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Interleukin-12 (IL-12) is a proinflammatory cytokine that links innate and adaptive immune responses against tumor cells. Single Nucleotide Polymorphisms (SNPs) in IL-12 genes have been associated with cancer risk. However, limited studies have assessed the role of IL-12 in breast cancer (BC) risk comprehensively, and these were done in European and Asian populations. Here, we evaluated the association of the IL-12 signaling pathway and BC risk in Puerto Rican women. A genetic association study was completed with 461 BC cases and 463 non-BC controls. By logistic regression, IL-12 signaling SNPs were associated with an increased BC risk, including rs2243123 (IL12A), rs3761041, rs401502 and rs404733 (IL12RB1), rs7849191 (JAK2), rs280500 (TYK2) and rs4274624 (STAT4). Conversely, other SNPs were associated with reduced BC risk including rs438421 (IL12RB1), rs6693065 (IL12RB2), rs10974947, and rs2274471 (JAK2), rs10168266 and rs925847 (STAT4), and rs2069718 (IFNG). Analyses based in hormone receptors such as estrogen (ER) and progesterone (PR) receptors also revealed protective (for SNPs rs3212227-IL12B; rs3024896 and rs3821236-STAT4) and predisposing (for rs2069705-IFNG SNP) BC associations. Haplotype analysis showed a decreased BC risk for IL12B and STAT4 SNPs, whereas increased risk for IL12RB1 SNPs. This study suggests a role of the IL-12 signaling axis and BC risk. SNPs in this pathway may alter IL-12 induced anti-tumor responses and modulate BC predisposition in a population-specific context. Functional studies will be necessary to confirm these findings, which potentially may benefit IL-12 related immunotherapeutic approaches towards BC.

Background: The interleukin-12 receptor β1 (IL-12Rβ1) deficiency is a primary immunodeficiency (PID), affecting the immunological pathway of interleukin 12/interferon- γ (IL12/IFN-γ) axis and interleukin 23 receptor (IL23R). Defect in this pathway is mainly affecting the cellular immunity-related disorders. IL-12Rβ1 is a receptor chain of both the IL-12 and the IL-23 receptors and thus, deficiency of IL-12Rβ1 abolishes both IL-12 and IL-23 signaling. Material and methods: In this study, we performed whole exon sequencing and confirmatory Sanger sequencing in IL-12Rβ1. Evaluation of the IL12/IFN-γ axis was performed by assessment of patients’ whole blood cell to IL12/IFN-γ responding. Total and surface IL-12Rβ1expression was evaluated, in peripheral blood mononuclear cells (PBMCs) and T cell- derived PBMCs, and Th17 count was assessed. Results: In the present study, we described a c.1791 + 2T > G mutation at a splicing site position in IL-12Rβ1, using whole exome sequencing, and confirmed with targeted Sanger sequencing in a 26- year-old patient with Mendelian susceptibility to mycobacterial disease (MSMD) and Crohn's disease (CD). Complete lack of IL-12Rβ1 protein expression was detected in patient's PBMCs, compared to the healthy control. Furthermore, no IL-12Rβ1 protein was expressed on the cell surface. Interestingly, IL-12Rβ1-mutant cells showed an impaired response to IL12, and Bacillus Calmette–Guérin stimulation, confirming that the mutation is causative in this patient. Conclusion: A 3′splicing site mutation in IL12Rβ1, can be corresponding to the abolished expression of IL12Rβ1 in patients' cells, and associated with an impaired IL12-mediated signaling, which may lead not only to MSMD, but also to inflammatory bowel disease (IBD). Keywords: MSMD, IBD, IL12RB1, Mutation

Mendelian Susceptibility to Mycobacterial Diseases (MSMD) is a primary immunodeficiency disease (PID) characterized by variable susceptibility to weakly virulent mycobacteria (Bacille Calmette-Guerin, BCG) and various intramacrophagic bacteria, fungi, parasites. Mycobacterial disease generally begins in childhood, more rarely during adolescence and adulthood. The pathogenesis of MSMD is the inherited impaired production of interferon gamma (IFN-γ) or inadequate response to it. Autosomal recessive IL12RB1 deficiency is the most common genetic etiology of MSMD. Here we identified three novel compound heterozygous mutations in IL12RB1 gene (c.635G>A, c.765delG; c.632G>C, c.847C>T; c.64G>A, c.1673insGAGCTTCCTGAG) in three Chinese families with MSMD.

Objective Interleukin 12 receptor beta 1 (IL-12Rβ1) deficiency is a primary immunodeficiency that exposes affected individuals to an augmented risk of intracellular pathogen-mediated infections. The paradoxical presence of autoimmune manifestations in immune-deficient patients has been recognized, but the basis of this phenomenon is unclear, with the role of frequent infections being a possible trigger to break tolerance. Our study aimed to analyze extensively a profile of autoantibodies in a clinically well-defined case series of patients with IL-12Rβ1 deficiency. Methods Eight patients with IL-12Rβ1 deficiency referred to Children's Medical Center in Tunis, Tunisia, during 1995-2012 were enrolled in the study. Sixteen age- and gender-matched blood donors served as controls. Serum, liver-related autoantibodies immunoglobulin (Ig)G, IgM, IgA were tested by ELISA and by standard indirect immunofluorescence on Hep-2 cells. Results We found a significant prevalence of liver autoantibodies in the study group. Regarding primary biliary cholangitis (PBC), two of eight patients were positive for MIT3 autoantibodies, both confirmed by immunofluorescence, and one patient was positive for PBC-specific antinuclear antibodies, sp100. Moreover, two patients had significantly increased gamma-glutamyltransferase levels and one had IgM levels twice the upper limit of normal. Intriguingly two patients were positive for anti-actin antibodies; a typical feature of autoimmune hepatitis type 1, along with a significant increase in IgG levels. Conclusions This is the first report of a serological analysis in patients with an IL-12Rβ1 deficiency. Despite the difficulty in interpreting the role of the IL-12, the evidence of liver-specific autoantibodies confirms the importance its signal in liver autoimmunity.

Introduction: Basal cell carcinoma (BCC) is an immunogenic neoplasm and the imbalance in Th1/Th2 cytokines expression seems to play the major role in pathogenesis and clinical behaviour of the tumour. Aim : To investigate the association of soluble interleukin 2α receptor (sIL-2Rα) and interleukin-2 (IL-2) serum concentrations with BCC. Material and methods : The study involved 110 individuals with BCC and 60 healthy age- and sex-matched volunteers. Serum levels of sIL-2Rα and IL-2 were measured using ELISA test. Results : We found significantly (p = 0.027) increased sIL-2Rα serum levels in BCC patients, in comparison to healthy controls. Statistically (p = 0.04) higher sIL-2Rα levels were observed in patients with more advanced tumours. Serum levels of sIL-2Rα showed a significant linear (r = 0.24, p = 0.018) correlation with tumour size. The average IL-2 serum levels in BCC patients were statistically (p = 0.039) decreased compared to controls. Significantly (p = 0.0454) lower median IL-2 levels were observed in patients with more advanced tumours. A negative correlation between sIL-2Rα and IL-2 serum concentrations was revealed (r = –0.22; p = 0.027). Conclusions : Our results testify to the importance of the IL-2/sIL-2Rα signalling pathway in pathogenesis of BCC, suggesting that IL-2 and sIL-2Rα might be considered as potential markers of disease and targets for immunotherapy in BCC patients.

Contains fulltext : 155337.pdf (Publisher’s version ) (Closed access) IL-12Rbeta1 deficiency, also known as immunodeficiency 30 (IMD30, OMIM 614891), is a rare immunodeficiency syndrome caused by biallelic mutations in IL12RB1. Three second-degree relatives of a patient with this syndrome, all women, developed intestinal-type gastric cancer (GC). In the Netherlands the incidence of non-cardia GC in women is only 7 per 100,000 person years. Both relatives that were available for testing proved to be heterozygous for the familial IL12RB1 mutation, suggesting there might be a causal relation. Testing 29 index patients from families with early onset and/or a familial history of GC for germline mutations in both IL12RB1 and IL12RB2, that encodes the binding partner of IL-12Rbeta1, did not reveal other germline mutations in these genes. Therefore heterozygous inactivating mutations in IL12RB1 and IL12RB2 are unlikely to be frequently involved in GC predisposition. Additional research in families with IL12RB1 mutations is required to determine whether carriers of IL12RB1 mutations have an increased (gastric) cancer risk.

Introduction: Interleukin 12 receptor beta 1 (IL12Rβ1) deficiency is a primary immunodeficiency resulting mainly in susceptibility to opportunistic infection by non-tuberculous, environmental mycobacteria and severe infection caused by Salmonella spp.. Till now less than three hundred patients with IL12Rβ1 deficiency have been reported. Among them, only three have been described to develop autoimmunity. Case presentation: We present the case of a 50-year-old male with IL12Rβ1 deficiency due to compound heterozygosity (c. 1623_1624delGCinsTT (pGln542Stop) and c.1791+2T>C (donor splice site), who – 18 months after diagnosis of disseminated BCGitis – presented with recurrent fever and sicca syndrome. No indication of an infectious origin of these symptoms could be found at that point. The diagnosis of a Sjogren’s syndrome was made on the basis of fulfilled American-European consensus classification criteria, including a positive minor salivary gland biopsy. Conclusions: Apart from persistent antigenic stimulation, which may drive autoimmune inflammation in primary immunodeficiency, evidence on the involvement of IL-12 in pathogenesis of Sjogren’s syndrome suggests, that the same immunological mechanism may underlie both defense against infection and the maintenance of tolerance. To our knowledge, this is the first report of a case of autoimmunity in form of Sjogren’s syndrome in a patient with a primary immunodeficiency and one of the rare cases of IL12Rβ1 deficiency with manifested autoimmunity.

Patients with Mendelian Susceptibility to Mycobacterial Diseases (MSMD) exhibit variable vulnerability to infections by mycobacteria and other intramacrophagic bacteria (e.g., Salmonella and Klebsiella) and fungi (e.g., Histoplasma, Candida, Paracoccidioides, Coccidioides, and Cryptococcus). The hallmark of MSMD is the inherited impaired production of interferon gamma (IFN-γ) or the lack of response to it. Mutations in the interleukin (IL)-12 receptor subunit beta 1 (IL12RB1) gene accounts for 38% of cases of MSMD. Most IL12RB1 pathogenic allele mutations, including ten known stop-gain variants, cause IL-12Rβ1 complete deficiency (immunodeficiency-30, IMD30) by knocking out receptor cell-surface expression. IL12RB1 loss-of-function genotypes impair both IL-12 and IL-23 responses. Here, we assess the health effects of a rare, novel IL12RB1 stop-gain homozygous genotype with paradoxical IL-12Rβ1 cell-surface expression. We appraise four MSMD children from three unrelated Brazilian kindreds by clinical consultation, medical records, and genetic and immunologic studies. The clinical spectrum narrowed down to Bacillus Calmette-Guerin (BCG) vaccine-related suppurative adenitis in all patients with one death, and recrudescence in two, histoplasmosis, and recurrence in one patient, extraintestinal salmonellosis in one child, and cutaneous vasculitis in another. In three patients, we established the homozygous Trp7Ter predicted loss-of-function inherited genotype and inferred it from the heterozygote parents of the fourth case. The Trp7Ter mutation maps to the predicted IL-12Rβ1 N-terminal signal peptide sequence. BCG- or phytohemagglutinin-blasts from the three patients have reduced cell-surface expression of IL-12Rβ1 with impaired production of IFN-γ and IL-17A. Screening of 227 unrelated healthy subjects from the same geographic region revealed one heterozygous genotype (allele frequency 0.0022) vs. one in over 841,883 public genome/exomes. We also show that the carriers bear European ancestry-informative alleles and share the extended CACCAGTCCGG IL12RB1 haplotype that occurs worldwide with a frequency of 8.4%. We conclude that the novel IL12RB1 N-terminal signal peptide stop-gain loss-of-function homozygous genotype confers IL-12Rβ1 deficiency with varying severity and early-onset age through diminished cell-surface expression of an impaired IL-12Rβ1 polypeptide. We firmly recommend attending to warning signs of IMD30 in children who are HIV-1 negative with a history of adverse effects to the BCG vaccine and presenting with recurrent Histoplasma spp. and extraintestinal Salmonella spp. infections.

WOS: 000413141200005, PubMed ID: 29062247, Aim: Susceptibility to mycobacterial diseases is observed in some primary immunodeficiency diseases. In this study, we aimed to evaluate mycobacterial infections in primary immunodeficiency diseases. Material and Methods: Patients under follow-up by Ege University Pediatric Immunology Department for severe combined and combined immunodeficiencies, interleukin 12/interferon gamma receptor deficiency, nuclear factor kappa-beta essential modulator deficiency and chronic granulomatosis disease were evaluated retrospectively in terms of the frequency and characteristics of mycobacterial infections using a questionnaire form for demographic properties, clinical features and laboratory tests. Results: A diagnosis of mycobacterial infection was made clinically in a total of 25 patients including five (11.3%) of 45 patients who had severe combined immune deficiency, 12 (52.3%) of 21 patients who had chronic granulomatous disease, four patients (100%) who had interferon gamma receptor 2 partical deficiency, two patients (100%) who had interleukin 12 receptor beta 1 deficiency and one patient (100%) who had nuclear factor kapa-beta essential modulator deficiency. Mycobacterium strain could be typed in 14 (33%) of these 25 patients including Mycobacterium bovis, Mycobacterium chelonea, Mycobacterium elephantis, Mycobacterium fortuitum, and Mycobacterium tuberculosis. All patients were treated with anti-tuberculosis therapy. Thirty-six percent of these 25 patients underwent hematopoietic stem cell transplantation. Eight patients (five before, three after transplantation) died. Conclusions: Non-tuberculosis mycobacteria including mainly Mycobacterium bovis were observed with a higher rate compared to Mycobacterium tuberculosis in primary immunodeficiencies, especially in those affecting the interleukin 12/interferon gamma pathway. Early diagnosis of primary immunodeficiencies with neonatal screening program and preventing administration of the Bacille Calmette-Guerin vaccine in these patients is important.

The immunopathology of paucibacillary and multibacillary sheep paratuberculosis is characterized by inflammatory T cell and macrophage responses respectively. IL-23 and IL-25 are key to the development of these responses by interaction with their complex receptors, IL-23R/IL-12RB1 and IL-17RA/IL-17RB. In humans, variations in structure, sequence and/or expression of these genes have been implicated in the different pathological forms of tuberculosis and leprosy, and in gastrointestinal inflammatory disorders such as Crohn’s disease. Sequencing has identified multiple transcript variants of sheep IL23R, IL12RB1 and IL17RB and a single IL17RA transcript. RT-qPCR assays were developed for all the identified variants and used to compare expression in the ileo-caecal lymph node of sheep with paucibacillary or multibacillary paratuberculosis and uninfected animals. With IL-23 receptor, only the IL12RB1v3 variant, which lacks the receptor activation motif was differentially expressed and was significantly increased in multibacillary disease; this may contribute to high Th2 responses. Of the IL17RB variants only full length IL17RB was differentially expressed and was significantly increased in multibacillary pathology; which may also contribute to Th2 polarization. IL17RA expression was significantly increased in paucibacillary disease. The contrast between the IL17RA and IL17RB results may indicate that, in addition to Th1 cells, Th17 T cells are also involved in paucibacillary pathology. Electronic supplementary material The online version of this article (doi:10.1186/s13567-016-0314-4) contains supplementary material, which is available to authorized users.

In spite of current standard therapies to target the major pathomechanisms in myocardial infarction (MI), inflammatory gene expression patterns have been consistently revealed in MI patients. In a multiethnic cohort, we aimed to identify MI-associated pathomechanisms that may be unresponsive to medical treatment to improve diagnosis and therapy. Gene expression profiles in whole blood were analyzed in medicated Asian, African American and Caucasian patients living in Hawaii with a history of early MI and age, ethnicity, risk factor and medication-matched controls. PANTHER ontological and Ingenuity Pathway analysis and functional evaluation of the consistently differentially expressed genes identified coordinated up-regulation of genes for inflammation (LGALS3, PTX3, ZBTB32, BCL2L1), T-cell activation (IL12RB1, VAV3, JAG1, CAMP), immune imbalance (IL-8, IL2RA, CCR7, AHNAK), and active atherosclerosis (NR1H4, BIN1, GSTT1, MARCO) that persist in MI patients in spite of concerted treatment efforts to control vascular pathology. Furthermore, significant ethnic differences appear to exist within the active disease mechanisms that need to be further investigated to identify key targets for effective medical intervention.

Background: Interferon beta (IFNb) reduces relapse frequency and disability progression in patients with multiple sclerosis (MS). Objectives: Early identification of prognostic biomarkers of IFNb-treated patients will allow more effective management of MS. Methods: The IMPROVE study evaluated subcutaneous IFNb versus placebo in 180 patients with relapsing–remitting MS. Magnetic resonance imaging scans, clinical assessments, and blood samples were obtained at baseline and every 4 weeks from every participant. Thirty-nine biomarkers (32 transcripts; seven proteins) were studied in 155 patients from IMPROVE. Therapeutic response was defined by absence of new combined unique lesions, relapses, and sustained increase in Expanded Disability Status Scale over 1 year. A machine learning approach was used to examine the association between biomarker expression and treatment response. Results: While baseline levels of individual genes were relatively poor predictors, combinations of three genes were able to identify subjects with sub-optimal therapeutic responses. The triplet CASP2/IRF4/IRF6, previously identified in an independent dataset, was tested among other combinations. This triplet showed acceptable predictive accuracy (0.68) and specificity (0.88), but had relatively low sensitivity (0.22) resulting in an area under the curve (AUC) of 0.63. Other combinations of biomarkers resulted in AUC of up to 0.80 (e.g. CASP2/IL10/IL12Rb1). Conclusions: Baseline expression, or induction ratios, of specific gene combinations correlate with future therapeutic response to IFNb, and have the potential to be prognostically useful.

WOS: 000329131300012, PubMed ID: 24186907, Background. Interleukin 12R beta 1 (IL-12R beta 1)-deficient patients are prone to clinical disease caused by mycobacteria, Salmonella, and other intramacrophagic pathogens, probably because of impaired interleukin 12-dependent interferon gamma production. About 25% of patients also display mucocutaneous candidiasis, probably owing to impaired interleukin 23-dependent interleukin 17 immunity. The clinical features and outcome of candidiasis in these patients have not been described before, to our knowledge. We report here the clinical signs of candidiasis in 35 patients with IL-12R beta 1 deficiency. Results. Most (n = 71) of the 76 episodes of candidiasis were mucocutaneous. Isolated oropharyngeal candidiasis (OPC) was the most common presentation (59 episodes, 34 patients) and was recurrent or persistent in 26 patients. Esophageal candidiasis (n = 7) was associated with proven OPC in 2 episodes, and cutaneous candidiasis (n = 2) with OPC in 1 patient, whereas isolated vulvovaginal candidiasis (VVC; n = 3) was not. Five episodes of proven invasive candidiasis were documented in 4 patients; 1 of these episodes was community acquired in the absence of any other comorbid condition. The first episode of candidiasis occurred earlier in life (median age +/- standard deviation, 1.5 +/- 7.87 years) than infections with environmental mycobacteria (4.29 +/- 11.9 years), Mycobacterium tuberculosis (4 +/- 3.12 years), or Salmonella species (4.58 +/- 4.17 years) or other rare infections (3 +/- 11.67 years). Candidiasis was the first documented infection in 19 of the 35 patients, despite the vaccination of 10 of these 19 patients with live bacille Calmette-Guerin. Conclusions. Patients who are deficient in IL-12R beta 1 may have candidiasis, usually mucocutaneous, which is frequently recurrent or persistent. Candidiasis may be the first clinical manifestation in these patients., Fondo de Investigaciones Sanitarias, Ministerio de Economia y Competitividad [PI06/1031, PI10/01718]; European Regional Development Fund-European Social Fund (FEDER-FSE); Fundacion Canaria de Investigacion y Salud (Canarian government) [INREDCAN 05/06]; Foundation Caja Rural de Canarias-Chil y Naranjo; Universidad de Las Palmas de Gran Canaria; INSERMInstitut National de la Sante et de la Recherche Medicale (Inserm); University Paris Descartes; Rockefeller University; National Center for Research ResourcesUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USANIH National Center for Research Resources (NCRR); National Center for Advancing Sciences of the National Institutes of Health [8UL1TR000043]; Laboratoire d'Excellence "Integrative Biology of Emerging Infectious Diseases" [ANR-10-LABX-62-IBEID]; European Research Council, Agence Nationale de la RechercheFrench National Research Agency (ANR) [GENCMCD 11-BSV3-005-01]; St Giles Foundation; Candidoser Association; Consejo Nacional de Ciencia y Tecnologia de MexicoConsejo Nacional de Ciencia y Tecnologia (CONACyT) [69992, 182817], This work was supported by Fondo de Investigaciones Sanitarias, Ministerio de Economia y Competitividad (grants PI06/1031 and PI10/01718), the European Regional Development Fund-European Social Fund (FEDER-FSE), Fundacion Canaria de Investigacion y Salud (Canarian government; INREDCAN 05/06), Foundation Caja Rural de Canarias-Chil y Naranjo (research prize 2004), Universidad de Las Palmas de Gran Canaria (fellowship to E. H. R), INSERM, University Paris Descartes, Rockefeller University, National Center for Research Resources and the National Center for Advancing Sciences of the National Institutes of Health (grant 8UL1TR000043), Laboratoire d'Excellence "Integrative Biology of Emerging Infectious Diseases" (grant ANR-10-LABX-62-IBEID), the European Research Council, Agence Nationale de la Recherche (grant GENCMCD 11-BSV3-005-01), the St Giles Foundation, the Candidoser Association, and Consejo Nacional de Ciencia y Tecnologia de Mexico (grants 69992 and 182817).

Tuberculosis (TB), an infectious disease caused by infection of Mycobacterium tuberculosis, is a major public health challenge globally. Genetic epidemiological evidence suggests a genetic basis for TB, but the molecular mechanism for a genetic predisposition to TB remains largely unknown. Thirty-five tag single-nucleotide polymorphisms (SNPs) across 11 candidate cytokines and related genes, including IL-12/IFN-γ axis genes (IL12B, IL12RB1, IL18R1, IL27, IFNGR1, IFNGR2 and STAT1), the TNF gene locus (TNF and LTA), IL10, and CCL2, were genotyped using Sequenom's iPLEX assays in 1,032 patients with TB and 1,008 controls of Chinese Han origin. We did not find that any of the 35 tag SNPs individually or as haplotypes was significantly associated with susceptibility to TB, on the basis of multivariable logistic regression analysis with adjustment for age and sex. However, stratification analyses showed that, in those with age 46 years or older, carrying the rs1974675 T allele in the IL18R1 gene had a significantly decreased susceptibility to TB occurrence compared with carrying the C/C genotype (OR = 0.57, P = 5.0×10(-4)). Further analysis indicated that a SNP in absolute linkage disequilibrium with rs1974675, rs3755276, is located within a CpG dinucleotide and showed hypomethylation in controls than in patients (19.6% vs. 31.4%; P = 1.0×10(-4)) and genotype-specific DNA methylation at the IL18R1 promoter and IL18R1 mRNA levels. In addition, DNA methylation levels were significantly inversely correlated with mRNA levels. Thus, decreased mRNA levels of IL18R1 due to rs3755276 may partially mediate the increased susceptibility to TB risk.

Mendelian susceptibility to mycobacterial disease (MSMD) is a rare disorder with predisposition to severe, sometimes lethal, disease caused by otherwise poorly virulent, non-tuberculous environmental mycobacteria and poorly virulent salmonellae. In patients with MSMD, mutations have been identified in five genes that encode for the proteins IL-12/IL-23p40, IL-12/ IL-23Rbeta1, IFN-R1, IFN-gammaR2 and STAT1. These proteins play important roles in the type-1 cytokine pathway, which is crucial for human host defence against intracellular pathogens such as mycobacteria and salmonellae. We report a girl with mild Mycobacterium bovis Bacille Calmette-Guérin (BCG) disease and Salmonella enteritidis cervical lymphadenitis. Despite treatment, she has remained a fecal carrier of S. enteritidis for the past 14 years. She was found to have complete IL-12/IL-23Rbeta1 deficiency. A homozygous r.518G>C IL12RB1 mutation was identified, leading to a non-functional R173P substitution in the IL-12/IL-23Rbeta1 protein. This mutation abrogated IL-12/IL-23Rbeta1 cell-surface expression and resulted in complete lack of T cell responsiveness to both IL-12 and IL-23.

IL-12R1 deficiency is an autosomal recessive disorder characterized by predisposition to recurrent and/or severe infections caused by otherwise poorly pathogenic mycobacteria and salmonella. IL-12R1 is a receptor chain of both the IL-12 and the IL-23 receptor and deficiency of IL-12R1 thus abolishes both IL-12 and IL-23 signaling. IL-12R1 deficiency is caused by bi-allelic mutations in the IL12RB1 gene. Mutations resulting in premature stop codons, such as nonsense, frame shift, and splice site mutations, represent the majority of IL-12R1 deficiency causing mutations (66%; 46/70). Also every other morbid mutation completely inactivates the IL-12R1 protein. In addition to disease-causing mutations, rare and common variations with unknown functional effect have been reported in IL12RB1. All these variants have been deposited in the online IL12RB1 variation database (www.LOVD.nl/IL12RB1). In this article, we review the function of IL-12R1 and molecular genetics of human IL12RB1. (C) 2013 Wiley Periodicals, Inc.

Palamaro et al. describe a child with recurrent bronchopneumonia and very high IgE levels in which a variation, R156H, was found in the IL12RB1 gene that encodes the IL-12Rβ1 chain. Based on the absence of this variation in 50 unrelated individuals they conclude it is a mutation. We (van de Vosse and van Dissel) feel there is no reason to suspect a defect in IL-12 signaling based on the clinical data, nor evidence for a functional defect in IL-12 signaling in this patient. In addition, the variation is not novel and known as a polymorphism. Without any functional evidence that R156H is a mutation, the current claim is not substantiated. Palamaro et al. respond to argue that the amino acid substitution, R156H described in the described case exerts a summatory effect, as a genetic cofactor, along with an additional and still unidentified molecular alteration of the same pathway.

Human granulocyte-macrophage colony-stimulating factor (hGM-CSF) receptor (hGMR) consists of alpha and beta subunits, and the precise stoichiometry of these subunits has remained to be determined. In this work, oligomerization of the beta subunit was studied using a chemical cross-linker. In Ba/F3, a mouse interleukin-3-dependent cell line expressing both subunits of hGMR (Ba/F3-alpha,beta), a protein with a molecular mass corresponding to that of a homodimer of the beta subunit (beta homodimer) was detected only when cells were treated with the cross-linker. Dimerization of the beta subunit was confirmed by coimmunoprecipitation of a tagged beta subunit with the wild type beta subunit COS7 cells. The beta homodimer had already formed in the absence of hGM-CSF, whereas stimulation with the ligand brought both alpha and beta subunits into a complex, the result being tyrosine phosphorylation of the beta homodimer. Tyrosine phosphorylation of the subunit was impaired by deletion of the cytoplasmic domain of the alpha subunit without interfering with the association of both subunits. These results indicate that the beta homodimer, which alone is insufficient for signaling, forms the functional hGMR with the alpha subunit in response to hGM-CSF.

The interleukin 3 (IL-3), IL-5, and granulocyte/macrophage colony-stimulating factor receptors consist of a cytokine-specific alpha subunit and the common beta subunit. Whereas IL-3 stimulates various lineages of hematopoietic cells, including multipotential progenitors, IL-5 acts mainly as an eosinophil lineage-specific factor. To investigate whether the lineage specificity of IL-5 is due to restricted expression of the IL-5 receptor alpha subunit (IL-5R alpha), we generated transgenic mice that express the mouse IL-5R alpha constitutively by phosphoglycerate kinase promoter. The transgenic mouse expressed IL-5R alpha ubiquitously, and the bone marrow cells formed various types of colonies, including multi-lineage colonies, in response to IL-5. IL-5 also supported formation of both multi-lineage and blast cell colonies from dormant progenitors of the 5-fluorouracil-treated transgenic mice. The cells composing the blast cell colony gave rise to many colonies including multi-lineage colonies when they were replated in secondary culture containing either Il-5 or IL-3. There was no significant difference in replating efficiency or in types of secondary colonies between IL-5- and IL-3-stimulated cultures. Conversely, the cells from the IL-3-induced blast cell colonies of the transgenic mice proliferated in response to either IL-3 or IL-5. Thus, the development of the progenitors can be equally supported by either IL-5 or IL-3, suggesting that intracellular signals from the IL-3R can be replaced by those from IL-5. These results strongly suggest that the lineage specificity of IL-5 is mainly due to the restricted expression of IL-5R alpha.

International audience; The long terminal repeat (LTR) sequences of endogenous retroviruses and retroelements contain promoter elements and are known to form chimeric transcripts with nearby cellular genes. Here we show that an LTR of the THE1D retroelement family has been domesticated as an alternative promoter of human IL2RB, the gene encoding the beta subunit of the IL-2 receptor. The LTR promoter confers expression specifically in the placental trophoblast as opposed to its native transcription in the hematopoietic system. Rather than sequence-specific determinants, DNA methylation was found to regulate transcription initiation and splicing efficiency in a tissue-specific manner. Furthermore, we detected the cytoplasmic signaling domain of the IL-2Rbeta protein in the placenta, suggesting that IL-2Rbeta undergoes preferential proteolytic cleavage in this tissue. These findings implicate novel functions for this cytokine receptor subunit in the villous trophoblast and reveal an intriguing example of ancient LTR exaptation to drive tissue-specific gene expression.

The increased incidence and severity of Clostridium difficile infection (CDI) in older adults (age, ⩾65 years) corresponds with the emergence of the BI/NAP1 strain, making elucidation of the host immune response extremely important. We therefore infected germ-free C57BL/6 mice aged 7–14 months with a BI/NAP1 strain and monitored the mice for response. Infected mice were moribund 48–72 h after infection and developed gross and histological cecitis and colitis and elevated concentrations of keratinocyte chemoattractant, interleukin 1β, monocyte chemotactic protein 1, and granulocyte colony-stimulating factor and decreased levels of interferon γ, interleukin 12 p40, interleukin 12 p70, and interleukin 10 compared with controls. We conclude that aged, germ-free C57BL/6 mice are susceptible to fulminant CDI from a BI/NAP1 strain and represent a novel model to further elucidate the host immune response to acute CDI.

This study investigated how CD8(+) T cell subsets respond to allo- and infectious immunity after living donor liver transplantation (LDLT). Early alloimmunity: 56 recipients were classified into three types according to the post-transplant course; type I demonstrated uneventful post-transplant course, type II developed severe sepsis leading to multiple organ dysfunction syndrome or retransplantation and type III with acute rejection. In 23 type I recipients, the interleukin (IL)-12 receptor beta-1 (R beta 1)(+) cells of central memory T cells (Il-12R beta 1(+) T(CM)) were increased above the pretransplant level. In 16 type II recipients, IL-12R beta 1(+) T(CM) was decreased markedly below the pretransplant level on postoperative day (POD) 5. In 17 type III recipients, IL-12R beta 1(+) T(CM) was decreased for a more prolonged period until POD 10. Along with down-regulation of IL-12R beta 1(+) T(CM), the IL-12R beta 1(+) cells of CCR7-negative subsets (CNS) as well as perforin, interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha decreased gradually, resulting in the down-regulation of effectors and cytotoxicity. The down-regulation of IL-12R beta 1(+) T(CM) was suggested to be due to the recruitment of alloantigen-primed T cells into the graft, and then their entry into the secondary lymphoid organ, resulting in graft destruction. Infectious immunity: immunocompetent memory T cells with the capacity to enhance effectors and cytotoxicity were generated in response to post-transplant infection along with both up-regulation of the IL-12R beta 1(+) T(CM) and an increase in the CNS showing the highest level of IL-12R beta 1(+) cells. In conclusion, this work demonstrated that the IL-12R beta 1(+) cells of T(CM) and CNS are regulated in a tightly coupled manner and that expression levels of IL-12R beta 1(+) T(CM) play a crucial role in controlling allo- and infectious immunity.

Mutations in IFNGR1, IFNGR2, IL12RB1, IL12B, STAT1 and NEMO result in a common clinical phenotype known as Mendelian Susceptibility to Mycobacterial Diseases (MSMD). Interleukin-12 receptor 01 (IL12R beta 1) deficiency is the most common genetic etiology for MSMD. Known mutations affecting IL12RB1 are recessively inherited and are associated with null response to both IL-12 and IL-23. Mutation IL12RB1 1623_1624delinsTT was originally described in 5 families from European origin (2 from Germany: I from Cyprus, France and Belgium). Interestingly, this same mutation was found in an unexpectedly high prevalence among IL-12R beta 1 deficient patients in Argentina: 5-out-of-6 individuals born to unrelated families carried this particular change. To determine whether mutation 1623_1624delinsTT represents a DNA mutational hotspot or a founder effect, 34 polymorphic markers internal or proximal to IL12RB1 were studied in the Argentinean and the Belgian patients. A common haplotype spanning 1.45-3.51 Mb was shared by all chromosomes carrying mutation 1623_1624delinsTT, and was not detected on 100 control chromosomes. Applying a modified likelihood-based method the age of the most recent common ancestor carrying mutation 1623_1624delinsTT was estimated in 475 years (95% CI, 175-1275), which is the time when the Spaniards initiated the colonization of the Americas. Mutation 1623_1624delinsTT represents the first founder effect described on IL-12R beta 1, the most frequently affected gene in MSMD, and affecting patients with European ancestors. The reason(s) behind the persistency of this mutation across multiple generations, its relative high prevalence, and any potential selective advantage are yet to be established.

Expression of metabotropic glutamate receptor 5 (mGluR5) protein is known to be plastic and to depend critically oil the astrocytes' microenvironment. In the present study we investigated whether interleukins, which are involved in the immune response following brain injury, could contribute to the regulation of mGluR5 protein in human astrocytes in culture. Using Western blotting and immunocytochemistry, no detectable changes in the expression of the mGluR5 protein were observed with both interleukin 1 beta and interleukin 6 in undifferentiated cultures (growing in serum free media). In contrast, in Cultures that had been morphologically differentiated by exposure to epidermal growth factor (EGF), addition of interleukin 1 beta (but not interleukin 6) reduced rnGluR5 protein expression. In addition, stimulation of phosphoinositide hydrolysis by the selective group 1 agonist (S)-3,5-dihydroxyphenylglycine (DHPG) was reduced after exposure to interleukin 1 beta. The suppressive effect on mGluR5 was prevented by the interleukin 1 receptor antagonist. Thus, interleukin 1 beta may represent an additional pathway through which mGluR5 expression and function call be modulated in astrocytes under different pathological conditions associated with an inflammatory response. (c) 2004 Elsevier B.V. All rights reserved

We have identified a cDNA from a human phytohemagglutinin-activated lymphoblast library encoding a protein that binds125I-labeled human interleukin 12 (125I-huIL-12) with aKdof about 5 nM when expressed in COS-7 cells. When coexpressed in COS-7 cells with the previously identified IL-12 β receptor (IL-12Rβ) protein, two classes of125I-huIL-12 binding sites were measured withKds of about 55 pM and 8 nM, corresponding to the high- and low-affinity binding sites seen on phytohemagglutinin-activated lymphoblasts. This newly identified huIL-12R subunit is a member of the cytokine receptor superfamily, with closest resemblance to the β-type cytokine receptor gp130 and the receptors for leukemia inhibitory factor and granulocyte colony-stimulating factor. Consequently, we have reclassified the previously identified IL-12Rβ subunit as huIL-12Rβ1 and designated the newly identified subunit as huIL-12Rβ2. huIL-12Rβ2 is an 862-amino acid type I transmembrane protein with a 595-amino-acid-long extracellular domain and a cytoplasmic tail of 216 amino acids that contains three tyrosine residues. A cDNA encoding the mouse homolog of the huIL12Rβ2 protein has also been isolated. Human and mouse IL-12Rβ2 proteins show a 68% amino acid sequence identity. When expressed in COS-7 cells, huIL-12Rβ2 exists as a disulfide-linked oligomer with an apparent monomeric molecular weight of 130 kDa. Coexpression of the two identified IL-12R subunits in Ba/F3 cells conferred IL-12 responsiveness, and clones of these cotransfected Ba/F3 cells that grew continuously in the presence of IL-12 were isolated and designated LJM-1 cells. LJM-1 cells exhibited dose-dependent proliferation in response to huIL-12, with an ED50of about 1 pM huIL-12. Interestingly, Ba/F3 cells transfected with IL-12Rβ2 alone proliferated in response to huIL-12 with an ED50of about 50 pM, although a role for endogenous mouse IL-12Rβ1 in IL-12 signal transduction in these transfectants cannot be ruled out. These results demonstrate that the functional high-affinity IL-12R is composed of at least two β-type cytokine receptor subunits, each independently exhibiting a low affinity for IL-12.

An amino-terminally truncated beta C receptor (beta C-R) subunit of the interleukin-3 (IL3)/granulocyte-macrophage colony-stimulating factor/IL5 receptor complex mediates factor-independent and tumorigenic growth in two spontaneous mutants of a promyelocytic cell line. The constitutive activation of the JAK2 protein kinase in these mutants confirms that signaling occurs through the truncated receptor protein. Noteworthily, in addition to a 10-kb deletion in the beta C-R subunit gene encoding the truncated receptor, several secondary and independent mutations that result in the deletion or functional inactivation of the allelic beta C-R subunit and the closely related beta IL3-R subunit genes were observed in both mutants, suggesting that such mutations are necessary for the full oncogenic penetrance of the truncated beta C-R subunit. Reversion of these mutations by the expression of the wild-type beta C-R in the two mutants resulted in a fivefold decrease in cloning efficiency of the mutants in the absence of IL3, confirming a functional interaction between the wild-type and truncated proteins. Furthermore, expression of the truncated beta C-R subunit in factor-dependent myeloid cells did not immediately render the cells autonomous but increased the spontaneous frequency to factor-independent growth by 4 orders of magnitude. Implications for both leukemogenic progression and receptor-subunit interaction and signaling are discussed.

International audience; To explore the possible involvement of STAT factors ("signal transducers and activators of transcription") in the interleukin 2 receptor (IL-2R) signaling cascade, murine HT-2 cells expressing chimeric receptors composed of the extracellular domain of the erythropoietin receptor fused to the cytoplasmic domains of the IL-2R beta or -gamma c chains were prepared. Erythropoietin or IL-2 activation of these cells resulted in rapid nuclear expression of a DNA-binding activity that reacted with select STAT response elements. Based on reactivity with specific anti-STAT antibodies, this DNA-binding activity was identified as a murine homologue of STAT-5. Induction of nuclear expression of this STAT-5-like factor was blocked by the addition of herbimycin A, a tyrosine kinase inhibitor, but not by rapamycin, an immunophilin-binding antagonist of IL-2-induced proliferation. The IL-2R beta chain appeared critical for IL-2-induced activation of STAT-5, since a mutant beta chain lacking all cytoplasmic tyrosine residues was incapable of inducing this DNA binding. In contrast, a gamma c mutant lacking all of its cytoplasmic tyrosine residues proved fully competent for the induction of STAT-5. Physical binding of STAT-5 to functionally important tyrosine residues within IL-2R beta was supported by the finding that phosphorylated, but not nonphosphorylated, peptides corresponding to sequences spanning Y392 and Y510 of the IL-2R beta tail specifically inhibited STAT-5 DNA binding.

IL-17 and related cytokines are direct and indirect targets of selective immunosuppressive agents for the treatment of autoimmune diseases and other diseases of pathologic inflammation. Insights into the potential adverse effects of IL-17 blockade can be drawn from the experience of patients with deficiencies in the IL-17 pathway. A unifying theme of susceptibility to mucocutaneous candidiasis is seen in both mice and humans with a variety of genetic defects that converge on this pathway. Mucocutaneous candidiasis is a superficial infection of mucosal, nail or skin surfaces usually caused by the fungal pathogen Candida albicans. The morbidity of the disease includes significant pain, weight loss and secondary complications, including carcinoma and aneurysms. This review describes the known human diseases associated with chronic mucocutaneous candidiasis (CMC) as well as the known and proposed connections to IL-17 signaling. The human diseases include defects in IL-17 signaling due to autoantibodies (AIRE deficiency), receptor mutations (IL-17 receptor mutations) or mutations in the cytokine genes (IL17F and IL17A). Hyper-IgE syndrome is characterized by elevated serum IgE, dermatitis and recurrent infections, including CMC due to impaired generation of IL-17-producing Th17 cells. Mutations in STAT1, IL12B and IL12RB1 result in CMC secondary to decreased IL-17 production through different mechanisms. Dectin-1 defects and CARD9 defects result in susceptibility to C. albicans because of impaired host recognition of the pathogen and subsequent impaired generation of IL-17-producing T cells. Thus, recent discoveries of genetic predisposition to CMC have driven the recognition of the role of IL-17 in protection from mucosal fungal infection and should guide counseling and management of patients treated with pharmacologic IL-17 blockade.

The expression of beta 2-adrenergic receptors is up-regulated by glucocorticoids. In contrast, beta 1-adrenergic receptors display glucocorticoid-induced down-regulation. In rat C6 glioma cells, which express both of these subtypes of beta-adrenergic receptors, the synthetic glucocorticoid dexamethasone stimulates no change in the total beta-adrenergic receptor content, but rather shifts the beta 1:beta 2 ratio from 80:20 to 50:50. Radioligand binding and immunoblotting demonstrate a sharp decline in beta 1-adrenergic receptor expression. Metabolic labelling of cells with [35S]-methionine in tandem with immunoprecipitation by beta 1-adrenergic-receptor-specific antibodies reveals a sharp decline in the synthesis of the receptor within 48 h for cells challenged with glucocorticoid. Steady-state levels of beta 1-adrenergic-receptor mRNA declined from 0.47 to 0.26 amol/microgram of total cellular RNA within 2 h of dexamethasone challenge, as measured by DNA-excess solution hybridization. The stability of receptor mRNA was not influenced by glucocorticoid; the half-lives of the beta 1- and beta 2-subtype mRNAs were 1.7 and 1.5 h respectively. Nuclear run-on assays revealed the basis for the down-regulation of receptor expression, i.e. a sharp decline in the relative rate of transcription for the beta 1-adrenergic-receptor gene in nuclei from dexamethasone-treated as compared with vehicle-treated cells. These data demonstrate transcriptional suppression as a molecular explanation for glucocorticoid-induced down-regulation of beta 1-adrenergic receptors.

The beta 2-adrenergic receptor (beta 2AR) can be constitutively activated by mutations in the third intracellular loop. Whereas the wild-type receptor exists predominantly in an inactive conformation (R) in the absence of agonist, the mutant receptor appears to spontaneously adopt an active conformation (R*). We now demonstrate that not only is the mutant beta 2AR constitutively active, it is also constitutively desensitized and down-regulated. To assess whether the mutant receptor can constitutively engage a known element of the cellular desensitization machinery, the receptor was purified and reconstituted into phospholipid vesicles. These preparations retained the essential properties of the constitutively active mutant receptor: agonist-independent activity [to stimulate guanine nucleotide-binding protein (Gs)-GTPase] and agonist-specific increase in binding affinity. Moreover, the purified mutant receptor, in the absence of agonist, was phosphorylated by recombinant beta AR-specific kinase (beta ARK) in a fashion comparable to the agonist-occupied wild-type receptor. Thus, the conformation of the mutated receptor is equivalent to the active conformation (R*), which stimulates Gs protein and is identical to the beta ARK substrate.

The 14-residue peptide (peptide 14) corresponding to Arg2410-Lys2423 of the insulin-like growth factor II receptor (IGF-IIR) can activate the adenylate cyclase-inhibitor guanine nucleotide-binding protein Gi, and the 15-residue beta III-2 peptide Arg259-Lys273 of the beta 2-adrenergic receptor (beta 2AR) can activate the stimulatory protein Gs. In phospholipid vesicles, IGF-IIR and beta 2AR activate Gi and Gs in response to IGF-II and isoproterenol, respectively. We constructed a chimeric IGF-II receptor (beta III-2/IGF-IIR) by converting its native peptide 14 sequence to the beta III-2 sequence. In cells expressing beta III-2/IGF-IIR, membrane adenylate cyclase activity markedly increased without IGF-II and was further promoted by IGF-II. This was verified by measuring chloramphenicol acetyltransferase (CAT) activity in beta III-2/IGF-IIR cells with cotransfection of a cAMP response element-CAT construct. This study shows not only the conversion of G-protein specificity of a receptor from Gi to Gs but also the simulation of G protein-coupled receptor signals by using a short receptor region and intact cells. These findings indicate that the G protein-activation signals are interchangeable, self-determined structural motifs that function in the setting of either a single-spanning or multiple-spanning receptor.

The interleukin 2 receptor (IL-2R) consists of at least two subunits, alpha and beta, both of which can bind interleukin 2 (IL-2). Recent studies have demonstrated the existence of a third subunit, a 64-kDa molecule termed IL-2R gamma chain, and have suggested that gamma chain functions to regulate the rate of IL-2 dissociation from the receptor. In the present report we have addressed whether the gamma chain modulates IL-2R affinity by contributing contact sites for IL-2 binding. Using reagents that allow the IL-2R complex to be immunoprecipitated through the IL-2 molecule itself, we demonstrate the existence of a stable IL-2-IL-2R gamma-chain complex. These studies thus establish that the IL-2R gamma chain directly contributes to the IL-2-binding site, consistent with the hypothesis that gamma chain influences IL-2R affinity through its direct interaction with IL-2.

International audience; In this report, we investigated the transforming properties of retinoic acid receptor beta (RAR beta). The v-erbA protein, which is the viral oncogenic homologue of the thyroid hormone receptor, was replaced by either the complete RAR beta (beta R) or a hepatitis B virus pre-S-RAR beta (H beta R) hybrid product in an avian erythroblastosis virus-based vector. In chicken hematopoietic cells, the H beta R protein was able to transform erythroid progenitor cells, whereas no such transformation was observed with the wild-type beta R protein. Moreover, the fully transformed phenotype was observed even in the absence of v-erbB, and H beta R-transformed erythroid cells grew independently of growth factors and transforming growth factor alpha. The analysis of erythrocytic-specific proteins revealed that the transformed cells were blocked at the colony-forming unit-erythroid stage and that the expression of the carbonic anhydrase II gene, a gene normally regulated by thyroid hormones, was repressed by the H beta R protein. Finally, hepatocarcinomas rapidly developed in some chickens infected in ovo with viruses encoding either the normal or the hybrid H beta R, suggesting that an inappropriate expression of the RAR beta gene may represent an important event in oncogenesis.

We have constructed a hybrid cDNA coding for a fusion protein between human interleukin 2 and a truncated heavy chain from human immunoglobulin M. The protein encoded by this cDNA contains the entire interleukin 2 sequence including its signal peptide, fused at its C terminus to domains 2 to 4 of the immunoglobulin heavy-chain constant region. Cells transfected with the hybrid cDNA secrete multimeric forms of the fusion protein, which bind specifically to cells bearing high-affinity interleukin 2 receptors. This binding leads either to T-cell proliferation or, if complement is added, to T-cell death. Multimeric forms of the fusion protein with a molecular mass above 500 kDa mediate complement-dependent lysis but trigger proliferation inefficiently when compared with forms with a low molecular mass (< 500 kDa). In contrast, the latter efficiently mediate T-cell proliferation without inducing complement-dependent lysis. The high molecular mass fusion proteins could thus constitute valuable tools for specific immunosuppression in humans.

The murine interleukin 3 receptor (mIL-3R) is a heterodimer consisting of a 70-kDa alpha subunit and one of two alternative 120-kDa beta subunits termed beta IL-3 and beta c. beta IL-3 (originally called Aic2A) is capable of binding mIL-3 by itself, whereas beta c (Aic2B) does not bind any ligand on its own but increases the affinity of mIL-3, murine granulocyte/macrophage-colony-stimulating factor, and mIL-5 for their respective alpha subunits. Interestingly, although the mIL-3R does not possess tyrosine kinase activity, its beta IL-3 subunit does become tyrosine phosphorylated upon binding mIL-3. To further investigate the properties of this subunit, we have purified it from the cell line B6SUtA1, which expresses a high level of mIL-3R. Intriguingly, studies comparing the stability of the 140-kDa, tyrosine-phosphorylated form of this subunit with its 120-kDa, non-tyrosine-phosphorylated form reveal that the former is far less stable and is rapidly degraded to a 70-kDa fragment. Mixing experiments demonstrate that the differential stability of the two forms is due to an intrinsic difference in protease susceptibility. Phosphatase studies indicate that the higher protease susceptibility of the tyrosine-phosphorylated beta IL-3 is due to the presence of both phosphotyrosine and phosphoserine residues. Western analyses using an anti-N-terminal mIL-3R beta IL-3 chain antibody reveal that this proteolytic cleavage also occurs rapidly in intact cells following stimulation with mIL-3 and occurs at the cell surface, since it takes place within minutes at 37 degrees C, is observed with purified plasma membranes, and is not inhibited by chloroquine. This degradative step may play an important role in the mechanism of action of mIL-3.

The high-affinity receptors for human granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 3 (IL-3) and interleukin 5 (IL-5) are composed of two distinct subunits, alpha and beta c. The alpha subunits are specific for each cytokine, whereas the beta subunit (beta c) is shared by the three receptors and is an essential component of signal transduction. We have made a series of mutant beta c cDNAs that delete various regions of the cytoplasmic domain and examined the function of these mutants by coexpressing them with the alpha subunit of the human GM-CSF receptor (hGMR) in an IL-3-dependent mouse pro-B cell line BaF3. Two domains in the membrane-proximal portion of beta c were found to be important for transducing the hGM-CSF-mediated growth signals: one domain between Arg456 and Phe487 appears to be essential for proliferation, and the second domain between Val518 and Asp544 enhances the response to GM-CSF, but is not absolutely required for proliferation. The region between Val518 and Leu626 was responsible for major tyrosine phosphorylation of 95 and 60 kDa proteins. Thus, beta c-mediated major tyrosine phosphorylation of these proteins was apparently separated from proliferation. However, the beta 517 mutant lacking residues downstream of Val518 transmitted a herbimycin-sensitive proliferation signal, suggesting that beta 517 still activates a tyrosine kinase(s). We also evaluated the role of the cytoplasmic domain of the GMR alpha subunit and the results suggest that it is involved in the hGM-CSF-mediated signal transduction, but is not essential.(ABSTRACT TRUNCATED AT 250 WORDS)

Interleukin 1 receptor antagonist (IL-1ra), a naturally occurring polypeptide with amino acid sequence homology to interleukin 1 alpha (IL-1 alpha) and interleukin 1 beta (IL-1 beta), prevents Escherichia coli-induced shock and death. Both IL-1 and IL-1ra are produced by monocytes stimulated with lipopolysaccharide (LPS). Because interleukin 4 (IL-4) suppresses IL-1 production, we investigated whether IL-4 modulated IL-1ra synthesis in LPS-stimulated human peripheral blood mononuclear cells. IL-1 beta and IL-1ra were measured by specific RIAs. IL-4 alone (0.01-100 ng/ml) did not stimulate IL-1 beta synthesis but rather induced IL-1ra (4.82 +/- 0.94 ng/ml). LPS induced synthesis of both IL-1 beta (6.67 +/- 1.06 ng/ml) and IL-1ra (10.77 +/- 2.79 ng/ml). IL-4 suppressed LPS-induced IL-1 beta mRNA accumulation and synthesis. However, IL-4 acted synergistically with LPS in inducing IL-1ra. IL-4 enhanced LPS-induced IL-1ra mRNA accumulation 4-fold and IL-1ra protein synthesis nearly 2-fold. Moreover, IL-1ra mRNA levels were maximal after 6 hr of exposure to LPS but peaked within the first 3 hr in the presence of IL-4. IL-4 added as late as 12 hr after LPS stimulation still enhanced IL-1ra synthesis. In human peripheral blood mononuclear cells stimulated with IL-1 alpha, IL-4 markedly suppressed IL-1 beta production but enhanced IL-1ra synthesis greater than 2-fold. Because IL-4 favors synthesis of the natural antagonist IL-1ra over synthesis of the agonist IL-1, IL-4 may exert potent antiinflammatory effects on host responses to Gram-negative infections.

The high-affinity receptor for human granulocyte/macrophage colony-stimulating factor (hGM-CSF) is composed of two subunits, alpha and beta. The alpha subunit binds GM-CSF with low affinity, whereas the beta subunit does not bind GM-CSF by itself. The alpha and beta subunits together form the high-affinity GM-CSF receptor. The beta subunit has extensive sequence homology with the mouse interleukin 3 (IL-3) receptor (AIC2A) and its homologue (AIC2B) that does not bind IL-3 or other cytokines including GM-CSF. To examine the function of these receptor components, we expressed the alpha subunit of the hGM-CSF receptor with the human beta subunit or the mouse AIC2A or AIC2B in a mouse IL-3-dependent pro-B-cell line, Ba/F3, and in a mouse IL-2-dependent T-cell line, CTLL2. Coexpression of the alpha and beta subunits in Ba/F3 and CTLL2 cells resulted in high-affinity hGM-CSF binding and growth response to low concentrations of hGM-CSF. Whereas Ba/F3 cells expressing the alpha subunit alone proliferated in response to high concentrations of hGM-CSF, CTLL2 cells expressing the alpha subunit alone did not respond to hGM-CSF at all. Since Ba/F3 cells express endogenous AIC2A and AIC2B whereas CTLL2 expresses neither of them, we examined the possibility that either AIC2A or AIC2B is involved in the formation of a functional GM-CSF receptor. The expression of the human alpha subunit with AIC2B, but not with AIC2A, in CTLL2 cells conferred a growth response to hGM-CSF. These results indicate that the beta subunit of the GM-CSF receptor is required for generation of growth signals and that AIC2B is likely the beta subunit of the mouse GM-CSF receptor.

Human interleukin 2 (IL-2) analogs with defined amino acid substitutions were used to identify specific residues that interact with the 55-kDa subunit (p55) or alpha chain of the human IL-2 receptor. Analog proteins containing specific substitutions for Lys-35, Arg-38, Phe-42, or Lys-43 were inactive in competitive binding assays for p55. All of these analogs retained substantial competitive binding to the intermediate-affinity p70 subunit (beta chain) of the receptor complex. The analogs varied in ability to interact with the high-affinity p55/p70 receptor. Despite the lack of binding to p55, all analogs exhibited significant biological activity, as assayed on the murine CTLL cell line. The dissociation constants of Arg-38 and Phe-42 analogs for p70 were consistent with intermediate-affinity binding; the Kd values were not significantly affected by the presence of p55 in binding to the high-affinity IL-2 receptor complex. These results confirm the importance of the B alpha-helix in IL-2 as the locus for p55-receptor binding and support a revised model of IL-2-IL-2 receptor interaction.

Two proteins that specifically bind the T-cell growth factor interleukin 2 (IL-2) have been identified previously on the surface of T cells; these proteins have been designated IL-2R alpha and IL-2R beta for the alpha and beta chains of the IL-2 receptor (IL-2R). The association of these independent binding proteins with each other on the surface of activated T cells correlates with the generation of high-affinity binding sites. These high-affinity sites transduce the major mitogenic signal of IL-2, yet the mechanisms of association of the alpha and beta chains with each other as well as signal transduction in response to IL-2 are unknown. Cotransfection experiments of cDNAs encoding the alpha and beta chains in T cells and fibroblasts have suggested functional requirements for other T cell-specific factor(s). We now provide biochemical evidence for a distinct 100-kDa protein that interacts with the alpha or beta chains, or both, on the surface of the IL-2-dependent cell line CTLL-2 as well as activated murine splenocytes. This same 100-kDa protein is capable of being chemically cross-linked to 125I-labeled IL-2.

Stimulation of a class II-restricted, antigen-specific T cell clone with interleukin 2 (IL-2) resulted in substantial increases in both cell surface IL-2 receptor (IL-2-R) and cytoplasmic IL-2-R messenger RNA (mRNA), whereas no increase was observed for cell-surface expression of Thy-1 and L3T4 antigens, and only a modest increase in Thy-1 mRNA was observed. These experiments demonstrate that, after initial acquisition of the IL-2-R, IL-2 as well as antigen is able to directly upregulate both the level of IL-2-R mRNA and cell surface IL-2-R molecules.

Objective: The interleukin 4 receptor has been demonstrated on the surface of human non–small cell lung carcinoma cell lines and tumor specimens. Interleukin 4 causes G1-phase cell-cycle arrest of non–small cell lung cancer cell lines expressing the interleukin 4 receptor; the effect directly correlates with the expression of the interleukin 4 receptor and is seen within 48 hours after treatment. We examined signal transduction pathways used by the interleukin 4 receptor that may account for growth arrest of the cell line LUst but had no effect on another non–small cell lung cancer cell line, SK-MES-1. Methods: Western blot analysis was performed on both LUst and SK-MES-1 cell lines cultured in the presence of interleukin 4 (500 U/mL). Cells were lysed, protein extracted, and electroblotted; blots were then probed with murine monoclonal antibodies to specific intracellular proteins. Results: Western blotting of the cell lines with antiphosphotyrosine antibody (4G10) demonstrated multiple (140 kd, 100-130 kd, and 65 kd) phosphoproteins seen only in the interleukin 4–treated LUst cell line and not observed in the SK-MES-1 cell lines. Immunoprecipitation and blotting of the LUst cell line with specific secondary antibodies demonstrated that the 140-kd phosphoprotein was the interleukin 4 receptor, the 130-kd phosphoprotein was Janus kinase 1, the 116-kd phosphoprotein was Janus kinase 3, and the 65-kd phosphoprotein was the interleukin 2 receptor γ-chain. Specific binding was not observed in the non–small cell lung cancer cell line SK-MES-1, suggesting that a functional interleukin receptor γ-chain was not present. Southern blotting with complementary DNA probes to interleukin 2 receptor γ-chain confirmed the absence of this receptor on cell line SK-MES-1. Conclusions: These results suggest that non–small cell lung cancer cells may express functional cytokine receptors, including the interleukin 2 receptor γ-chain commonly found in association with the lymphocyte interleukin 2 receptor. These receptors may be novel targets for directing cytokine-based immune therapy. (J Thorac Cardiovasc Surg 2000;119:10-20)

Among receptors that bind the Fc region of immunoglobulins ("Fc receptors"), only the one with high affinity for immunoglobulin E (IgE) is known to consist of more than a single polypeptide. In addition to the IgE-binding alpha chain, the receptor contains a single beta chain and two, disulfide-linked, gamma chains. From a cDNA library of a rat mucosal mast cell tumor, from which we recently cloned cDNAs coding for the alpha chain, we have now isolated cDNAs coding for the beta subunit. In vitro transcription-translation of the cDNA directed the synthesis of a polypeptide reactive with two distinctive anti-beta monoclonal antibodies and whose molecular weight was identical to that of authentic beta chains. Polyclonal antibodies to beta peptides expressed in Escherichia coli reacted with intact receptors and isolated beta chains. The gene encodes a protein of 243 residues with no leader sequence. A hydropathicity plot suggests that the polypeptide crosses the plasma membrane four times. The epitope recognized by one of the monoclonal antibodies was localized to the NH2 terminus; that by the other was localized to the COOH terminus. Since those antibodies react with membranes and not with intact cells, we suggest that both ends of the beta subunit are cytoplasmic. RNA transfer blots at high stringency failed to reveal mRNA for beta chains in a variety of cells (in particular, monocytes) that do not contain the high-affinity receptor for IgE.

Transcription of the interleukin 1 beta (IL-1 beta) gene was studied by mRNA hybridization with a cDNA probe in the human promonocytic cell line U-937. Phorbol ester and lipopolysaccharide increased the steady-state level of IL-1 beta mRNA. Glucocorticoids markedly decreased IL-1 beta mRNA levels by two mechanisms. Transcription of the IL-1 gene was inhibited, as shown by in vitro transcription assays with nuclei isolated from glucocorticoid-treated cells. Moreover, kinetic analyses and pulse-labeling of mRNAs showed that glucocorticoids selectively decrease the stability of IL-1 beta mRNA, without affecting the stability of beta-actin and FOS mRNAs. Inhibition of the formation and effects IL-1 is a mechanism by which glucocorticoids can exert antiinflammatory and immunosuppressive effects.

Cloned cDNAs encoding two new beta subunits of the rat and bovine GABAA receptor have been isolated using a degenerate oligonucleotide probe based on a highly conserved peptide sequence in the second transmembrane domain of GABAA receptor subunits. The beta 2 and beta 3 subunits share approximately 72% sequence identity with the previously characterized beta 1 polypeptide. Northern analysis showed that both beta 2 and beta 3 mRNAs are more abundant in the brain than beta 1 mRNA. All three beta subunit encoding cDNAs were also identified in a library constructed from adrenal medulla RNA. Each beta subunit, when co-expressed in Xenopus oocytes with an alpha subunit, forms functional GABAA receptors. These results, together with the known alpha subunit heterogeneity, suggest that a variety of related but functionally distinct GABAA receptor subtypes are generated by different subunit combinations.

More than 150 different mycobacterial strains are known, of which only a few are considered pathogens in humans. The most pathogenic strains are the mycobacteria from the Mycobacterium tuberculosis complex which cause the globally spread disease tuberculosis and the Mycobacterium leprae and Mycobacterium lepromatosis strains that cause leprosy. In addition to these well-known mycobacteria, there are many non-tuberculous mycobacteria (NTM) that are present in the environment and that seldom cause severe disease in healthy individuals. NTM infection can lead to severe disease in individuals with a failing immune system due to a primary or secondary immunodeficiency. Mendelian susceptibility to mycobacterial disease (MSMD) is a rare primary immunodeficiency characterized by a predisposition to severe, sometimes lethal disease caused by otherwise poorly virulent NTM, as well as the vaccine strain Mycobacterium bovis BCG. In these patients the mycobacterial infection is often disseminated to various parts of the body and is difficult to treat. Interestingly, MSMD patients do not, in general, develop pulmonary tuberculosis. MSMD is usually caused by mutations in genes involved in the IL-12/IFN-γ pathway, such as: IL12RB1 which encodes the IL-12Rβ1 chain of the IL-12 and the IL-23 receptor, IL12B which encodes the IL-12p40 subunit of IL-12 and IL-23, IFNGR1 and IFNGR2 which encode the two chains of the IFN-γ receptor, and STAT1 which encodes one of the proteins that signal in response to IFN-γ. The aim of our research is to identify the immunological and genetic defects in MSMD patients. Hereto the integrity of the IL-12/IFN-γ pathway is analysed in blood and isolated cells from the patients, as well as expression of receptor chains on the membrane of the cells. Mutations are subsequently identified by sequencing the genes involved. The effect of novel mutations that lead to amino acid changes can be analysed in retroviral expression models, as we have done for amongst others IL12RB1 , IFNGR1 and IFNGR2 . To facilitate the analysis of variations identified by researchers around the world, databases have been set up that contain all reported MSMD patients and mutations (see for instance: www.lovd.nl/IL12RB1). Thus far, just over 400 patients have been reported worldwide with MSMD and this is probably only the tip of the iceberg. Also, other genes are still expected to be found to cause MSMD; no genes have been reported so far in which mutations specifically lead to susceptibility to tuberculosis.