Interleukin-2 Receptor Beta Research Articles

Activation of JAK2-V617F by Heterodimeric Receptor Components.

Abstract 2955Poster Board II-931Mutational activation of Janus Kinase 2 (JAK2) is an important etiologic factor for the development of myeloproliferative neoplasms (MPNs). JAK2 is mutated in nearly all patients with polycythemia vera and about half of patients with essential thrombocythemia and primary myelofibrosis. The most prevalent mutation of JAK2 is valine 617 mutated to phenylalanine (V617F). The normal function of JAK2 is to interact with and become activated by cytokine receptors following their interaction with ligand. Interestingly, while JAK2-V617F is considered a constitutively activated kinase, there is evidence that it still requires interaction with a cytokine receptor to elicit its transforming signal. When expressed at less than or near endogenous JAK2 levels in hematopoietic cells, JAK2-V617F requires co-expression of a homodimeric receptor in order to become activated, transduce downstream signals, and induce transformation. When expressed at high levels in hematopoietic cells, co-expression of a homodimeric cytokine receptor is not needed. However, a functional cytokine receptor interacting domain is still required, suggesting that even when expressed at high levels JAK2-V617F requires interaction with a receptor. Also, a functional cytokine receptor interacting domain in JAK2-V617F is required for it to induce MPN-like disease in mouse models. The ability of cytokine receptor expression to activate JAK2-V617F has focused on homodimeric receptors. In this study we demonstrate that single components of heterodimeric receptors can also activate JAK2-V617F. Expression of interleukin-27 receptor alpha (IL27Ra), the ligand-binding component of the IL-27 receptor, enhances phosphorylation of JAK2-V617F on tyrosines 1007 and 1008. This activation of JAK2 also leads to tyrosine phosphorylation of signal transducers and activators of transcription-5 (STAT5), a main downstream effector of JAK2 activation. We obtain similar results when we utilize interleukin-12 receptor beta 1 (IL12RB1), a receptor belonging to the same family as IL27Ra. To extend these studies to other heterodimeric cytokine receptor components, we utilized the components of the interleukin-3 receptor, interleukin-3 receptor alpha (IL3Ra) and the beta common chain, which is also utilized in the receptors for interleukin-5 and granulocyte macrophage colony stimulating factor. Expression of each of these receptor subunits activates JAK2-V617F as well as STAT5. Importantly, we demonstrate that expression of IL27Ra can functionally replace the expression of a homodimeric cytokine receptor to support the activation of JAK2-V617F in BaF3 cells as well as promote the transforming activity of JAK2-V617F in these cytokine dependent hematopoietic cells. Interestingly, while IL12RB1 expression activates JAK2-V617F in 293T cells, it is not capable of enhancing the transforming signals of JAK2-V617F in BaF3 cells. Expression of IL3Ra or the beta common chain in BaF3 cells also enhances the ability of JAK2-V617F to transform these cells to cytokine independence. However, this enhancement is not immediate as it only becomes evident at later time points. Together our data demonstrate that in addition to homodimeric receptors, some heterodimeric receptor components may contribute to JAK2-V617F activation. It should also be considered that such receptors may play a role in JAK2-V617F-negative MPNs, perhaps through altered expression or activating receptor mutations, analogous to mutated thrombopoietin receptor proteins that play a role in the development of disease in a fraction of these MPN patients. Disclosures:No relevant conflicts of interest to declare.

IL-12 Enhances Tumor Immunity Via Differential Effects On Regulatory T Cells and Cytotoxic Lymphocytes.

Abstract 1656Poster Board I-682To study the roles of IL-12 and Interferon-gamma (IFNg) in tumor immunity, we used RMAS lymphoma cells to challenge IL-12 receptor beta 2-deficient (IL-12Rb2-/-) and IFNg receptor 1-deficient (IFNgR1-/-) mice that are in the syngeneic C57BL/6J background. We intravenously injected mice with a dose of 1 × 104 RMAS cells that caused death in about 50% of wild-type (WT) mice. As shown in the Figure below, all of the WT mice treated with exogenous IL-12 were rescued from death caused by tumor growth; endogenous IL-12 was not sufficient to impact tumor growth since IL-12Rb2-/- mice showed a survival rate similar to that of WT mice. However, all of the IFNgR1-/- mice succumbed to tumor growth, indicating that endogenous IFNg is required for tumor immunity in this system. Furthermore, IL-12 treatment did not improve the survival of the IFNgR1-/- mice, suggesting that IFNg signaling is required for IL-12's anti-tumor effect. [Display omitted] We previously showed that an IL-12/IFNg axis can inhibit tumor-induced regulatory T cell (Treg) proliferation in vitro (Cao et al, 2008 ASH Annual Meeting). We have subsequently examined their effects on Treg cells in vivo. Compared to naive mice, significant Treg expansion (4.9 ± 2.1 fold, n=5, p=0.025) was observed in the peritoneal cavity of WT mice within 2 weeks after an intraperitoneal injection of 1 × 104 RMAS cells. This expansion was completely blocked by treatment with exogenous IL-12. Treg cells in the IL-12Rb2-/- mice expanded to levels comparable to that in WT animals, suggesting that endogenous IL-12 was not sufficient to control Treg expansion. In contrast, significantly higher Treg expansion was observed in IFNgR1-/- mice (36.8 ± 11.8 fold, n=5, p=0.002), which was partially inhibited by IL-12 treatment (13.2 ± 3.5 fold, n=5, p=0.002), suggesting that an IFNg-independent mechanism may also account for IL-12's anti-Treg effect.To further study the effects of IL-12 and IFNg on cytotoxic T lymphocyte (CTL) function, we performed mixed lymphocyte reactions (MLR) and used flow-based killing assays (FloKA) to measure cell contact-dependent killing of allogeneic P815 tumor cells. MLR-activated CTLs were found to kill tumor targets via perforin/granzyme-mediated cytotoxicity. At a 10:1 (effector:target) ratio, granzyme AxB-deficient CTLs and perforin-deficient CTLs displayed significantly reduced killing (8.6 ± 1.2% and 4.5 ± 0.9%, respectively) compared to WT CTLs (36.1 ± 3.5%). IL-12 supplement (2ng/ml) to the MLR significantly increased the killing activity of WT CTLs (65.3 ± 4.2%), but had no significant effect on granzyme AxB-deficient CTLs or perforin-deficient CTLs. In contrast, IFNg supplement (10ng/ml) to the MLR had no significant effect on the killing activity of CTLs. Conversely, MLR-activated IFNgR1-/- CTLs killed P815 cells as efficiently as WT CTLs and responded to IL-12 treatment as efficiently as WT CTLs.Taken together, these data suggest that IL-12 treatment inhibits tumor-induced Treg expansion and stimulates IFNg-dependent anti-tumor immune responses. In addition, IL-12 also activates perforin/granzyme-dependent function of cytotoxic T lymphocytes. These differential effects on diverse immune components may collectively result in enhanced tumor immunity. DisclosuresNo relevant conflicts of interest to declare.

Jak2 Phosphorylates Tyr 177 of Bcr-Abl Activating the Ras and PI-3 Kinase Pathways and Maintains Functional Levels of Bcr-Abl in Chronic Myelogenous Leukemia.

Abstract 39We have shown that Jak2 is activated in Bcr-Abl+ hematopoietic cells. Our findings indicate that Jak2 kinase inhibition overcomes drug-resistance in CML cells (Samanta et al., 2006). Recently, we showed that Jak2 inhibition deactivates the Lyn kinase in Bcr-Abl+ cells by inhibiting SET expression; SET inhibits the PP2A-SHP1 (Samanta et al., 2009). Because of the critical effects of Jak2 in Bcr-Abl+ cells, we explored the mechanism of Jak2 action in CML cells. Bcr-Abl is known to be phosphorylated on a number of tyrosine residues including tyrosine 177. Phosphorylation of Tyr 177 leads to binding to Grb2, activation of the SOS-Ras pathway, and activation of the PI-3 kinase through the Grb2-Gab2 pathway. Here, we show that Jak2 and not Bcr-Abl phosphorylates Tyr 177 of Bcr-Abl. Tyr 177 of Bcr-Abl (YVNV) has the Jak2 consensus target sequence (YxxV/L/I) (Argetsinger et al., 2004) as do a number of other Tyr residues in the Bcr portion of Bcr-Abl (b3a2). Inhibition of Jak2 by a specific Jak2 kinase inhibitor TG101209 (TargeGen Inc., San Diego, CA) and a new Jak2 kinase inhibitor (WP1193, synthesized by Dr. Priebe) in Bcr-Abl+ cells, but not inhibition of Bcr-Abl by imatinib mesylate (IM), rapidly reduced phosphorylation of Tyr 177 of Bcr-Abl in IM-sensitive and –resistant cell lines including K562, K562-R, Bcr-Abl+ 32D, Bcr-Abl+ BaF3, Bcr-Abl+T315I BaF3 cells and cells from blast crisis CML patients. Jak2 inhibitors but not IM also strongly inhibited the phosphorylation of a synthetic Bcr peptide containing Tyr 177 (and the surrounding Bcr sequences) catalyzed by either recombinant purified Jak2 or Jak2 immune complexes. Jak2 inhibition of Bcr-Abl+ 32D cells rapidly reduced Grb2 binding to Bcr-Abl, and diminished activation of Ras and PI-3 kinase pathways within two h. Importantly, knockdown of Jak2 with Jak2-specific siRNA also reduced levels of pTyr 177 Bcr-Abl and total Bcr-Abl protein. Rescue experiments reversed Jak2 knockdown and stimulated pTyr 177 levels. In whole cells the total levels of the Bcr-Abl protein also decreased upon Jak2 inhibition with the above chemical inhibitors but at a rate slower than the rate observed in the inhibition of the Tyr 177 phosphorylation of Bcr-Abl. In kinase assays with Bcr-Abl network complexes from Bcr-Abl+ cells, phosphorylation of Tyr 177 of Bcr-Abl was drastically reduced by Jak2 inhibition but Bcr-Abl protein levels remained stable. Reduction of Bcr-Abl protein levels in Bcr-Abl+ cells by Jak2 inhibition also caused a severe reduction of tyrosine phosphorylation of STAT5 and STAT3. We have shown by gel filtration column chromatography of lysates isolated from Bcr-Abl+ cells that Bcr-Abl is physically associated with the IL-3 receptor beta chain, Jak2, Akt and HSP90 in high molecular weight complexes. GSK3β, STAT5 and STAT3 also emerge from the column in the high molecular weight fractions and suggested that several high molecular weight complexes may exist. Nevertheless, incubation of Bcr-Abl+ cells with WP1193 caused disruption of these large molecular network complexes within three h. Nude mice, injected subcutaneously with IM-resistant K562-R cells developed solid tumors, which were reduced in weight by i.p. injection of WP1193 every 48 h at 30 mg/Kg over 10 days. WP1193 at a similar dosage strongly reduced leukemic and tumor effects of Bcr-Abl+ 32D cells expressing T315I Bcr-Abl injected i.v. into nude mice. WP1193 injections into nude mice did not cause significant toxicity over a 10 day period. In summary, Jak2 inhibitors TG101209/WP1193 rapidly reduced levels of pTyr 177 Bcr-Abl, reduced levels of Bcr-Abl protein and induced high levels of apoptosis in IM-sensitive and –resistant Bcr-Abl+ cell lines including T315I cells, CML cell lines, cells from CML patients in blast crisis. In addition Jak2 inhibition reduced tumor formation in mice injected with IM-resistant K562-R and T315I Bcr-Abl+ mouse cells. These findings establish: 1) Jak2 as a major driver of Bcr-Abl signaling pathways in Bcr-Abl+ CML cells, which were previously thought to be activated directly by Bcr-Abl. 2) Although Jak2 is activated by Bcr-Abl, Jak2 is the dominant target molecule in CML and Jak2 becomes even more dominant in CML cells that become resistant to IM and other tyrosine kinase inhibitors. These findings indicate that CML cells, because of the dominant role of Jak2, resemble leukemia cells from Bcr-Abl negative myeloproliferative diseases. Disclosures:No relevant conflicts of interest to declare.

Association study of IFNAR2 and IL10RB genes with the susceptibility and interferon response in HBV infection

A recent genome-wide association study discovered that two polymorphisms, interferon (IFN) alpha receptor 2 (IFNAR-2) F8S and interleukin 10 receptor (IL10RB) K47E, were associated with susceptibility to hepatitis B virus (HBV) infection in Africa. Here, we reevaluate the effects of the two polymorphisms on HBV susceptibility in the Chinese Han population, and extended the study to look at their association with IFN response in chronic hepatitis B (CHB). We included 341 patients with CHB and 341 unrelated controls presenting with asymptotic HBV self-limited infection, who were well matched in age and sex. In the CHB group, 101 patients had been treated with peg-IFN-alpha-2a for 48 weeks and followed up for 24 weeks to determine the clinical response, resulting 34 individuals with sustained virological response (SVR) and 67 individuals with nonsustained response (NR). Subgroups in the CHB group were divided according to the viral loads, HBeAg and maternal HBsAg status. The association with the susceptibility to HBV infection was only observed for IL10RB K47E when we compared the individuals with persistent HBV infection through nonmaternal transmission to the controls with asymptomatic self-limited HBV infection. Further, we found that the IFNAR2-8SS genotype was associated with HBeAg negative patients (OR = 0.316, 95% CI: 0.121-0.825, P = 0.019) and that the IFNAR2-8F allele was associated with the risk to high viral loads (OR = 1.667, 95% CI: 1.148-2.420, P = 0.007). In addition, the IFNAR2-8FF genotype predisposed to higher MxA gene induction and correlated with sustained IFN response (OR = 0.348, 95% CI: 0.129-0.935, P = 0.036). Haplotype analysis based on polymorphisms of three single-nucleotide polymorphisms, MxA - 88 G/T, IFNAR-2 F8S and IL10RB K47E showed that the haplotype distribution was significantly different between the SVR and NR groups (P = 0.040). This study suggests that IFNAR2 may play an important role in determining IFN response and clinical phenotypes of HBV infection in the Chinese Han population.

Interleukin‐12 is sufficient to promote antigen‐independent interferon‐γ production by CD8 T cells in old mice

Numerous functional defects have been identified in naive T cells from aged mice, including deficiencies in proliferation, cytokine production and signal transduction. It is well documented that the ratio of naïve to memory T cells significantly decreases with age resulting in the majority of T cells from aged hosts expressing activated/memory T-cell markers (CD44(hi)), yet it is unclear whether T cells with a CD44(hi) phenotype in aged hosts are functionally equivalent to T cells with a similar phenotype in young hosts. We have identified a population of CD44(hi) CD8 T cells in old mice that are capable of secreting interferon-gamma (IFN-gamma) in response to interleukin-12 (IL-12) stimulation. This occurred in the absence of T-cell receptor engagement, a function that was not observed in CD8 T cells from young mice. This phenotype was associated with increased IL-12 receptor beta2 gene expression and IL-12 induced signal transducer and activator of transcription 4 (STAT-4) activation, even when CD8 T-cell numbers from young and old mice were normalized for CD44(hi) expression. Furthermore, we demonstrate that IL-12-induced STAT-4 activation was required for T helper type 1 (Th1) cytokine-induced IFN-gamma production in CD8 T cells. These data illustrate that old mice possess a specialized subset of CD44(hi) CD8 T cells with an enhanced responsiveness to IL-12, enabling these cells to produce substantial amounts of IFN-gamma in response to Th1 cytokine stimulation. We have therefore identified a functional difference in the populations of CD44(hi) CD8 T cells from young and old mice, and believe that understanding age-associated immunological changes is essential for helping the elderly combat deadly diseases.

Interleukin-2 receptor gene C627T polymorphism in Korean women with endometriosis: a case-control study

The purpose of this study was to investigate the potential association of the C627T polymorphism in the interleukin-2 receptor beta gene (IL-2R beta) with the risk of endometriosis in Korean women. Two hundred and thirty-seven women with surgically or histologically diagnosed endometriosis of stages III and IV were recruited for this study, and 164 patients with no evidence of endometriosis diagnosed by laparoscopy or laparotomy served as controls. The C627T polymorphism of the IL-2R beta was assessed using the TaqMan allelic discrimination assay. Chi2 analysis was used to examine any differences in genotype distributions and allele frequencies of the IL-2R beta C627T polymorphism between the endometriosis cases and the controls. There was no statistically significant difference in the frequency of the IL-2R beta C627T polymorphism between the endometriosis patients and the controls (28.7% C/C, 48.1% C/T and 23.2% T/T versus 29.3, 44.5 and 26.2%, respectively, P = 0.72) or in the T allele frequencies (47.3 versus 48.5%, respectively, P = 0.73). Even when the endometriosis cases were subdivided into stages III and IV, no statistically significant differences in genotype distributions or allele frequencies were observed among the three groups. Contrary to the recent data reported in a Taiwanese population, our results suggest that the C627T polymorphism of the IL-2R beta gene may not be associated with the risk of endometriosis in the Korean population.

Interleukin-1 cluster gene polymorphisms in childhood IgA nephropathy

We have carried out a study with the aim of investigating the association between single nucleotide polymorphisms (SNPs) of the IL-1 gene cluster and childhood IgA nephropathy (IgAN). SNPs of the IL-1 alpha, IL-1 beta, and IL-1 receptor antagonist (RN) genes (IL1A, IL1B, and IL1RN, respectively) were analyzed in 182 patients with childhood IgAN and in 500 healthy controls. The IgAN patients were also dichotomized and compared with respect to proteinuria (<4 mg and >or=4 mg/m(2) per hour, respectively), the presence or absence of podocyte foot process effacement, and the presence of pathologically early and advanced disease markers, such as interstitial fibrosis, tubular atrophy, or global sclerosis. Significant differences in SNP frequencies were observed for the IL1B and IL1RN genes (rs1143627, rs3917356, and rs1143633 in the IL1B gene, and rs928940, rs439154, and rs315951 in the IL1RN gene). Moreover, rs1143627, rs3917356, and rs1143633 of IL1B were found to be significantly associated with the presence of podocyte foot process effacement. Our results suggest that the IL1B and IL1RN genes are associated with increased susceptibility to IgAN in children. They also suggest that the development of proteinuria in IgAN is related to IL1A and that podocyte foot process effacement is associated with IL1B.

Primary Biliary Cirrhosis Associated withHLA, IL12A,andIL12RB2Variants

Primary biliary cirrhosis is a chronic granulomatous cholangitis, characteristically associated with antimitochondrial antibodies. Twin and family aggregation data suggest that there is a significant genetic predisposition to primary biliary cirrhosis, but the susceptibility loci are unknown. To identify genetic loci conferring a risk for primary biliary cirrhosis, we carried out a genomewide association analysis in which DNA samples from 2072 Canadian and U.S. subjects (536 patients with primary biliary cirrhosis and 1536 controls) were genotyped for more than 300,000 single-nucleotide polymorphisms (SNPs). Sixteen of the SNPs most strongly associated with primary biliary cirrhosis were genotyped in two independent replication sets. We carried out fine-mapping studies across three loci associated with primary biliary cirrhosis. We found significant associations between primary biliary cirrhosis and 13 loci across the HLA class II region; the HLA-DQB1 locus (encoding the major histocompatibility complex class II, DQ beta chain 1) had the strongest association (P=1.78x10(-19); odds ratio for patients vs. controls, 1.75). Primary biliary cirrhosis was also significantly and reproducibly associated with two SNPs at the IL12A locus (encoding interleukin-12alpha), rs6441286 (P=2.42x10(-14); odds ratio, 1.54) and rs574808 (P=1.88x10(-13); odds ratio, 1.54), and one SNP at the IL12RB2 locus (encoding interleukin-12 receptor beta2), rs3790567 (P=2.76x10(-11); odds ratio, 1.51). Fine-mapping analysis showed that a five-allele haplotype in the 3' flank of IL12A was significantly associated with primary biliary cirrhosis (P=1.15x10(-34)). We found a modest genomewide association (P<5.0x10(-5)) with the risk of disease for SNPs at the STAT4 locus (encoding signal transducer and activator of transcription 4) and the CTLA4 locus (encoding cytotoxic T-lymphocyte-associated protein 4) and 10 other loci. Our data show significant associations between primary biliary cirrhosis and common genetic variants at the HLA class II, IL12A, and IL12RB2 loci and suggest that the interleukin-12 immunoregulatory signaling axis is relevant to the pathophysiology of primary biliary cirrhosis. (ClinicalTrials.gov number, NCT00242125.)

Convergence of IL-1β and VDR Activation Pathways in Human TLR2/1-Induced Antimicrobial Responses

Antimicrobial effector mechanisms are central to the function of the innate immune response in host defense against microbial pathogens. In humans, activation of Toll-like receptor 2/1 (TLR2/1) on monocytes induces a vitamin D dependent antimicrobial activity against intracellular mycobacteria. Here, we report that TLR activation of monocytes triggers induction of the defensin beta 4 gene (DEFB4), requiring convergence of the IL-1β and vitamin D receptor (VDR) pathways. TLR2/1 activation triggered IL-1β activity, involving the upregulation of both IL-1β and IL-1 receptor, and downregulation of the IL-1 receptor antagonist. TLR2/1L induction of IL-1β was required for upregulation of DEFB4, but not cathelicidin, whereas VDR activation was required for expression of both antimicrobial genes. The differential requirements for induction of DEFB4 and cathelicidin were reflected by differences in their respective promoter regions; the DEFB4 promoter had one vitamin D response element (VDRE) and two NF-κB sites, whereas the cathelicidin promoter had three VDREs and no NF-κB sites. Transfection of NF-κB into primary monocytes synergized with 1,25D3 in the induction of DEFB4 expression. Knockdown of either DEFB4 or cathelicidin in primary monocytes resulted in the loss of TLR2/1-mediated antimicrobial activity against intracellular mycobacteria. Therefore, these data identify a novel mechanism of host defense requiring the induction of IL-1β in synergy with vitamin D activation, for the TLR-induced antimicrobial pathway against an intracellular pathogen.

Novel Association of the Interleukin 2–Interleukin 21 Region With Inflammatory Bowel Disease

Genome-wide association studies have reported the role of the interleukin (IL) 2-IL21 chromosomal region at 4q27 in several autoimmune conditions. Mice deficient in IL-2 develop a disease with clinical and histological similarity to ulcerative colitis (UC) in humans. Modest evidence of linkage with UC was tentatively proposed for the IL2 gene more than a decade ago. Therefore, we decide to investigate the association of polymorphisms in the IL-2 axis (IL2, IL2RA, and IL2RB genes) with inflammatory bowel diseases (IBDs). Seven hundred and twenty-eight white Spanish unrelated IBD patients (356 Crohn's disease (CD) and 372 UC) and 549 ethnically matched controls were included in a case-control study. In addition, a Spanish replication cohort with 562 CD and 430 UC patients and 1,310 controls were analyzed. Eight single-nucleotide polymorphisms previously associated with different autoimmune diseases were analyzed using TaqMan chemistry. The IL2-rs6822844 polymorphism modified CD predisposition (P=0.002; odds ratio, OR (95% confidence interval, CI)=0.61 (0.44-0.84)); this was replicated in the other Spanish cohort, resulting in a strong protective effect of the minor allele in the merged samples (P=0.0002; OR (95% CI)=0.70 (0.58-0.85)). A similar effect of rs6822844 was detected for UC. Another marker, rs11938795, also showed evidence of an association with CD (P=0.006; OR (95% CI)=0.73 (0.58-0.92)). Polymorphisms within the IL2-IL21 linkage disequilibrium (LD) block show a novel association with IBD, this is concordant with suggestive previous results of whole genome analyses in CD and type 1 diabetes. Our data agree with the effect previously observed for other conditions and delineate a shared underlying mechanism.

Sequential Polarization and Imprinting of Type 1 T Helper Lymphocytes by Interferon-γ and Interleukin-12

Differentiation of naive T lymphocytes into type I T helper (Th1) cells requires interferon-gamma and interleukin-12. It is puzzling that interferon-gamma induces the Th1 transcription factor T-bet, whereas interleukin-12 mediates Th1 cell lineage differentiation. We use mathematical modeling to analyze the expression kinetics of T-bet, interferon-gamma, and the IL-12 receptor beta2 chain (IL-12Rbeta2) during Th1 cell differentiation, in the presence or absence of interleukin-12 or interferon-gamma signaling. We show that interferon-gamma induced initial T-bet expression, whereas IL-12Rbeta2 was repressed by T cell receptor (TCR) signaling. The termination of TCR signaling permitted upregulation of IL-12Rbeta2 by T-bet and interleukin-12 signaling that maintained T-bet expression. This late expression of T-bet, accompanied by the upregulation of the transcription factors Runx3 and Hlx, was required to imprint the Th cell for interferon-gamma re-expression. Thus initial polarization and subsequent imprinting of Th1 cells are mediated by interlinked, sequentially acting positive feedback loops of TCR-interferon-gamma-Stat1-T-bet and interleukin-12-Stat4-T-bet signaling.

Anti-inflammatory effects of cannabinoid receptor-2-selective agonists in immune cells (98.12)

Abstract A few studies have been published showing effects of cannabinoids in modulating immune response from T helper cell type 1 (Th1) to T helper cell type 2 (Th2) in a model of Legionella pneumophila infection. Cannabinoids act through two cloned receptors, CB1 and CB2. The CB1 receptor is expressed primarily in the CNS, whereas the CB2 receptor is highly expressed in immune cells in the periphery. Previous results from our laboratories indicated that the CB2 agonist had an anti-inflammatory effect on dendritic cells (DCs) and T cells. DCs differentiated in the presence of CB2 agonist produced less IL-12 upon LPS stimulation and T cells produced less IFN-gamma when co-cultured with these DCs in syngeneic culture in the presence of specific antigen. Here, we report that CB2 agonist treatment of DCs resulted in reduced STAT-1 phosphorylation. Also, treatment of polyclonally activated CD4+ T cells resulted in reduced expression of IL-12 receptor beta 2 chain, which correlated with decreased IFN-gamma production. Our data lead us to believe that CB2 agonists can modulate immune responses by acting directly on CD4+ T cells or through their effects on DCs. This work is supported by NIH/NIAID R01AI05230, and training grant T32 DA07237 (SA)

Transport of interleukin‐1 across cerebromicrovascular endothelial cells

The inflammatory cytokine interleukin-1 (IL-1) has profound actions in the brain, causing neuronal cell death and exacerbating brain damage. While circulating levels are normally low, IL-1 can be produced on the vascular side of the brain endothelium, and within the brain. The naturally occurring IL-1 receptor antagonist has been administered peripherally in a Phase II trial in acute stroke patients; understanding how IL-1 and IL-1 receptor antagonist penetrate the brain is, therefore, of considerable importance. An in vitro blood-brain barrier model was generated by co-culture of porcine brain microvascular endothelial cells with astrocytes. The mechanisms of transcellular transport of IL-1beta and IL-1 receptor antagonist were characterized in this model, using endocytosis inhibitors and IL-1 receptor-blocking antibodies. Transcellular IL-1beta and IL-1 receptor antagonist transport was temperature-dependent and IL-1beta was transported with higher affinity than IL-1 receptor antagonist. IL-1beta inhibited IL-1 receptor antagonist transport more potently than IL-1 receptor antagonist inhibited IL-1beta transport. Transport of IL-1beta and IL-1 receptor antagonist was not via adsorptive-mediated endocytosis, although inhibition of microtubule assembly significantly attenuated transport of both cytokines. An antibody directed to the type II IL-1 receptor significantly reduced IL-1beta transport. These results are consistent with IL-1 and IL-1 receptor antagonist being transported across cultured cerebromicrovascular endothelial cells and suggest that IL-1beta transport may occur via a type II IL-1 receptor-dependent mechanism. Understanding IL-1 transport into the brain may have benefits, particularly in enhancing penetration of IL-1 receptor antagonist into the brain.

IL10 and IL10 receptor gene variation and outcomes after unrelated and related hematopoietic cell transplantation.

Results of a previous study with human leukocyte antigen (HLA)-identical siblings showed individual and synergistic associations of single nucleotide polymorphisms in the promoter region of the recipient's IL10 gene and the donor's IL10 receptor beta (IL-10RB) gene with development of grades III-IV acute graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation. In this study of 936 patients who had unrelated donors, genotypes of single nucleotide polymorphisms in the IL10 gene and the IL-10RB gene were evaluated as correlates with outcomes after transplantation. We found no statistically significant associations of polymorphisms at positions -3575, -2763, -1082, and -592 of the IL10 gene or codon 238 of the IL10RB gene with severe acute GVHD, extensive chronic GVHD or nonrelapse mortality after hematopoietic cell transplantation. Among HLA-matched unrelated pairs, the patient's IL10/-592 genotype and donor's IL10RB/c238 genotype showed trends suggesting individual and combined associations with grades III-IV acute GVHD similar to those observed among patients with HLA-identical sibling donors. Although genetic variation in IL10 pathway affects risk of acute GVHD and non-relapse mortality in HLA-identical sibling transplants, the current results indicate that genetic variation in the IL10 pathway does not significant affect these outcomes in unrelated donor transplants suggesting that the strength of the alloimmune response in the latter exceeds the anti-inflammatory activity of IL10.

IL1β‐ and LPS‐induced serotonin secretion is increased in EC cells derived from Crohn’s disease

Gut mucosal enterochromaffin (EC) cells are regarded as key regulators of intestinal motility and fluid secretion via secretion of serotonin (5HT), are increased in numbers in mucosal inflammation and located in close proximity to immune cells. We examined whether interleukin (IL)1beta and Escherichia coli lipopolysaccharide (LPS) induced EC cell 5HT release through Toll-like/IL-1 (TIL) receptor activation, nuclear factor kappa B (NFkappaB) and mitogen-activated protein kinase (MAPK) phosphorylation and evaluated whether somatostatin could inhibit this phenomenon. Pure (>98%) human intestinal EC cells were isolated by fluorescent activated cell sorting from preparations of normal (n = 5) and Crohn's colitis (n = 6) mucosa. 5HT release was measured (ELISA), and NFkappaB and ERK phosphorylation quantitated (ELISA) in response to IL1beta and LPS. 5HT secretion was increased by both E. coli LPS (EC(50) = 5 ng mL(-1)) and IL1beta (EC(50) = 0.05 pmol L(-1)) >2-fold (P < 0.05) in Crohn's EC cells compared with normal EC cells. Secretion was reversible by the TLR4 antagonist, E. coli K12 LPS (IC(50) = 12 ng mL(-1)) and the IL1beta receptor antagonist (ILRA; IC(50) = 3.4 ng mL(-1)). IL1beta caused significant (P < 0.05) NFkappaB and MAPK phosphorylation (40-55%). The somatostatin analogue, lanreotide inhibited IL1beta-stimulated secretion in Crohn's (IC(50) = 0.61 nmol L(-1)) and normal EC cells (IC(50) = 1.8 nmol L(-1)). Interleukins (IL1beta) and bacterial products (E. coli LPS) stimulated 5HT secretion from Crohn's EC cells via TIL receptor activation (TLR4 and IL1beta). Immune-mediated alterations in EC cell secretion of 5HT may represent a component of the pathogenesis of abnormal bowel function in Crohn's disease. Inhibition of EC cell-mediated 5HT secretion may be an alternative therapeutic strategy in the amelioration of inflammatory bowel disease symptomatology.

A Functional Interleukin-1 Receptor Antagonist Polymorphism Influences Atherosclerosis Development The Interleukin-1.BETA.:Interleukin-1 Receptor Antagonist Balance in Atherosclerosis

Interleukin (IL)-beta plays a central role in inflammation and atherosclerosis, but levels of IL-1beta, its natural antagonist, IL-1Ra, and their balance in human atherosclerotic lesions, are unknown. Knowledge of protein levels in atherosclerosis and the influence of a functional IL-1Ra polymorphism would increase the understanding of atherosclerosis pathogenesis. Fresh and endotoxin-stimulated explanted human atherosclerotic and normal arteries were analyzed for IL-1beta, IL-1Ra and IL-1 receptor 1 (IL-1R1) using TaqMan PCR and enzyme-linked immunosorbent assay. Two hundred forty-three survivors of a first myocardial infarction were genotyped for a polymorphism in IL-1Ra and their coronary atherosclerosis analyzed by using coronary angiography. Levels of IL-1beta, IL-1Ra and IL-1R1 mRNA were significantly increased in atherosclerotic arteries compared with normal arteries. Endotoxin stimulation increased IL-1beta levels more than IL-1Ra levels (ie, promoted a pro-inflammatory state). A polymorphism in IL-1Ra known to increase levels of IL-1Ra was associated with decreased mean coronary artery plaque area. Activation of innate immunity changed the balance between IL-1beta and IL-1Ra in atherosclerotic arteries towards a more pro-inflammatory state. In line with this, the presence of an IL-1Ra intron 2 polymorphism known to increase IL-1Ra levels, and possibly the IL-1Ra:IL-1beta ratio, was associated with reduced coronary atherosclerosis.

Activation of Interleukin-1beta receptor suppresses the voltage-gated potassium currents in the small-diameter trigeminal ganglion neurons following peripheral inflammation

The glial cytokine, interleukin-1beta (IL-1beta), potentiates the excitability of nociceptive trigeminal ganglion (TRG) neurons via membrane depolarization following peripheral inflammation. Perforated patch-clamp technique was used this study to show that the mechanism underlying the excitability of small-diameter TRG neurons following inflammation is due to IL-1beta. Inflammation was induced by injection of complete Freund's adjuvant (CFA) into the whisker pad. The TRG neurons innervating the site of inflammation were identified by fluorogold (FG) labeling. The threshold for escape from mechanical stimulation applied to the orofacial area in inflamed rats was significantly lower than observed for control rats. IL-1beta at 1nM suppressed total voltage-gated K(+) currents in most TRG neurons (70%) under voltage-clamp conditions in control and inflamed rats. IL-1beta significantly decreased the total, transient (I(A)) and sustained (I(K)) currents in FG-labeled small-diameter TRG neurons in both groups. The IL-1beta-induced suppression of TRG neuron excitability was abolished by co-administration of ILra, an IL-1beta receptor blocker. The magnitude of inhibition of I(A) and I(K) currents by IL-1beta was significantly greater in inflamed rats than in controls. IL-1beta inhibited I(A) to a significantly greater extent than I(K). These results suggest that the inhibitory effect of I(A) and I(K) currents by IL-1beta in small-diameter TRG neurons potentiates neuronal excitability thereby contributing to trigeminal inflammatory hyperalgesia. These findings provide evidence for the development of voltage-gated K(+) channel openers and IL-1beta antagonists as therapeutic agents for the treatment of trigeminal inflammatory hyperalgesia.

Stronger inflammatory/cytotoxic T cell response in women identified by microarray analysis

AbstractWomen develop chronic inflammatory autoimmune diseases like lupus more often than men. The mechanisms causing the increased susceptibility are incompletely understood, although estrogen is believed to contribute. Chronic immune stimulation characterizes many autoimmune disorders. We hypothesized that repeated stimulation may cause a different T cell immune response in women than men. Microarray approaches were used to compare gene expression in T cells from healthy men and women with and without repeated stimulation. Four days following a single stimulation only 25% of the differentially expressed, gender-biased genes were expressed at higher levels in the women. In contrast, following restimulation 72% were more highly expressed in women. Immune response genes were significantly over-represented among the genes upregulated in women, and among the immune response genes, the inflammatory/cytotoxic effector genes interferon gamma (IFNG), lymphotoxin beta (LTB), granzyme A (GZMA), interleukin-12 receptor beta2 (IL12RB2), and granulysin (GNLY) were among those overexpressed to the greatest degree. In contrast, IL17A was the only effector gene more highly expressed in men. Estrogen response elements were identified in the promoters of half of the overexpressed immune genes in women, and in &amp;lt;10% of the male biased genes. The differential expression of inflammatory/cytotoxic effector molecules in restimulated female T cells may contribute to the differences in autoimmune diseases between women and men.

Lipid and Protein Substrates of PI3K in Cytokine Receptor Survival Signalling: Deregulation in Leukemia

New therapeutic approaches to acute myeloid leukemia (AML) must ultimately target cell survival pathways in leukemic cells in order to be effective. We have identified a serine residue (Ser585) in the cytoplasmic domain of the common GM-CSF and IL-3 receptor beta subunit which is phosphorylated in response to sub-picomolar concentrations of growth factor and is involved in signalling cytokine-mediated survival via 14-3-3 zeta phosphoserine adaptor. While Serine 585 is tightly controlled in non-transformed haematopoietic cells from normal donors, Serine 585 is constitutively phosphorylated in AML blasts suggesting a role in AML cell survival and a novel target for anti-leukaemic therapy. We attempted to isolate Ser585 kinase activity from leukemic blasts and characterise this activity in response to serine/threonine kinase inhibitors in biochemical and biological assays.Results: Cell extracts from primary AML blasts (>99% blasts by flow/morphology) obtained from adult patients were fractionated and assayed for intrinsic serine 585 peptide (13-mer) kinase activity via 32P gamma-ATP in vitro kinase assay. A single peak of Ser585 kinase activity was isolated and tested against a panel of serine/threonine kinase inhibitors. Kinase activity was selectively sensitive to LY294002, wortmannin and quercelin suggesting a role for the PI3K family of kinases in activating this residue. Ser585 kinase activity was also directly present in both p85 and p110 alpha PI3K immunoprecipitates from AML blasts and leukemic cell lines tested on both Ser585 peptide and recombinant beta cytoplasmic domain protein substrates. Serine 585 phosphorylation induced by sub-picomolar concentrations of GM-CSF in TF1.8 cells was inhibited by three different isoform selective p110 alpha inhibitors used at low nanomolar ranges consistent with reported IC50s. These results suggest a novel role for protein kinase rather than lipid kinase activity of PI3K alpha subunit in low dose cytokine signalling. We also show induction of serine phosphorylation of p85 PI3K regulatory subunit on Ser608 by GM-CSF, a previously reported protein substrate of PI3K. Furthermore, p110 alpha and delta inhibitors abrogate GM-CSF dependent survival of murine lineage negative bone marrow progenitor cells and also exert apoptotic activity on flow-sorted CD34+CD38−CD123+ sub-populations of primary AML blasts.Conclusions: Inhibition of Ser585 phosphorylation by targetting PI3K protein kinase activity by isoform selective inhibitors represents a novel approach toward the eradication of residual leukemic stem cells.

Disseminated Mycobacterium avium Infection in a Patient with a Novel Mutation in the Interleukin-12 Receptor-β1 Chain

A girl with relapsing cervical lymphadenopathy due to Mycobacterium avium subsequently developed abdominal adenopathy and intestinal inflammation. 1 known (c.1623_1624delGCinsTT) and 1 novel mutation (c.65_68delCTGC of exon2) of the Interleukin-12 Receptor-beta1 (IL-12Rbeta1) gene was detected. Unlike reports of a more favorable outcome in these patients, our patient died of severe intestinal involvement.