Heather L. Wilson

Rapid Secretion of Interleukin-1β by Microvesicle Shedding

The proinflammatory cytokine interleukin-1beta (IL-1beta) is a secreted protein that lacks a signal peptide and does not follow currently known pathways of secretion. Its efficient release from activated immune cells requires a secondary stimulus such as extracellular ATP acting on P2X(7) receptors. We show that human THP-1 monocytes shed microvesicles from their plasma membrane within 2-5 s of activation of P2X(7) receptors. Two minutes after such stimulation, the released microvesicles contained bioactive IL-1beta, which only later appeared in the vesicle-free supernatant. We conclude that microvesicle shedding is a major secretory pathway for rapid IL-1beta release from activated monocytes and may represent a more general mechanism for secretion of similar leaderless secretory proteins.

Proteomic and functional evidence for a P2X7 receptor signalling complex.

P2X receptors are ATP‐gated ion channels in the plasma membrane, but activation of the P2X7 receptor also leads to rapid cytoskeletal re‐arrangements such as membrane blebbing. We identified 11 proteins in human embryonic kidney cells that interact with the rat P2X7 receptor, by affinity purification followed by mass spectroscopy and immunoblotting [laminin α3, integrin β2, β‐actin, α‐actinin, supervillin, MAGuK, three heat shock proteins, phosphatidylinositol 4‐kinase and receptor protein tyrosine phosphatase‐β (RPTPβ)]. Activation of the P2X7 receptor resulted in its dephosphorylation. Whole‐cell recordings from cells expressing P2X7 receptors showed that this markedly reduced subsequent ionic currents and it also slowed membrane bleb formation. By mutagenesis, we identified Tyr343 in the putative second transmembrane domain as the site of phosphorylation. Thus, we have identified a P2X7 receptor signalling complex, some members of which may initiate cytoskeletal rearrangements following receptor activation. Others, such as RPTPβ, might exert feedback control of the channel itself through its dephosphorylation.

Secretion of Intracellular IL-1 Receptor Antagonist (Type 1) Is Dependent on P2X7 Receptor Activation

Inflammatory mechanisms are critical in the arterial response to injury. Both IL-1 and the naturally occurring inhibitor of IL-1, IL-1R antagonist (IL-1ra), are expressed in the arterial wall, and in particular in the endothelium. Previous studies suggest that endothelial cells only make the intracellular type I isoform of IL-1ra (icIL-1ra1), an isoform known to lack a secretory signal peptide. It is unclear how icIL-1ra is released from the endothelial cell to act as an antagonist on cell surface IL-1 type I receptors. IL-1β, which also lacks a secretory signal peptide, may be released by ATP stimulation of the P2X7R. Therefore, we examined whether icIL-1ra1 release occurs in an analogous manner, using both the mouse macrophage cell line RAW264.7 and HUVECs. P2X7R activation caused icIL-1ra1 release from LPS-primed RAW264.7 macrophages and from HUVECs. This release was inhibited in the absence of extracellular calcium, and attenuated by preincubation with oxidized ATP, KN62, and apyrase. Endogenous ATP release, which also facilitated release of icIL-1ra1, was detected during LPS treatment of both RAW264.7 macrophages and HUVECs. Annexin V assays showed that ATP stimulation resulted in a rapid phosphatidylserine (PS) exposure on the cell surface of RAW264.7 macrophages, and that PS-exposed microvesicles contained icIL-1ra1. However, PS flip and microvesicle shedding was not apparent in ATP-treated HUVECs. These data support a general role for the P2X7R in the release of leaderless cytokines into the extracellular medium, and indicate how icIL-1ra1 may act upon its extracellular target, the IL-1R.

Functional properties of heteromeric P2X1/5 receptors expressed in HEK cells and excitatory junction potentials in guinea-pig submucosal arterioles

P2X receptors are ATP-gated cation channels; they form as homomers or heteromers from a family of seven related subunits. In particular, heteromeric channels comprising P2X(2) and P2X(3) subunits, or P2X(1) and P2X(5) subunits, show distinctive physiological and pharmacological properties in heterologous expression systems. There is substantial evidence that one of the native P2X receptors in sensory neurones corresponds to the P2X(2/3) heteromer, but there is no evidence for P2X(1/5) heteromers in native tissue. We recorded currents in response to activation of heteromeric P2X(1/5) receptors expressed in HEK293 cells to characterize further their functional properties. The ATP concentration-response curve had a threshold concentration of 1 nM, and a Hill slope of one. TNP-ATP was a weak partial agonist, and a non-competitive antagonist which inhibited maximal ATP currents by 60%. Increasing or decreasing pH from 7.3 shifted the ATP concentration-response curves to the right by fivefold and decreased the maximum current by 40%. Calcium permeability was lower than that observed for other P2X receptors (P(Ca)/P(Na) ratio=1.1). The nanomolar sensitivity of this receptor revealed a steady release of ATP from HEK293 cells, providing an extracellular concentration which ranged from 3 to 300 nM. Noradrenaline (0.3-30 microM) increased ATP-evoked currents by 35%; this facilitation occurred within 20 ms. We also recorded excitatory junction potentials (EJPs) from guinea-pig submucosal arterioles. EJPs were inhibited by suramin and PPADS (IC(50)s of 0.2 microM and 20 microM) but TNP-ATP (0.1-10 microM) inhibited EJPs by <30%. Noradrenaline (0.3-30 microM in the presence of phentolamine and propranolol) decreased EJPs in control preparations but facilitated EJPs by 5-20% in submucosal arterioles from reserpinized guinea-pigs. These properties are discussed in relation to P2X receptors underlying EJPs at autonomic neuroeffector junctions.

Characterization of a 5‐HT1B receptor on CHO cells: functional responses in the absence of radioligand binding

1 Chinese hamster ovary (CHO) cells have been reported to be devoid of 5‐HT receptors and have frequently been used as hosts for the expression of cloned 5‐HT receptors. Unexpectedly, 5‐HT was found to induce profound inhibition of forskolin‐stimulated cyclic AMP production in these cells and the aim of this study was to classify the 5‐HT receptor involved. 1 In CHO(dhfr‐) cells 5‐HT was a potent agonist and caused 80–100% inhibition of forskolin stimulated cyclic AMP production. A study using several 5‐HT1 receptor agonists revealed the following potencies (p[A50]): RU24969 (9.09±0.17) > 5‐carboxamidotryptamine (8.86±0.20) > 5‐HT (8.07±0.05) > CP‐93,129 (7.74±0.10) > sumatriptan (5.93±0.04). All five agonists achieved a similar maximum effect. Irreversible receptor alkylation studies yielded a pKA estimate of 7.04±0.34 for 5‐HT. 3 The 5‐HT1A/1B antagonist, (±)‐cyanopindolol (4–100 nM), caused parallel rightward shifts of the 5‐HT concentration‐effect curve with no change in asymptote. Schild analysis yielded a pKB estimate of 8.69±0.09 (Schild slope 1.13±0.10). (±)‐Cyanopindolol actually behaved as a partial agonist with an intrinsic activity of 0.2‐0.5 and a p[A50] of 8.55. 4 5‐HT (0.01–10 μm) also elicited a concentration‐dependent increase in intracellular [Ca2+] in CHO(dhfr‐) cells thus demonstrating that dual coupling is not a phenomenon restricted to systems in which there is overexpression of transfected receptors. 5 This agonist and antagonist profile is consistent with the presence of a 5‐HT1B receptor. 8‐OH‐DPAT (1 μm) and renzapride (3 μm) were without effect on forskolin‐stimulated cyclic AMP production and ketanserin (0.3 μm) did not antagonize the inhibition produced by 5‐HT, thus excluding the involvement of 5‐HT1A, 5‐HT4, and 5‐HT2 receptors. 6 The possibility that expression of a 5‐HT1B receptor was associated with the dhfr‐ mutation was excluded since RU24969, 5‐HT and CP‐93,129 were also potent agonists in unmutated, CHO‐K1 cells: p[A50] 9.03±0.03, 8.34±0.05, 7.69±0.07 respectively, and (±)‐cyanopindolol (0.1 μm) shifted the 5‐HT curve to the right and yielded a pA2 estimate of 8.70±0.06. 7 Little or no specific binding of [3H]‐5‐HT (0.1–200 nM) or of the high affinity ligand [125I]‐iodocyanopindolol (0.01–3 nM) to CHO(dhfr‐) cell membranes could be detected. 5‐HT also failed to elicit any increase in the binding of [35S]‐GTPγS to CHO membranes. 8 In conclusion, cultured CHO cells express 5‐HT1B receptors which are negatively coupled to adenylyl cyclase and positively coupled to increases in intracellular calcium. The absence of radioligand binding was unexpected in view of the high potency of 5‐HT and the partial agonist activity of the normally ‘silent’ competitive antagonist, (±)‐cyanopindolol. This implies very efficient receptor‐effector coupling of a low density of 5‐HT1B receptors. Clearly, the absence of detectable radioligand binding cannot be assumed to mean the absence of receptors capable of eliciting a significant functional response.

P2X receptor characterization and IL‐1/IL‐1Ra release from human endothelial cells

The pro‐inflammatory cytokine, interleukin‐1β (IL‐1β), has been implicated in the pathogenesis of atherosclerosis, potentially via its release from vascular endothelium. Endothelial cells (EC) synthesize IL‐1β in response to inflammatory stimuli, but the demonstration and mechanism of release of IL‐1 from ECs remains unclear. In activated monocytes, efficient release of bioactive IL‐1β occurred via activation of ATP‐gated P2X7 receptors (P2X7Rs). Activation of P2X7R in ECs from human umbilical vein (HUVECs) released IL‐1 receptor antagonist (IL‐1Ra). The purpose of this study was to provide a quantitative investigation of P2XR expression and function, in parallel with IL‐1β and IL‐1Ra synthesis, processing and release, in HUVECs under pro‐inflammatory conditions.

Temporal Interleukin-1β Secretion from Primary Human Peripheral Blood Monocytes by P2X7-independent and P2X7-dependent Mechanisms

The processing and regulated secretion of IL-1β are critical points of control of the biological activity of this important pro-inflammatory cytokine. IL-1β is produced by both monocytes and macrophages, but the rate and mechanism of release differ according to the differentiation status and the origin of these cells. We aimed to study the control of processing and release in human blood monocytes and human monocyte-derived macrophages. Toll-like receptor (TLR)-induced IL-1β production and release were investigated for dependence upon caspase-1, P2X7 receptor activation, and loss of membrane asymmetry associated with microvesicle shedding. TLR agonists induced P2X7 receptor-dependent IL-1β release in both monocytes and macrophages; however, only monocytes also showed P2X7 receptor-independent release of mature IL-1β. Furthermore, in monocytes ATP-mediated PS exposure could be activated independently of IL-1β production. Release of IL-1β from monocytes showed selectivity for specific TLR agonists and was accelerated by P2X7 receptor activation. Human monocytes released more IL-1β/cell than macrophages. These data have important implications for inflammatory diseases that involve monocyte activation and IL-1 release.

Translational Mini-Review Series on Immunology of Vascular Disease: Inflammation, infections and Toll-like receptors in cardiovascular disease

Cardiovascular disease, in which atherosclerosis is the major underlying cause, is currently the largest cause of death in the world. Atherosclerosis is an inflammatory disease characterized by the formation of arterial lesions over a period of several decades at sites of endothelial cell dysfunction. These lesions are composed of endothelial cells, vascular smooth muscle cells, monocytes/macrophages and T lymphocytes (CD4+). As the lesions progress some can become unstable and prone to disruption, resulting in thrombus formation and possibly a myocardial infarction or stroke depending upon the location. Although the exact triggers for plaque disruption remain unknown, much recent evidence has shown a link between the incidence of myocardial infarction and stroke and a recent respiratory tract infection. Interestingly, many reports have also shown a link between a family of pattern recognition receptors, the Toll‐like receptors, and the progression of atherosclerosis, suggesting that infections may play a role in both the progression of atherosclerosis and in inducing the more severe complications associated with the disease.

Zebrafish tissue injury causes upregulation of interleukin-1 and caspase-dependent amplification of the inflammatory response

ABSTRACT Interleukin-1 (IL-1), the ‘gatekeeper’ of inflammation, is the apical cytokine in a signalling cascade that drives the early response to injury or infection. Expression, processing and secretion of IL-1 are tightly controlled, and dysregulated IL-1 signalling has been implicated in a number of pathologies ranging from atherosclerosis to complications of infection. Our understanding of these processes comes from in vitro monocytic cell culture models as lines or primary isolates, in which a range and spectra of IL-1 secretion mechanisms have been described. We therefore investigated whether zebrafish embryos provide a suitable in vivo model for studying IL-1-mediated inflammation. Structurally, zebrafish IL-1β shares a β-sheet-rich trefoil structure with its human counterpart. Functionally, leukocyte expression of IL-1β was detectable only following injury, which activated leukocytes throughout zebrafish embryos. Migration of macrophages and neutrophils was attenuated by inhibitors of either caspase-1 or P2X7, which similarly inhibited the activation of NF-κB at the site of injury. Zebrafish offer a new and versatile model to study the IL-1β pathway in inflammatory disease and should offer unique insights into IL-1 biology in vivo.

Cyclic ADP-ribose and the regulation of calcium-induced calcium release in eggs and cardiac myocytes.

Cyclic ADP-ribose (cADPR) is a cyclic metabolite of NAD+ synthesised in cells and tissues expressing ADP-ribosyl cyclases. Although it was first discovered in sea-urchin egg extracts as a potent calcium mobilizing agent, subsequent studies have indicated that it may have a widespread action in the activation of calcium-release channels in such diverse systems as mammalian neurones, myocytes, blood cells, eggs, and plant microsomes. In this review we focus on recent work suggesting that cADPR enhances the sensitivity of ryanodine-sensitive calcium-release channels (RyRs) to activation by calcium, a phenomenon termed calcium-induced calcium release (CICR). Two roles for cADPR in calcium signaling are discussed. The first is as a classical second messenger where its levels are controlled by extracellular stimuli, and the second mode of cellular regulation is that the levels of intracellular cADPR may set the sensitivity of RyRs to activation by an influx of calcium in excitable cells. These two possible actions of cADPR are illustrated by considering the signal transduction events during the fertilization of the sea-urchin egg and the modulation of CICR during excitation-coupling in isolated guinea-pig ventricular myocytes, respectively.