not a requirement for signaling human B cells through CD72: simple occupancy by univalent antibody was sufficient both to trigger the rise in basal [Ca2+]i and to promote DNA synthesis.