A His-155 to Tyr polymorphism confers gain-of-function to the human P2X7 receptor of human leukemic lymphocytes

The P2X(7)R is an ATP-gated cation channel expressed in hemopoietic cells that participates in both cell proliferation and apoptosis. Expression and function of the P2X(7)R have been associated with the clinical course of patients affected by chronic lymphocytic leukemia (CLL). Functional variants causing loss-of-function of the P2X(7)R have been identified, namely, polymorphisms 1513A>C (E496A), 1729T>A (I568N), and 946G>A (R307Q). Here we investigated other nonsynonymous polymorphisms located either in the extracellular portion of the receptor, such as the 489C>T (H155Y) variant, or in the long cytoplasmic tail of the receptor, such as the 1068G>A (A348T), 1096C>G (T357S), and 1405A>G (Q460R) variants. P2X(7)R function was monitored by measuring ATP-induced Ca(2+) influx in PBL of patients affected by CLL and in recombinant human embryonic kidney (HEK) 293 cells stably transfected with each single P2X(7) allelic variant. Ca(2+) influx was markedly reduced in association with the 1513C allele, whereas variants located in the same intracellular domain, such as the 1068A, 1096G, or 1405G variants, were associated with a minor functional decrease. Significant Ca(2+) flux increase was observed in lymphocytes from CLL patients bearing the 489C/T and 489T/T genotypes in association with the 1513A/A genotype. Functional analysis in recombinant HEK293 cells expressing P2X(7)R confirmed an increased ATP-dependent activation of the P2X(7) 489T mutant with respect to the wild type receptor, as assessed by both by [Ca(2+)](i) influx and ethidium uptake experiments. These data identify the 489C>T as a gain-of-function polymorphism of the P2X(7)R.     

Loss of P2X7 nucleotide receptor function leads to abnormal fat distribution in mice

The P2X7 receptor is an ATP-gated cation channel expressed by a number of cell types. We have shown previously that disruption of P2X7 receptor function results in downregulation of osteogenic markers and upregulation of adipogenic markers in calvarial cell cultures. In the present study, we assessed whether loss of P2X7 receptor function results in changes to adipocyte distribution and lipid accumulation in vivo. Male P2X7 loss-of-function (KO) mice exhibited significantly greater body weight and epididymal fat pad mass than wild-type (WT) mice at 9 months of age. Fat pad adipocytes did not differ in size, consistent with adipocyte hyperplasia rather than hypertrophy. Histological examination revealed ectopic lipid accumulation in the form of adipocytes and/or lipid droplets in several non-adipose tissues of older male KO mice (9–12 months of age). Ectopic lipid was observed in kidney, extraorbital lacrimal gland and pancreas, but not in liver, heart or skeletal muscle. Specifically, lacrimal gland and pancreas from 12-month-old male KO mice had greater numbers of adipocytes in perivascular, periductal and acinar regions. As well, lipid droplets accumulated in the renal tubular epithelium and lacrimal acinar cells. Blood plasma analyses revealed diminished total cholesterol levels in 9- and 12-month-old male KO mice compared with WT controls. Interestingly, no differences were observed in female mice. Moreover, there were no significant differences in food consumption between male KO and WT mice. Taken together, these data establish novel in vivo roles for the P2X7 receptor in regulating adipogenesis and lipid metabolism in an age- and sex-dependent manner.     

Regulation of P2X(7) nucleotide receptor function in human monocytes by extracellular ions and receptor density

P2X receptors function as ATP-gated cation channels. The P2X(7) receptor subtype is distinguished from other P2X family members by a very low affinity for extracellular ATP (millimolar EC50) and its ability to trigger induction of nonselective pores on repeated or prolonged stimulation. Previous studies have indicated that certain P2X(7) receptor-positive cell types, such as human blood monocytes and murine thymocytes, lack this pore-forming response. In the present study we compared pore formation in response to P2X(7) receptor activation in human blood monocytes with that in macrophages derived from these monocytes by in vitro tissue culture. ATP induced nonselective pores in macrophages but not in freshly isolated monocytes when both cell types were identically stimulated in standard NaCl-based salines. However, ion substitution studies revealed that replacement of extracellular Na+ and Cl- with K+ and nonhalide anions strongly facilitated ATP-dependent pore formation in monocytes. These ionic conditions also resulted in increased agonist affinity, such that 30-100 microM ATP was sufficient for activation of nonselective pores by P2X(7) receptors. Comparison of P2X(7) receptor expression in blood monocytes with that in macrophages indicated no differences in steady-state receptor mRNA levels but significant increases (up to 10-fold) in the amount of immunoreactive P2X(7) receptor protein at the cell surface of macrophages. Thus ability of ATP to activate nonselective pores in cells that natively express P2X(7) receptors can be modulated by receptor subunit density at the cell surface and ambient levels of extracellular Na+ and Cl-. These mechanisms may prevent adventitious P2X(7) receptor activation in monocytes until these proinflammatory leukocytes migrate to extravascular sites of tissue damage.     

P2X7 receptor regulation of non‐classical secretion from immune effector cells

P2X7 receptors (P2X7R) are extracellular ATP‐gated ion channels expressed in the immune effector cells that carry out critical protective responses during the early phases of microbial infection or acute tissue trauma. P2X7R‐positive cells include monocytes, macrophages, dendritic cells and T cells. Given its presence in all host and pathogen cell types, ATP can be readily released into extracellular compartments at local sites of tissue damage and microbial invasion. Thus, extracellular ATP and its target receptors on host effector cells can be considered as additional elements of the innate immune system. In this regard, stimulation of P2X7R rapidly triggers a key step of the inflammatory response: induction of NLRP3/caspase‐1 inflammasome signalling complexes that drive the proteolytic maturation and secretion of the proinflammatory cytokines interleukin‐1β (IL‐1β) and interleukin‐18 (IL‐18). IL‐1β (and IL‐18) lacks a signal sequence for compartmentation within the Golgi and classical secretory vesicles and the proIL‐1β precursor accumulates within the cytosol following translation on free ribosomes. Thus, ATP‐induced accumulation of the mature IL‐1β cytokine within extracellular compartments requires non‐classical mechanisms of export from the cytosolic compartment. Five proposed mechanisms include: (i) exocytosis of secretory lysosomes that accumulate cytosolic IL‐1β via undefined protein transporters; (ii) release of membrane‐delimited microvesicles derived from plasma membrane blebs formed by evaginationsof the surface membrane that entrap cytosolic IL‐β; (iii) release of membrane‐delimited exosomes secondary to the exocytosis of multivesicular bodies formed by invaginations of recycling endosomes that entrap cytosolic IL‐β; (iv) exocytosis of autophagosomes or autophagolysosomes that accumulate cytosolic IL‐1β via entrapment during formation of the initial autophagic isolation membrane or omegasome and (v) direct release of cytosolic IL‐1β secondary to regulated cell death by pyroptosis or necroptosis. These mechanisms are not mutually exclusive and may represent engagement of parallel or intersecting membrane trafficking responses to P2X7R activation.     

Absence of the P2X7 receptor alters leukocyte function and attenuates an inflammatory response

When challenged with extracellular ATP, leukocytes respond and activate processes attributed to the P2X(7) receptor (P2X(7)R), an unusual ligand-gated ion channel. To prove P2X(7)R involvement, blood samples from P2X(7)R-deficient mice were characterized. Monocytes and lymphocytes associated with wild-type blood responded to ATP and underwent volume/shape changes and shed L-selectin. In contrast, leukocytes from P2X(7)R-deficient animals demonstrated no change in physical properties or L-selectin expression following ATP challenge. Blood stimulated with LPS or ATP individually generated minimal quantities of the leaderless polypeptide IL-1 beta, but sequential treatment of wild-type, but not P2X(7)R-deficient, blood with LPS and ATP yielded large amounts of cell-free cytokine. Based on these differences, wild-type and P2X(7)R-deficient animals were compared following induction of monoclonal anti-collagen-induced arthritis. Ab-treated wild-type animals subsequently challenged with LPS developed inflamed, swollen paws; their joint cartilage demonstrated lesions, loss of proteoglycan content, and the presence of collagen degradation products. P2X(7)R-deficient animals subjected to the same challenge were markedly less affected; both the incidence and severity of disease were reduced. These data indicate that ATP does act via the P2X(7)R to affect leukocyte function and that the P2X(7)R can serve as an important component of an in vivo inflammatory response.     

A Glu-496 to Ala polymorphism leads to loss of function of the human P2X7 receptor

The P2X(7) receptor is a ligand-gated cation-selective channel that mediates ATP-induced apoptosis of cells of the immune system. We and others have shown that P2X(7) is nonfunctional both in lymphocytes and monocytes from some subjects. To study a possible genetic basis we sequenced DNA coding for the carboxyl-terminal tail of P2X(7). In 9 of 45 normal subjects a heterozygous nucleotide substitution (1513A-->C) was found, whereas 1 subject carried the homozygous substitution that codes for glutamic acid to alanine at amino acid position 496. Surface expression of P2X(7) on lymphocytes was not affected by this E496A polymorphism, demonstrated both by confocal microscopy and immunofluorescent staining. Monocytes and lymphocytes from the E496A homozygote subject expressed nonfunctional receptor, whereas heterozygotes showed P2X(7) function that was half that of germline P2X(7). Results of transfection experiments showed that the mutant P2X(7) receptor was nonfunctional when expressed at low receptor density but regained function at a high receptor density. This density dependence of mutant P2X(7) function was also seen on differentiation of fresh monocytes to macrophages with interferon-gamma, which up-regulated mutant P2X(7) and partially restored its function. P2X(7)-mediated apoptosis of lymphocytes was impaired in homozygous mutant P2X(7) compared with germline (8.6 versus 35.2%). The data suggest that the glutamic acid at position 496 is required for optimal assembly of the P2X(7) receptor.     

An Ile-568 to Asn polymorphism prevents normal trafficking and function of the human P2X7 receptor

The P2X(7) receptor is a ligand-gated channel that is highly expressed on mononuclear cells and that mediates ATP-induced apoptosis of these cells. Wide variations in the function of the P2X(7) receptor have been observed, in part because of a loss-of-function polymorphism that changes Glu-496 to Ala without affecting the surface expression of the receptor on lymphocytes. In this study a second polymorphism (Ile-568 to Asn) has been found in heterozygous dosage in three of 85 normal subjects and in three of 45 patients with chronic lymphocytic leukemia. P2X(7) function was measured by ATP-induced fluxes of Rb(+), Ba(2+), and ethidium(+) into various lymphocyte subsets and was decreased to values of approximately 25% of normal. The expression of the P2X(7) receptor on lymphocytes was approximately half that of normal values as measured by the binding of fluorescein-conjugated monoclonal antibody. Transfection experiments showed that P2X(7) carrying the Ile-568 to Asn mutation was non-functional because of the failure of cell surface expression. The differentiation of monocytes to macrophages with interferon-gamma up-regulated P2X(7) function in cells heterozygous for the Ile-568 to Asn mutation to a value around 50% of normal. These data identify a second loss-of-function polymorphism within the P2X(7) receptor and show that Ile-568 is critical to the trafficking domain, which we have shown to lie between residues 551 and 581.     

Residues 155 and 348 contribute to the determination of P2X7 receptor function via distinct mechanisms revealed by single-nucleotide polymorphisms

P2X(7) receptors are important in mediating the physiological functions of extracellular ATP, and altered receptor expression and function have a causative role in the disease pathogenesis. Here, we investigated the mechanisms determining the P2X(7) receptor function by following two human single-nucleotide polymorphism (SNP) mutations that replace His-155 and Ala-348 in the human (h) P2X(7) receptor with the corresponding residues, Tyr-155 and Thr-348, in the rat (r) P2X(7) receptor. H155Y and A348T mutations in the hP2X(7) receptor increased ATP-induced currents, whereas the reciprocal mutations, Y155H and T348A, in the rP2X(7) receptor caused the opposite effects. Such a functional switch is a compelling indication that these residues are critical for P2X(7) receptor function. Additional mutations of His-155 and Ala-348 in the hP2X(7) receptor to residues with diverse side chains revealed a different dependence on the side chain properties, supporting the specificity of these two residues. Substitutions of the residues surrounding His-155 and Ala-348 in the hP2X(7) receptor with the equivalent ones in the rP2X(7) receptor also affected ATP-induced currents but were not fully reminiscent of the H155Y and A348T effects. Immunofluorescence imaging and biotin labeling assays showed that H155Y in the hP2X(7) receptor increased and Y155H in the rP2X(7) receptor decreased cell-surface expression. Such contrasting effects were not obvious with the reciprocal mutations of residue 348. Taken together, our results suggest that residues at positions 155 and 348 contribute to P2X(7) receptor function via determining the surface expression and the single-channel function, respectively. Such interpretations are consistent with the locations of the residues in the structural model of the hP2X(7) receptor.     

Differential assembly of rat purinergic P2X7 receptor in immune cells of the brain and periphery

ATP-gated P2X(7) purinoceptors are found in most immune cells of the periphery and the brain where their activation leads to multiple downstream events such as cell permeabilization, apoptosis, and/or cytokine release. P2X(7) receptors do not form heteromeric receptors with any of the other six P2X subunits, and it is not known what type of homomeric assemblies the P2X(7) subunit makes. We constructed and purified an ectodomain protein of the rat P2X(7) receptor (amino acids 60-323) and used this to generate a monoclonal antibody (Ab) with which to probe P2X(7) receptors in central and peripheral immune cells. In HEK cells expressing rat P2X(7) receptors, the Ab increased the maximum current evoked by BzATP by 3-8-fold with a 5-fold leftward shift in EC(50) concentration. This Ab recognized only a non-denatured, multimeric form of the receptor on blue native-PAGE but did not recognize the denatured form on SDS-PAGE. A C-terminal polyclonal P2X(7) Ab recognized both monomeric subunits on SDS-PAGE and a multimeric complex on blue native-PAGE in this heterologous expression system. With Western blotting using these two Abs, native P2X(7) receptors in peritoneal macrophage and bone marrow cells are shown to exist as a strongly bound multimeric complex, whereas P2X(7) receptors in brain glia and/or astrocytes appear to form only as monomeric subunits.     

P2X7 nucleotide receptor activation enhances IFN gamma-induced type II nitric oxide synthase activity in BV-2 microglial cells

Under normal and pathological conditions, brain cells release nucleotides that regulate a wide range of cellular responses due to activation of P2 nucleotide receptors. In this study, the effect of extracellular nucleotides on IFN gamma-induced NO release in murine BV-2 microglial cells was investigated. BV-2 cells expressed mRNA for metabotropic P2Y and ionotropic P2X receptors. Among the P2 receptor agonists tested, ATP, ADP, 2',3'-O-(4-benzoylbenzoyl)-ATP (BzATP), and 2-methylthio-ATP (2-MeSATP), but not UTP, enhanced IFN gamma-induced iNOS expression and NO production, suggesting that the uridine nucleotide receptors P2Y2 and P2Y6 are not involved in this response. U0126, an antagonist for MEK1/2, a kinase that phosphorylates the extracellular signal-regulated kinases ERK1/2, decreased IFN gamma-induced NO production. BzATP, a potent P2X7 receptor agonist, was more effective than ATP, ADP, or 2-MeSATP at enhancing IFN gamma-induced ERK1/2 phosphorylation. Consistent with activation of the P2X7 receptor, periodate-oxidized ATP, a P2X7 receptor antagonist, and suramin, a non-specific P2 receptor antagonist, inhibited the effect of ATP or BzATP on IFN gamma-induced NO production, whereas pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), an antagonist of several P2X receptor subtypes, was ineffective. These results suggest that activation of P2X7 receptors may contribute to inflammatory responses in microglial cells seen in neurodegenerative diseases.     

An Arg307 to Gln polymorphism within the ATP-binding site causes loss of function of the human P2X7 receptor

The P2X(7) receptor is a ligand-gated channel that is highly expressed on mononuclear cells of the immune system and that mediates ATP-induced apoptosis. Wide variations in the function of the P2X receptor have been observed, explained in part by (7)loss-of-function polymorphisms that change Glu(496) to Ala (E496A) and Ile(568) to Asn (I568N). In this study, a third polymorphism, which substitutes an uncharged glutamine for the highly positively charged Arg(307) (R307Q), has been found in heterozygous dosage in 12 of 420 subjects studied. P2X(7) function was measured by ATP-induced fluxes of Rb(+), Ba(2+), and ethidium(+) into peripheral blood monocytes or various lymphocyte subsets and was either absent or markedly decreased. Transfection experiments showed that P2X(7) carrying the R307Q mutation lacked either channel or pore function despite robust protein synthesis and surface expression of the receptor. The monoclonal antibody (clone L4) that binds to the extracellular domain of wild type P2X(7) and blocks P2X(7) function failed to bind to the R307Q mutant receptor. Differentiation of monocytes to macrophages up-regulated P2X(7) function in cells heterozygous for the R307Q to a value 10-40% of that for wild type macrophages. However, macrophages from a subject who was double heterozygous for R307Q/I568N remained totally non-functional for P2X(7), and lymphocytes from the same subject also lacked ATP-stimulated phospholipase D activity. These data identify a third loss-of-function polymorphism affecting the human P2X(7) receptor, and since the affected Arg(307) is homologous to those amino acids essential for ATP binding to P2X(1) and P2X(2), it is likely that this polymorphism abolishes the binding of ATP to the extracellular domain of P2X(7).     

Glial cells,but not interstitial cells,express P2X7, an ionotropic purinergic receptor,in rat gastrointestinal musculature

Purinergic (ATP) neurotransmission is a component of the inhibitory response of the musculature in various regions of the gastrointestinal tract. So far, seven ionotropic purinergic receptors (P2X1-7) have been cloned. As specific antibodies become available, their respective distribution in the gastrointestinal tract can be elucidated. Here, we used high-resolution tricolor confocal microscopy, to study the distribution of P2X7-immunoreactive (-ir) cells in the muscularis propria of the rat stomach, small intestine, and colon. Smooth muscle cells, KIT-ir interstitial cells of Cajal, and CD34/SK3-ir fibroblastlike cells were P2X7-negative, whereas P2X7 immunoreactivity was observed in nerves and S100-ir glial cells. In all regions studied, P2X7 immunoreactivity was also observed in myenteric and submucosal ganglia, where perineuronal nerve endings appeared brightly labeled. Our observations suggest that purinergic signaling could influence the enteric glia through P2X7 receptors.     

Mutation of a dibasic amino acid motif within the C terminus of the P2X7 nucleotide receptor results in trafficking defects and impaired function

Activation of the P2X(7) receptor by extracellular nucleotides modulates multiple immune functions, including inflammatory mediator production, membrane fusion events, and apoptosis. Previous studies have revealed that the C terminus of this multimeric cation channel possesses a lipid-interaction motif that has been proposed to regulate receptor function. This domain is homologous to the LPS binding region of the LPS binding protein, and we demonstrated that two basic residues (Arg(578), Lys(579)) within this motif are essential for LPS binding to P2X(7) in vitro. Because P2X(7) can influence LPS action, and because lipid interaction motifs modulate the trafficking of other ion channel-linked receptors, we hypothesized that this motif of P2X(7) is critical for receptor function and trafficking. In these studies we mutated Arg(578) and Lys(579) of P2X(7), and the expression profile, channel activity, and pore formation of the mutant were characterized in transfected human embryonic kidney 293 cells. In contrast with the wild-type receptor, the P2X(7)-R578E/K579E mutant fails to demonstrate surface immunoreactivity despite normal levels of total protein expression. This effect on the mutant receptor is unlikely to result from widespread defects in protein folding, because surface localization, determined using conformation-specific Abs, can be restored by growing the cells at 25 degrees C, conditions that slow receptor recycling. Despite surface expression at reduced temperatures, at 25 degrees C the P2X(7)-R578E/K579E mutant still exhibits greatly reduced sodium, potassium, and calcium channel activity when compared with the wild-type receptor, and cannot induce pore formation. These data suggest that the lipid interaction motif of the P2X(7) C terminus controls receptor trafficking and modulates channel activity.     

Identification of an intracellular microdomain of the P2X7 receptor that is crucial in basolateral membrane targeting in epithelial cells

We investigated membrane targeting of the P2X₇ receptor (P2X₇R) in polarized epithelial cells using immunofluorescent confocal imaging. The wild-type receptor was targeted to the basolateral membrane, independently of adaptor protein μ1B. Deletion of the majority of the intracellular C-terminus, or the last 26 residues (P570-Y595), conferred targeting of the protein to the apical membrane. Alanine substitution in the microdomain P582-Q587 caused similar apical membrane targeting without major effect on the receptor function and surface expression. Our results show basolateral membrane targeting of the P2X₇R in epithelial cells and that the intracellular C-terminal microdomain P582-Q587 is crucial in this process.

Structured summary

MINT-8055849:Beta-catenin (uniprotkb:B6V8E6) and P2X7R (uniprotkb:Q64663) colocalize (MI:0403) by fluorescence microscopy (MI:0416)     

Essential role for Ca2+ in regulation of IL-1beta secretion by P2X7 nucleotide receptor in monocytes,macrophages, and HEK-293 cells

Interleukin (IL)-1beta is a proinflammatory cytokine that elicits the majority of its biological activity extracellularly, but the lack of a secretory signal sequence prevents its export via classic secretory pathways. Efficient externalization of IL-1beta in macrophages and monocytes can occur via stimulation of P2X7 nucleotide receptors with extracellular ATP. However, the exact mechanisms by which the activation of these nonselective cation channels facilitates secretion of IL-1beta remain unclear. Here we demonstrate a pivotal role for a sustained increase in cytosolic Ca2+ to potentiate secretion of IL-1beta via the P2X7 receptors. Using HEK-293 cells engineered to coexpress P2X7 receptors with mature IL-1beta (mIL-1beta), we show that activation of P2X7 receptors results in a rapid secretion of mIL-1beta by a process(es) that is dependent on influx of extracellular Ca2+ and a sustained rise in cytosolic Ca2+. Moreover, reduction in extracellular Ca2+ attenuates approximately 90% of P2X7 receptor-mediated IL-1beta secretion but has no effect on enzymatic processing of precursor IL-1beta (proIL-1beta) to mIL-1beta by caspase-1. Similar experiments with THP-1 human monocytes and Bac1.2F5 murine macrophages confirm the unique role of Ca2+ in P2X7 receptor-mediated secretion of IL-1beta. In addition, we report that cell surface expression of P2X7 receptors in the absence of external stimulation also results in enhanced release of IL-1beta and that this can be repressed by inhibitors of P2X7 receptors. We clarify an essential role for Ca2+ in ATP-induced IL-1beta secretion and indicate an additional role of P2X7 receptors as enhancers of the secretory apparatus by which IL-1beta is released.     

Expression and function of P2X(7) receptor and CD39/Entpd1 in patients with type 2 diabetes and their association with biochemical parameters

Chronic inflammation is an important contributor to the insulin resistance observed in type 2 diabetes (T2D). We evaluated the expression and function of the P2X₇ receptor and CD39/Entpd1, molecules involved in the cellular regulation of inflammation, in peripheral blood mononuclear cells from T2D patients, and their correlation with the concentration of HbA1c in blood. T2D patients with deficient metabolic control (DC) showed increased proportion of P2X₇⁺ cells compared with healthy individuals; T2D-DC subjects also displayed higher proportion of CD14⁺, CD4⁺ and CD19⁺ subpopulations of P2X₇⁺ cells when compared with T2D patients with acceptable metabolic control. A significant association was observed between the proportion of P2X₇⁺CD14⁺ cells and blood concentration of LDL-c. In addition, the percentages of CD39⁺ cells and CD39⁺CD19⁺ cells were significantly associated with HbA1c and fasting plasma glucose levels. No changes were observed in the function of P2X₇⁺ cells from T2D patients; however, enhanced CD39/Entpd1 enzyme activity and low serum levels of IL-17 were detected. Therefore, CD39⁺ cells could have a balancing regulatory role in the inflammatory process observed in patients with T2D.     

Redirection of T cell effector function in vivo and enhanced collagen-induced arthritis mediated by an IL-2R beta/IL-4R alpha chimeric cytokine receptor transgene

Chronic inflammatory autoimmune diseases such as diabetes, experimental autoimmune encephalomyelitis, and collagen-induced arthritis (CIA) are associated with type 1 (Th1, Tc1) T cell-dependent responses against autoantigens. Immune deviation toward type 2 (Th2, Tc2) response has been proposed as a potential means of gene therapy or immunomodulation to treat autoimmune diseases based on evidence that type 2 cytokines can prevent or alleviate these conditions. In this report we assessed the effects of elevated type 2 responses on CIA using transgenic mice expressing an IL-2R beta/IL-4R alpha chimeric cytokine receptor transgene specifically in T cells. In response to IL-2 binding, this chimeric receptor transduces IL-4-specific signals and dramatically enhances type 2 responses. In contrast to published reports of Th2-mediated protection, CIA was exacerbated in IL-2R beta/IL-4R alpha chimeric receptor transgenic mice, with increased disease incidence, severity, and earlier disease onset. The aggravated disease in transgenic mice was associated with an increase in type 2 cytokines (IL-4, IL-5, IL-10) and an increase in collagen-specific IgG1 levels. However, IFN-gamma production is not affected significantly in the induction phase of the disease. There is also an extensive eosinophilic infiltration in the arthritic joints of the transgenic animal, suggesting a direct contribution of type 2 response to joint inflammation. Taken together, our findings provide novel evidence that enhancement of a polyclonal type 2 response in immunocompetent hosts may exacerbate an autoimmune disease such as CIA, rather than serving a protective role. This finding raises significant caution with regard to the potential use of therapeutic approaches based on immune deviation toward type 2 responses.     

Association studies of 33 single nucleotide polymorphisms (SNPs) in 29 candidate genes for bronchial asthma: positive association of a T924C polymorphism in the thromboxane A2 receptor gene

Although intensive studies have attempted to elucidate the genetic background of bronchial asthma (BA), one of the most common of the chronic inflammatory diseases in human populations, genetic factors associated with its pathogenesis are still not well understood. We surveyed 29 possible candidate genes for this disease for single nucleotide polymorphisms (SNPs), the most frequent type of genetic variation, in genomic DNAs from Japanese BA patients. We identified 33 SNPs, only four of which had been reported previously, among 14 of those genes. We also performed association studies using 585 BA patients and 343 normal controls for these SNPs. Of the 33 SNPs tested, 32 revealed no positive association with BA, but a T924C polymorphism in the thromboxane A2 receptor gene showed significant association (chi2=4.71, P=0.030), especially with respect to adult patients (chi2=6.20, P=0.013). Our results suggest that variants of the TBXA2R gene or some nearby gene(s) may play an important role in the pathogenesis of adult BA.