ATP-induced Release Of IL-1β Research Articles

P2X7 receptor knockout does not alter renal function or prevent angiotensin II-induced kidney injury in F344 rats

P2X7 receptors mediate immune and endothelial cell responses to extracellular ATP. Acute pharmacological blockade increases renal blood flow and filtration rate, suggesting that receptor activation promotes tonic vasoconstriction. P2X7 expression is increased in kidney disease and blockade/knockout is renoprotective. We generated a P2X7 knockout rat on F344 background, hypothesising enhanced renal blood flow and protection from angiotensin-II-induced renal injury. CRISPR/Cas9 introduced an early stop codon into exon 2 of P2rx7, abolishing P2X7 protein in kidney and reducing P2rx7 mRNA abundance by ~ 60% in bone-marrow derived macrophages. The M1 polarisation response to lipopolysaccharide was unaffected but P2X7 receptor knockout suppressed ATP-induced IL-1β release. In male knockout rats, acetylcholine-induced dilation of the renal artery ex vivo was diminished but not the response to nitroprusside. Renal function in male and female knockout rats was not different from wild-type. Finally, in male rats infused with angiotensin-II for 6 weeks, P2X7 knockout did not reduce albuminuria, tubular injury, renal macrophage accrual, and renal perivascular fibrosis. Contrary to our hypothesis, global P2X7 knockout had no impact on in vivo renal hemodynamics. Our study does not indicate a major role for P2X7 receptor activation in renal vascular injury.

Anti-Inflammatory Lindenane Sesquiterpenoid Dimers from the Roots of Sarcandra glabra.

Sarglaroids A-H (1-8), eight new lindenane dimers, and a monomer sarglaroid I (9), along with fourteen known analogues (10-23), were isolated from the roots of Sarcandra glabra. The planar structures and the absolute configurations were elucidated by HR-MS, NMR, ECD calculations, and X-ray diffraction crystallography. Sarglaroid A (1) was identified as a rare 8,9-seco lindenane dimer with a unique 5/5/5 tricyclic system. The biological evaluation showed that compounds 1 and 13 potently inhibited NO production with IC50 values at 19.8 ± 1.06 and 10.7 ± 0.25 μM, respectively, and had no cytotoxicity to RAW264.7 cells. Compound 6 significantly inhibited the LPS-/ATP-induced IL-1β release by inactivating the NLRP3 inflammasome through inhibiting the initiation and assembly by affecting the K+ efflux. Compounds 2 and 3 inhibited the proliferation of MCF-7 and MDA-MB-231 with IC50 values ranging from 5.4 to 10.2 μM.

Indirect effect of alpha-1-antitrypsin on endotoxin-induced IL-1β secretion from human PBMCs.

Human alpha-1-antitrypsin (AAT) encoded by the SERPINA1 gene, is an acute phase glycoprotein that regulates inflammatory responses via both protease inhibitory and non-inhibitory activities. We previously reported that AAT controls ATP-induced IL-1β release from human mononuclear cells by stimulating the release of small bioactive molecules. In the current study, we aimed to elucidate the identity of these putative effectors released from human PBMCs in response to AAT, which may inhibit the LPS-induced release of IL-1β. We pre-incubated human PBMCs alone or with different preparations of AAT (4 mg/ml) for 30 min at 37°C, 5% CO2, and collected cell supernatants filtered through centrifugal filters (cutoff 3 kDa) to eliminate AAT and other high molecular weight substances. Supernatants passed through the filters were used to culture PBMCs isolated from the autologous or a heterologous donors with or without adding LPS (1 μg/ml) for 6 h. Unexpectedly, supernatants from PBMCs pre-incubated with AAT (Zemaira®), but not with other AAT preparations tested or with oxidized AAT (Zemaira®), lowered the LPS-induced release of IL-1β by about 25%–60% without affecting IL1B mRNA. The reversed-phase liquid chromatography coupled with mass spectrometry did not confirm the hypothesis that small pro-resolving lipid mediators released from PBMCs after exposure to AAT (Zemaira®) are responsible for lowering the LPS-induced IL-1β release. Distinctively from other AAT preparations, AAT (Zemaira®) and supernatants from PBMCs pre-treated with this protein contained high levels of total thiols. In line, mass spectrometry analysis revealed that AAT (Zemaira®) protein contains freer Cys232 than AAT (Prolastin®). Our data show that a free Cys232 in AAT is required for controlling LPS-induced IL-1β release from human PBMCs. Further studies characterizing AAT preparations used to treat patients with inherited AAT deficiency remains of clinical importance.

Comparison of the Anti-inflammatory Properties of Two Nicotinic Acetylcholine Receptor Ligands, Phosphocholine and pCF3-diEPP.

Activation of nicotinic acetylcholine receptors (nAChRs) expressed by innate immune cells can attenuate pro-inflammatory responses. Silent nAChR agonists, which down-modulate inflammation but have little or no ionotropic activity, are of outstanding clinical interest for the prevention and therapy of numerous inflammatory diseases. Here, we compare two silent nAChR agonists, phosphocholine, which is known to interact with nAChR subunits α7, α9, and α10, and pCF3-N,N-diethyl-N′-phenyl-piperazine (pCF3-diEPP), a previously identified α7 nAChR silent agonist, regarding their anti-inflammatory properties and their effects on ionotropic nAChR functions. The lipopolysaccharide (LPS)-induced release of interleukin (IL)-6 by primary murine macrophages was inhibited by pCF3-diEPP, while phosphocholine was ineffective presumably because of instability. In human whole blood cultures pCF3-diEPP inhibited the LPS-induced secretion of IL-6, TNF-α and IL-1β. The ATP-mediated release of IL-1β by LPS-primed human peripheral blood mononuclear leukocytes, monocytic THP-1 cells and THP-1-derived M1-like macrophages was reduced by both phosphocholine and femtomolar concentrations of pCF3-diEPP. These effects were sensitive to mecamylamine and to conopeptides RgIA4 and [V11L; V16D]ArIB, suggesting the involvement of nAChR subunits α7, α9 and/or α10. In two-electrode voltage-clamp measurements pCF3-diEPP functioned as a partial agonist and a strong desensitizer of classical human α9 and α9α10 nAChRs. Interestingly, pCF3-diEPP was more effective as an ionotropic agonist at these nAChRs than at α7 nAChR. In conclusion, phosphocholine and pCF3-diEPP are potent agonists at unconventional nAChRs expressed by monocytic and macrophage-like cells. pCF3-diEPP inhibits the LPS-induced release of pro-inflammatory cytokines, while phosphocholine is ineffective. However, both agonists signal via nAChR subunits α7, α9 and/or α10 to efficiently down-modulate the ATP-induced release of IL-1β. Compared to phosphocholine, pCF3-diEPP is expected to have better pharmacological properties. Thus, low concentrations of pCF3-diEPP may be a therapeutic option for the treatment of inflammatory diseases including trauma-induced sterile inflammation.

Amyloid beta peptide (Aβ 1–42 ) antagonizes nicotinic acetylcholine receptors of monocytes and enables ATP‐mediated release of interleukin‐1β

Amyloid-β peptide (Aβ1-42) plays a pathogenic role in Alzheimer´s disease. Its physiological functions are, however, still debated. Aβ1-42 can induce the secretion of the pro-inflammatory cytokine intereukin-1β (IL-1β) by immune cells. Known interaction partners of Aβ1-42 are α7 nicotinic acetylcholine receptors (nAChRs). Recently, we identified a cholinergic mechanism that inhibits the ATP-associated release of IL-1β by human monocytes via nAChRs containing α7, α9 and/or α10 subunits. Moreover, we discovered novel nAChR agonists that efficiently inhibit monocytic IL-1β release via a metabotropic mechanism. The objective of this study was to test if Aβ1-42 can modulate this cholinergic mechanism. Human monocytic U937 cells and freshly isolated human peripheral blood mononuclear cells (approved by the ethics committee of the medical faculty Giessen, Germany, and performed in accordance with the Helsinki Declaration) were primed with lipopolysaccharide and ATP-induced IL-1β release in the presence or absence nAChR agonists and Aβ1-42 was measured by enzyme-linked immunosorbent assay. We found that Aβ1-42 dose-dependently (IC50= 2.5 µM) antagonized the inhibitory effect of canonical and novel non-canonical nicotinic agonists like phosphocholine (100 µM), C-reactive protein (5 µg/mL) and glycerophosphocholine (10 µM), indicating a novel pro-inflammatory Aβ1-42 function that enables monocytic IL-1β release in the presence of nAChR agonists. In whole-cell patch-clamp and calcium imaging experiments on human P2X7 receptor overexpressing HEK293 cells, no direct effect of Aβ1-42 on P2X7 receptor activity was observed. In blood plasma samples from major surgery patients, IL-1β was mostly detected together with increased Aβ1-42 levels, which is in line with our hypothesis. Taken together, our results suggest that Aβ1-42 can antagonize the cholinergic control of ATP-mediated monocytic IL-1β-release and, thus, provide evidence for a novel function of Aβ1-42 that also might be considered in situations of systemic inflammation.

Commercial α1-antitrypsin preparations markedly differ in their potential to inhibit the ATP-induced release of monocytic interleukin-1β

The acute phase protein α1-antitrypsin (AAT) inhibits numerous proteases, specifically neutrophil elastase. Patients with an AAT deficiency due to mutations frequently develop early onset emphysema. The commercial preparations of human plasma AAT are clinically used as biopharmaceuticals to protect the lung tissue of AAT-deficient patients from damage caused by neutrophil elastase. Accordingly, preparations of AAT are validated for their anti-elastase activity. However, several anti-inflammatory effects of AAT were described, some of them being independent from its anti-protease function. We recently demonstrated that AAT isolated from the blood of healthy persons efficiently inhibits the ATP-induced release of interleukin-1β by human monocytes. This finding is of therapeutic relevance, because IL-1β plays an important role in numerous debilitating and life-threatening inflammatory diseases. As anti-inflammatory functions of AAT are of increasing clinical interest, we compared the potential of two widely used AAT preparations, Prolastin® and Respreeza®, to inhibit the ATP-induced release of IL-1β using human monocytic U937 cells. We detected marked functional differences between both medicaments. The AAT preparation Respreeza® is less active compared to Prolastin® regarding the inhibition of the ATP-induced release of monocytic IL-1β. Chemical oxidation of Respreeza® restored this anti-inflammatory activity, while destroying its anti-protease function. Our data suggest that the anti-inflammatory potential and the anti-protease function of AAT can be fully uncoupled. In the light of the increasing clinical interest in anti-inflammatory functions of AAT, commercial AAT preparations should be carefully reinvestigated and optimized to preserve the dual anti-protease and anti-inflammatory activity of native AAT.

P2X7 receptor inhibition by 2-amino-3-aryl-1,4-naphthoquinones

Extracellular ATP activates purinergic receptors such as P2X7, cationic channels for Ca2+, K+, and Na+. There is robust evidence of the involvement of these receptors in the immune response, so P2X7 receptors (P2X7R) are considered a potential therapeutic target for the development of anti-inflammatory drugs. Although there are many studies of the anti-inflammatory properties of naphthoquinones, these molecules have not yet been explored as P2X7 antagonists. In previous work, our group prepared 3-substituted (halogen or aryl) 2-hydroxy-1,4-naphthoquinones and studied their action on P2X7R. In this paper, eight 2-amino-3-aryl-1,4-naphthoquinones were evaluated to identify the inhibitory activity on P2X7R and the toxicological profile. Three analogues (AD-4CN, AD-4Me, and AD-4F) exhibited reduced toxicity for mammalian cells with CC50 values higher than 500 µM. These three 3-substituted 2-amino-1,4-naphthoquinones inhibited murine P2X7R (mP2X7R) in vitro. However, the analogues AD-4CN and AD-4Me showed low selectivity index values. AD-4F inhibited both mP2X7R and human P2X7R (hP2X7R) with IC50 values of 0.123 and 0.93 µM, respectively. Additionally, this analogue exhibited higher potency than BBG at inhibiting the ATP-induced release of IL-1β in vitro. Carrageenan-induced paw edema in vivo was reversed for AD-4F with an ID50 value of 11.51 ng/kg. Although AD-4F was less potent than previous 3-substituted (halogen or aryl) 2-hydroxy-1,4-naphthoquinones such as AN-04in vitro, this 3-substituted 2-amino-1,4-naphthoquinone revealed higher potency in vivo to reduce the edematogenic response. In silico analysis suggests that the binding site of the novel 2-amino-3-aryl-1,4-naphthoquinone derivatives, including all the tautomeric forms, is located in the pore area of the hP2X7R model. Based on these results, we considered AD-4F to be a satisfactory P2X7R inhibitor. AD-4F might be used as a scaffold structure to design a novel series of inhibitors with potential inhibitory activity on murine (mP2X7R) and human (hP2X7R) P2X7 receptors.

Conopeptides [V11L;V16D]ArIB and RgIA4: Powerful Tools for the Identification of Novel Nicotinic Acetylcholine Receptors in Monocytes.

Venomous marine snails of the genus Conus employ small peptides to capture prey, mainly osteichthyes, mollusks, and worms. A subset of these peptides known as α-conotoxins, are antagonists of nicotinic acetylcholine receptors (nAChRs). These disulfide-rich peptides provide a large number of evolutionarily refined templates that can be used to develop conopeptides that are highly selective for the various nAChR subtypes. Two such conopeptides, namely [V11L;V16D]ArIB and RgIA4, have been engineered to selectively target mammalian α7∗ and α9∗ nAChRs, respectively, and have been used to study the functional roles of these subtypes in immune cells. Unlike in neurons and cochlear hair cells, where α7∗ and α9∗ nAChRs, respectively, function as ligand-gated ion channels, in immune cells ligand-evoked ion currents have not been demonstrated. Instead, different metabotropic functions of α7∗ and α9∗ nAChRs have been described in monocytic cells including the inhibition of ATP-induced ion currents, inflammasome activation, and interleukin-1β (IL-1β) release. In addition to conventional nAChR agonists, diverse compounds containing a phosphocholine group inhibit monocytic IL-1β release and include dipalmitoyl phosphatidylcholine, palmitoyl lysophosphatidylcholine, glycerophosphocholine, phosphocholine, phosphocholine-decorated lipooligosaccharides from Haemophilus influenzae, synthetic phosphocholine-modified bovine serum albumin, and the phosphocholine-binding C-reactive protein. In monocytic cells, the effects of [V11L;V16D]ArIB and RgIA4 suggested that activation of nAChRs containing α9, α7, and/or α10 subunits inhibits ATP-induced IL-1β release. These results have been corroborated utilizing gene-deficient mice and small interfering RNA. Targeted re-engineering of native α-conotoxins has resulted in excellent tools for nAChR research as well as potential therapeutics. ∗indicates possible presence of additional subunits.

Alpha-1 Antitrypsin Inhibits ATP-Mediated Release of Interleukin-1β via CD36 and Nicotinic Acetylcholine Receptors.

While interleukin (IL)-1β is a potent pro-inflammatory cytokine involved in host defense, high levels can cause life-threatening sterile inflammation including systemic inflammatory response syndrome. Hence, the control of IL-1β secretion is of outstanding biomedical importance. In response to a first inflammatory stimulus such as lipopolysaccharide, pro-IL-1β is synthesized as a cytoplasmic inactive pro-form. Extracellular ATP originating from injured cells is a prototypical second signal for inflammasome-dependent maturation and release of IL-1β. The human anti-protease alpha-1 antitrypsin (AAT) and IL-1β regulate each other via mechanisms that are only partially understood. Here, we demonstrate that physiological concentrations of AAT efficiently inhibit ATP-induced release of IL-1β from primary human blood mononuclear cells, monocytic U937 cells, and rat lung tissue, whereas ATP-independent IL-1β release is not impaired. Both, native and oxidized AAT are active, suggesting that the inhibition of IL-1β release is independent of the anti-elastase activity of AAT. Signaling of AAT in monocytic cells involves the lipid scavenger receptor CD36, calcium-independent phospholipase A2β, and the release of a small soluble mediator. This mediator leads to the activation of nicotinic acetylcholine receptors, which efficiently inhibit ATP-induced P2X7 receptor activation and inflammasome assembly. We suggest that AAT controls ATP-induced IL-1β release from human mononuclear blood cells by a novel triple-membrane-passing signaling pathway. This pathway may have clinical implications for the prevention of sterile pulmonary and systemic inflammation.

β-Nicotinamide Adenine Dinucleotide (β-NAD) Inhibits ATP-Dependent IL-1β Release from Human Monocytic Cells.

While interleukin-1β (IL-1β) is a potent pro-inflammatory cytokine essential for host defense, high systemic levels cause life-threatening inflammatory syndromes. ATP, a stimulus of IL-1β maturation, is released from damaged cells along with β-nicotinamide adenine dinucleotide (β-NAD). Here, we tested the hypothesis that β-NAD controls ATP-signaling and, hence, IL-1β release. Lipopolysaccharide-primed monocytic U937 cells and primary human mononuclear leukocytes were stimulated with 2′(3′)-O-(4-benzoyl-benzoyl)ATP trieethylammonium salt (BzATP), a P2X7 receptor agonist, in the presence or absence of β-NAD. IL-1β was measured in cell culture supernatants. The roles of P2Y receptors, nicotinic acetylcholine receptors (nAChRs), and Ca2+-independent phospholipase A2 (iPLA2β, PLA2G6) were investigated using specific inhibitors and gene-silencing. Exogenous β-NAD signaled via P2Y receptors and dose-dependently (IC50 = 15 µM) suppressed the BzATP-induced IL-1β release. Signaling involved iPLA2β, release of a soluble mediator, and nAChR subunit α9. Patch-clamp experiments revealed that β-NAD inhibited BzATP-induced ion currents. In conclusion, we describe a novel triple membrane-passing signaling cascade triggered by extracellular β-NAD that suppresses ATP-induced release of IL-1β by monocytic cells. This cascade links activation of P2Y receptors to non-canonical metabotropic functions of nAChRs that inhibit P2X7 receptor function. The biomedical relevance of this mechanism might be the control of trauma-associated systemic inflammation.

Surfactant inhibits ATP-induced release of interleukin-1β via nicotinic acetylcholine receptors

Interleukin (IL)-1β is a potent pro-inflammatory cytokine of innate immunity involved in host defense. High systemic IL-1β levels, however, cause life-threatening inflammatory diseases, including systemic inflammatory response syndrome. In response to various danger signals, the pro-form of IL-1β is synthesized and stays in the cytoplasm unless a second signal, such as extracellular ATP, activates the inflammasome, which enables processing and release of mature IL-1β. As pulmonary surfactant is known for its anti-inflammatory properties, we hypothesize that surfactant inhibits ATP-induced release of IL-1β. Lipopolysaccharide-primed monocytic U937 cells were stimulated with an ATP analog in the presence of natural or synthetic surfactant composed of recombinant surfactant protein (rSP)-C, palmitoylphosphatidylglycerol, and dipalmitoylphosphatidylcholine (DPPC). Both surfactant preparations dose-dependently inhibited IL-1β release from U937 cells. DPPC was the active constituent of surfactant, whereas rSP-C and palmitoylphosphatidylglycerol were inactive. DPPC was also effective in primary mononuclear leukocytes isolated from human blood. Experiments with nicotinic antagonists, siRNA technology, and patch-clamp experiments suggested that stimulation of nicotinic acetylcholine receptors (nAChRs) containing subunit α9 results in a complete inhibition of the ion channel function of ATP receptor, P2X7. In conclusion, the surfactant constituent, DPPC, efficiently inhibits ATP-induced inflammasome activation and maturation of IL-1β in human monocytes by a mechanism involving nAChRs.

Chemokines (CCL3, CCL4, and CCL5) Inhibit ATP-Induced Release of IL-1β by Monocytic Cells.

Chemokines and ATP are among the mediators of inflammatory sites that can enter the circulation via damaged blood vessels. The main function of chemokines is leukocyte mobilization, and ATP typically triggers inflammasome assembly. IL-1β, a potent inflammasome-dependent cytokine of innate immunity, is essential for pathogen defense. However, excessive IL-1β may cause life-threatening systemic inflammation. Here, we hypothesize that chemokines control ATP-dependent secretion of monocytic IL-1β. Lipopolysaccharide-primed human monocytic U937 cells were stimulated with the P2X7 agonist BzATP for 30 min to induce IL-1β release. CCL3, CCL4, and CCL5 dose dependently inhibited BzATP-stimulated release of IL-1β, whereas CXCL16 was ineffective. The effect of CCL3 was confirmed for primary mononuclear leukocytes. It was blunted after silencing CCR1 or calcium-independent phospholipase A2 (iPLA2) by siRNA and was sensitive to antagonists of nicotinic acetylcholine receptors containing subunits α7 and α9. U937 cells secreted small factors in response to CCL3 that mediated the inhibition of IL-1β release. We suggest that CCL chemokines inhibit ATP-induced release of IL-1β from U937 cells by a triple-membrane-passing mechanism involving CCR, iPLA2, release of small mediators, and nicotinic acetylcholine receptor subunits α7 and α9. We speculate that whenever chemokines and ATP enter the circulation concomitantly, systemic release of IL-1β is minimized.

A soluble factor mediates alpha-1 antitrypsin-induced inhibition of ATP-induced IL-1β release by monocytic cells

Introduction: Alpha-1 antitrypsin (AAT), a major antiprotease of the lung, exerts anti-inflammatory functions via poorly defined pathways. At previous DZL meetings, we provided evidence that AAT is a potent inhibitor of ATP-induced release of IL-1β from human monocytes, and that CD36, calcium-independent phospholipase A2 (iPLA2) and nicotinic acetylcholine receptor (nAChR) subunit α9 participate in this mechanism. Here we test the hypothesis that AAT induces the release of a soluble factor X from U937 cells, which then inhibits ATP-dependent inflammasome activation via nAChR. As glycerophosphocholine (GPC) is a metabolite of membrane phospholipids cleaved by iPLA2, we tested whether GPC functionally resembles factor X.

Alpha-1 antitrypsin inhibits ATP-induced release of IL-1β in human monocytes via a cholinergic pathway

Alpha-1 antitrypsin inhibits ATP-induced release of IL-1β in human monocytes via a cholinergic pathway

Extracellular adenosine 5′-triphosphate and lipopolysaccharide induce interleukin-1β release in canine blood

Binding of extracellular adenosine 5′-triphosphate (ATP) or lipopolysaccharide (LPS) to the damage-associated molecular pattern receptor P2X7 or the pathogen-associated molecular pattern receptor Toll-like receptor (TLR)4, respectively, can induce the release of the pleiotropic cytokine interleukin (IL)-1β in humans and mice. However, the release of IL-1β in dogs remains poorly defined. Using a canine IL-1β enzyme-linked immunosorbent assay, this study investigated whether ATP or LPS could induce IL-1β release in a canine blood-based assay. Short-term incubations (30min) with ATP induced IL-1β release in LPS-primed canine blood, and this process could be near-completely impaired by the P2X7 antagonist, A438079. In contrast, ATP failed to induce IL-1β release from blood not primed with LPS. ATP-induced IL-1β release was observed with LPS-primed blood from eight different pedigrees or cross breeds. Long-term incubations (24h) with LPS induced IL-1β release in canine blood in a concentration-dependent manner. This process was not altered by co-incubation with A438079. LPS-induced IL-1β release was observed with blood from 10 different pedigrees or cross breeds. These results demonstrate that both extracellular ATP and LPS can induce IL-1β release in dogs, and that ATP- but not LPS-induced IL-1β release in blood is dependent on P2X7 activation. These findings support the role of both P2X7 and TLR4 in IL-1β release in dogs.

Inflammasome activation in bovine monocytes by extracellular ATP does not require the purinergic receptor P2X7

Extracellular adenosine triphosphate (ATP) is a second signal for the assembly of the NLR family, pyrin domain-containing 3 (NLRP3) inflammasome, which form a framework to activate caspase 1, leading to the processing and secretion of the pro-inflammatory cytokine interleukin-1β (IL-1β). The aim of the present study was to investigate the role of the ATP-gated ion channel subtype P2X7 receptor in the inflammasome activation of bovine monocytes. ATP-induced inflammasome assembly in bovine monocytes was shown by caspase-1 activation and the release of IL-1β by LPS/ATP-stimulated bovine cells. The IL-1β release depended on potassium efflux but was independent of reactive oxygen generation of bovine monocytes. Unlike in the human system, a P2X7 receptor antagonist did not block the ATP-induced release of IL-1β of LPS-primed bovine cells. P2X7 mediated pore formation was observed in subsets of bovine T lymphocytes (CD4+>CD8+) but not in monocytes. In addition, ATP and 2-MeSATP but not the high affinity P2X7 agonist BzATP induced calcium influx in bovine monocytes. The data indicate that ROS generation plays no role in the ATP-induced activation of inflammasome in bovine monocytes and that P2X7-mediated pore formation is not necessary for the release of Interleukin-1β.

Functional P2X7 Receptors in Myeloma Cell Lines and Myeloma Patient Bone Marrow Samples Can Be Blocked by Specific P2X7 Receptor Antagonists: Implications for Myeloma Therapy and Chemotherapy Induced Inflammation.

Abstract 1845Inflammation is a driving factor in the pathogenesis of many cancers including multiple myeloma (MM). The active role that cytokines play, IL-1β in particular, during the early stages of MM has been extensively characterized. Myeloma is a plasma cell tumor whose early stages are characterized by cytokine dependent growth, predominantly driven by IL-6. We have demonstrated that IL-1β is a key inducer of IL-6 expressed by bone marrow stromal cells in a paracrine fashion during the progression of multiple myeloma from smoldering myeloma (SMM) to active disease (Mayo Clinic Proc 84:114 (Feb. 2009)). Elevated ATP levels have been documented in vivo in tumor micro-environments and is a potential contributing factor to IL-1β induced inflammation. The P2X7 receptor (P2X7R) is an ATP-gated ion channel expressed by cells of the hematopoetic lineage that has been shown to play a critical role in IL-1b processing and secretion. We investigated the role this receptor may have in the release of IL-1β in the bone marrow microenvironment using bone marrow samples from both chemotherapy treated and untreated patients.Myeloma cell lines were evaluated by RT-PCR for the expression of P2X7R mRNA. Expression was detected and confirmed by sequencing in ANBL-6, MM1.S, U266, RPMI-8226 and KAS-6/1 and was comparable to U937, a previously characterized P2X7R positive cell line. A newly derived plasmacytoma cell line, PCYT3, showed very low levels of message expression. The ability of ATP, the P2X7R agonist, to trigger the processing and release of IL-1β by activation of the P2X7 channel was studied using KAS-Pro, a myeloma cell line stably transfected with a pro- IL-1β gene. IL-1β, measured by ELISA, was released in a dose dependent fashion in response to increasing amounts of ATP ranging from.6mM to 5mM. Four P2X7R specific antagonists were tested for their effect on ATP stimulated IL- 1β release from KAS-Pro; all 4 compounds demonstrated inhibition in a dose dependent manner with varying degrees of potency correlating with P2X7R inhibition.In addition fresh patient samples (8 MGUS/7SMM/9 newly diagnosed MM as well as 12 Treated MM) were evaluated for receptor expression and function. All stages of disease expressed the receptor by RT-PCR. Analysis of samples from all stages of MM, as measured directly by IL-1β ELISA as well as indirectly by measurement of IL-1β induced IL-6 production from normal bone marrow stromal cells, showed functional receptor activation in response to ATP stimulation. IL-6 production detected from MGUS patient samples had a mean result of 2,253 pg/ml, the mean for SMM was 5,495 pg/ml, MM were 6,875 pg/ml while treated MM samples showed a mean result of 15,219 pg/m. Greater than 90% inhibition of IL-1β release by the P2X7R antagonists was seen for all patients who demonstrated a positive ATP induced IL-1β release (see figure). We observed a greater than 2 fold increase in IL-1β release between the treated MM samples and the newly diagnosed MM samples, suggesting a chemotherapy induced inflammation process may be ongoing in these patients. [Display omitted] These results confirm the activity of functional P2X7 receptors on myeloma cell lines and in fresh patient samples and suggest a role for the use of P2X7 receptor antagonists in the therapy of myeloma, particularly in patients undergoing chemotherapy. Disclosures:Hromockyj:Pfizer Corporation: Employment. Meyer:Pfizer Corporation: Employment.

Translocator protein (18 kDa)/peripheral benzodiazepine receptor specific ligands induce microglia functions consistent with an activated state

In the brain, translocator protein (18 kDa) (TSPO), previously called peripheral benzodiazepine receptor (PBR), is a glial protein that has been extensively used as a biomarker of brain injury and inflammation. However, the functional role of TSPO in glial cells is not well characterized. In this study, we show that the TSPO-specific ligands R-PK11195 (PK) and Ro5-4864 (Ro) increased microglia proliferation and phagocytosis with no effect on migration. Both ligands increased reactive oxygen species (ROS) production, and this effect may be mediated by NADPH-oxidase. PK and Ro also produced a small but detectable increase in IL-1β release. We also examined the effect of PK and Ro on the expression of proinflammatory genes and cytokine release in lipopolysaccharide (LPS) and adenosine triphosphate (ATP) activated microglia. PK or Ro had no effect on LPS-induced increase of pro-inflammatory genes, but they both decreased the ATP-induced increase of COX-2 gene expression. Ro, but not PK, enhanced the LPS-induced release of IL-1β. However, Ro decreased the ATP-induced release of IL-1β and TNF-α, and PK decreased the ATP-induced release of TNF-α. Exposure to Ro in the presence of LPS increased the number of apoptotic microglia, an effect that could be blocked by PK. These findings show that TSPO ligands modulate cellular functions consistent with microglia activation. Further, when microglia are activated, these ligands may have therapeutic potential by reducing the expression of pro-inflammatory genes and cytokine release. Finally, Ro-like ligands may be involved in the elimination of activated microglia via apoptosis.