IL-2R Gene Research Articles

Los Polimorfismos en la Uteroglobina y en IL2R están asociados con el daño articular y/o la Cirugía articular en pacientes con Artritis Reumatoide

Introduction and Objectives. Of all clinical manifestations that mark prognosis of a patient with RA, radiological damaged is considered one of the most important variables. In addition to clinical and laboratory markers are increasingly studying the influence of genetic factors in the predictive ability of the RA forecast. Previous studies have reported misleading results about polymorphisms in different genes associated with radiological damage in RA. The aim of this study was to assess the involvement of the clinical, analytical and genetic variables in the radiological damage in a Spanish RA population. Methods. Six hundred thirty-two Spanish RA patients were included. SNP genotyping was performed using an oligonucleotide microarray. Allele and genotype association analyses with articular damage and a haplotype association test were performed. Results. This study includes retrospectively 604 Spanish RA patients with articular erosions and 616 with surgery due to RA. The A587G polymorphism (rs3741240) in uteroglobin gene was associated with increased radiological damage defined by phenotypes RX2 (multiple erosions on hands and feet) and surgery due to RA (OR 1.6 and 1.7 respectively). IL2R polymorphism was associated with RX2 phenotype (OR 2.95). Disease duration, painful joints, ESR, RF and ACPA are the analytical and clinical variables significantly associated with articular damage. Conclusions. These results demonstrate that the A587G and C4599T polymorphism in uteroglobin and IL2R genes respectively are associated with radiological damage in a Spanish RA population.

Effect of Colchicine on Th1 and Th17 Cytokines, Cytokine Receptors, and Chemokine Gene Expression Profiles in Behçet's Disease

Objective: Colchicine has been used in recent years as an effective drug for controlling attacks in Behcet’s disease. In the present study, we investigated expression levels of IL1R, IL2R, IL12RB, IL23R, IL17, CXCR3, CXCR10 and IL8 genes in patients with active and inactive Behcet’s disease. We also evaluated how colchicine use in patients with active and inactive disease affected these genes and evaluated their role in the etiopathogenesis of the disease. Methods: Thirty-five patients who were diagnosed with Behcet’s disease according to the International Working Group criteria (28 with active disease, 7 inactive) and were taking colchicine were enrolled in the study. Twenty healthy subjects were included as a control group. Expression levels of the IL1R, IL2R, IL12RB, IL23R, IL17, CXCR3 ,CXCR10 and IL8, genes were evaluated. Results: Expression levels of CXCR3 and IL23R were significantly lower in patients with active Behcet's disease when compared with the inactive disease and control groups. However, the differences in CXCR3 and IL23R expression between the inactive Behcet’s patient group and the control group were nonsignificant. Expression levels of the other genes did not differ statistically between the active Behcet’s patients, inactive Behcet’s patients, and control subjects. Conclusion: While the expression levels of the CXCR3 and IL23R genes in active Behcet’s patients were statistically lower than controls, there was no statistical difference between active and inactive Behcet’s patients or controls in terms of IL1R, IL2R, IL17, IL12RB, CXCR10 and IL8, gene expression levels. This study may form the basis for further studies to determine the molecular mechanism of colchicine in the treatment of Behcet's disease.

Selective IL-2 responsiveness of regulatory T cells through multiple intrinsic mechanisms supports the use of low-dose IL-2 therapy in type 1 diabetes.

Low-dose interleukin-2 (IL-2) inhibited unwanted immune responses in several clinical settings and is currently being tested in patients with type 1 diabetes (T1D). Low-dose IL-2 selectively targets regulatory T cells (Tregs), but the mechanisms underlying this selectivity are poorly understood. We show that IL-2-dependent STAT5 activation in Tregs from healthy individuals and patients with T1D occurred at an ∼10-fold lower concentration of IL-2 than that required by T memory (TM) cells or by in vitro-activated T cells. This selective Treg responsiveness is explained by their higher expression of IL-2 receptor subunit α (IL-2Rα) and γ chain and also endogenous serine/threonine phosphatase protein phosphates 1 and/or 2A activity. Genome-wide profiling identified an IL-2-dependent transcriptome in human Tregs. Quantitative assessment of selected targets indicated that most were optimally activated by a 100-fold lower concentration of IL-2 in Tregs versus CD4(+) TM cells. Two such targets were selectively increased in Tregs from T1D patients undergoing low-dose IL-2 therapy. Thus, human Tregs possess an IL-2-dependent transcriptional amplification mechanism that widens their selective responses to low IL-2. Our findings support a model where low-dose IL-2 selectively activates Tregs to broadly induce their IL-2/IL-2R gene program and provide a molecular underpinning for low-dose IL-2 therapy to enhance Tregs for immune tolerance in T1D.

Impact of IL-2 and IL-2R SNPs on proliferation and tumor- killing activity of lymphokine-activated killer cells from healthy chinese blood donors.

One of the goals of tumor immunotherapy is to generate immune cells with potent anti-tumor activity through in vitro techniques using peripheral blood collected from patients. However, cancer patients generally have poor immunological function. Thus using patient T cells, which have reduced in vitro proliferative capabilities and less tumor cell killing activity to generate lymphokine-activated killer (LAK) cells, fails to achieve optimal clinical efficacy. Interleukin-2 (IL-2) is a potent activating cytokine for both T cells and natural killer cells. Thus, this study aimed to identify optimal donors for allogeneic LAK cell immunotherapy based on single nucleotide polymorphisms (SNP) in the IL-2 and IL-2R genes. IL-2 and IL-2R SNPs were analyzed using HRM- PCR. LAK cells were derived from peripheral blood mononuclear cells by culturing with IL-2. The frequency and tumor-killing activity of LAK cells in each group were analyzed by flow cytometry and tumor cell killing assays, respectively. Regarding polymorphisms at IL-2-330 (rs2069762) T/G, LAK cells from GG donors had significantly greater proliferation, tumor-killing activity, and IFN-γ production than LAK cells from TT donors (P<0.05). Regarding polymorphisms at IL-2R rs2104286 A/G, LAK cell proliferation and tumor cell killing were significantly greater in LAK cells from AA donors than GG donors (P<0.05). These data suggest that either IL- 2-330(rs2069762)T/G GG donors or IL-2R rs2104286 A/G AA donors are excellent candidates for allogeneic LAK cell immunotherapy.

Study on the association between IL-2R and IL-7R gene polymorphism and idiopathic demyelinating optic neuritis

To analyze the relationship between the gene polymorphism of IL-2R (rs2104286) and IL-7R (rs6897932) and the susceptibility to idiopathic demyelinating optic neuritis (IDON). A case-control study was performed in 72 IDON patients and 81 healthy individuals as the control group. DNA was extracted from peripheral blood leukocytes. PCR was used to amplify the aim genes; the SNP of IL-2R were analyzed by restriction fragment length polymorphism (RFLP) assay and the SNP of IL-7R were analyzed by gene sequencing. The χ2 test was used to compare genotype and allele frequencies between the IDON and control populations. Odds ratio (OR) was calculated using the approximation of Woolf for the 95% confidence interval (CI). No significant difference of genotype (χ2=0.410, P=0.815) and allele frequency(χ2=0.413, P=0.520) of IL-2R gene existed in IDON group(AA:54/AG:15/GG:3;A:123/G:21) as compared with the control group (AA:57/AG:20/GG:4;A:134/G:28). No significant difference of genotype frequency (χ2=3.787, P=0.150) of IL-7R existed in IDON group (CC:56/CT:13/TT:3) as compared with the control group (CC:52/CT:21/TT:8). However, there was a significant difference in IL-7R allele (C/T) between IDON group (C:125/T:19) and the controls (C:125/T:37) (χ2=4.743, P=0.029), OR=1.95 (95%CI: 1.07-3.55). There is no significant difference in IL-2R rs2104286 polymorphism between IDON group and the controls; the frequencies of IL-7R rs6897932 C allele in IDON group was significantly greater than that of the controls. Polymorphism of IL-7R gene (rs6897932) may contribute to the risk of IDON.

High Resolution Molecular Analysis of 5q- Syndrome Patients at Diagnosis and Following Lenalidomide Therapy

The ‘5q- syndrome’ is characterized by macrocytic anaemia, normal to elevated platelet count, dyserythropoiesis, hypolobulated megakaryocytes and an interstitial deletion of the long arm of chromosome 5(q31–q32) as the sole karyotypic abnormality. In this study we have used the Affymetrix 250K high resolution SNP arrays to refine the 5q- syndrome commonly deleted region (CDR) and to explore undetected chromosomal aberrations in CD34+ cells and in constitutional tissue from 5q- syndrome patients at diagnosis (n=17) and following Lenalidomide treatment at haematological remission (n=9). To correlate genomic data with gene expression, genome-wide gene expression analysis on CD34+ cells was also performed using the Affymetrix U133plus2 arrays. We detected 35 chromosomal aberrations (3 deletions, 1 amplification and 31 regions of uniparental disomy (UPD) >2Mb) in 8 (35%) of patients unidentified by metaphase cytogenetic analysis. The median size of UPD was (4.03MB) (range: 2.49 – 30.31). Copy number changes identified were acquired aberrations, whereas UPD's were also present in constitutional DNA. There were no common regions of overlap between any of the UPDs and no obvious change in gene expression correlating with the sites of UPD. SNP analysis precisely mapped the proximal and distal break points of the 5q deletion and clearly identified the proximal end of the common deleted region (CDR) to extend from 5q23.3 to the distal breakpoint 5q33.1. Our results did not identify any patients with isolated minimal deletions of 5q31 or the 5q32/33.1 regions as defined by Ebert et al, 2008. Furthermore, there were no regions of UPD identified on chromosome 5q in any of our patients. There were 3 genes located on the deleted 5q region that were >3 fold down-regulated; LOX (5q23–q31, Kif20a (5q31) and SLC22A4 (5q31.2). Gene set enrichment analysis (GSEA) of CD34 expression data from presentation samples identified the IL2 (p<0.001), IL2R (p<0.02), IL3 (p<0.03), IL6 (p<0.03) and TCR (p<0.04) gene sets to be significantly up-regulated in these cells compared to normal controls, emphasizing the contribution of aberrant immune regulation in this disease. Previously identified gene sets (erythroid, megakaryocytic and neutrophil (Ebert et al, 2008)) were not significant in our analysis, suggesting that the 5q deletion in CD34 cells does not pre-program differentiation along these lineages. Nine patients had paired analysis at presentation and following treatment with Lenalidomide. Interestingly, the 5q- deletion was detected in two patients by SNP arrays which were undetected by cytogenetic analysis. The proximal and distal breakpoints of the detected 5q deletion in these two patients post Lenalidomide therapy were identical to their corresponding presentation sample. Furthermore, in three other patients cytogenetic and SNP array analysis showed loss of the 5q- deletion following Lenalidomide therapy, however, deletions observed at presentations on chromosome 21q21, 7q22.1-36.3 and 12q21.2-q22 were retained in CD34+ cells post treatment respectively, suggesting the presence of additional abnormal clones in the bone marrow. GSEA of presentation CD34 cells and post Lenalidomide treated cells identified the IL4 (p<0.04) and IL6 (p<0.04) gene sets as being significant pathways affected by Lenalidomide therapy. Our SNP array data clearly defines the 5q- syndrome CDR and shows a high frequency of UPD in these patients. The identification of clonal aberrations not removed by Lenalidomide treatment suggests the presence of additional abnormal clones which may play a role in transformation of this disease. GSEA analysis of gene expression data in CD34+ cells clearly shows the important role of immune modulatory networks both at presentation of the disease and following Lenalidomide therapy.

IL-2Rβ Links IL-2R Signaling with Foxp3 Expression

Background: Inflammatory bowel disease is associated with enhanced activation of T cells, but the genes responsible for this state are not well characterized. Methods: T-cell activation genes were studied in peripheral blood and intestinal mucosal mononuclear cells of control, Crohn's disease, and ulcerative colitis patients. Results: In all groups the expression of interleukin-2 (IL-2), IL-2 receptor α (IL-2Rα), and IL-2Rβ messenger RNA (mRNA) was significantly higher in intestinal than circulating cells, and it correlated well with protein levels. Both IL-2Rα and IL-2Rβ mRNA were abundant in mucosal cells, suggesting that a substantial number of them displays high affinity IL-2R. This would explain why intestinal cells proliferate more, express more IL-2 transcripts, and secrete more IL-2 than peripheral cells. Inflammatory bowel disease cells produced similar or higher IL-2Rα and IL-2Rβ mRNA than controls but generated significantly lower IL-2 mRNA. Thus, the reported defect of IL-2 activity in Crohn's disease and ulcerative colitis is probably related to decreased IL-2 transcription. Crohn's disease intestinal cells had the highest expression of IL-2R gene products. This provides a mechanism for their increased response to IL-2 and supports claims that elevated soluble IL-2Rα serum levels reflect gut T-cell hyperactivity in this disease. Conclusions: These findings underscore the importance of T cells in mucosal immunity and indicate that abnormal T-cell activation is intimately associated to the pathogenesis of inflammatory bowel disease.

Lymph node cells from BALB/c mice with chronic visceral leishmaniasis exhibiting cellular anergy and apoptosis: Involvement of Ser/Thr phosphatase

Visceral leishmaniasis (VL) produced in BALB/c mice through intracardial administration of Leishmania donovani amastigotes was accompanied by hepatosplenomegaly with high organ parasite load and lymphadenopathy when followed up to 4-months or so. To elucidate the mechanism of immunosuppression associated with VL, we report here progressive impairment of the proliferative response of lymph node cells (lymphocytes) from infected animals (I-LNC) to in vitro stimulation with the combination of phorbol 12-myristate 13-acetate (PMA) and ionomycin (Io) that could be related to the downregulation of PKC and MAP kinase (ERK 1/2) activation process. Further, pretreatment of I-LNC with the protein phosphatase inhibitor okadaic acid (OA), but not with calyculin A or sodium orthovanadate, significantly restored their proliferative response as well as PMA-induced activation of PKC. A population of LNC (primarily T-lymphocytes) from chronically infected animals was shown to undergo apoptosis, the number of which increased considerably following PMA+ Io stimulation. The apoptotic pathway, which was followed through binding of cells to Annexin V, activation of caspase-3 and fragmentation of DNA, involved destabilization of mitochondria, probably as a result of downregulation of PKC and Bcl-2. Interestingly, prior incubation of I-LNC with OA reversed the state of cell cycle arrest (anergy) and apoptosis through progression of cells from G0/G1 to S and G2/M phases with transcriptional activation of IL-2 and IL-2R genes. Our results suggest that the cellular (immune) dysfunction in VL could be attributed to dephosphorylation of key molecules in the T-lymphocyte signaling pathway by Ser/Thr phosphatase leading to their inactivation.

Immunosuppressive properties of flavonoids isolated from Boerhaavia diffusa Linn.

Plant extracts have been widely evaluated for possible immunomodulatory properties. We have earlier reported that ethanolic extract of Boerhaavia diffusa root, a plant used in Indian traditional medicine, has significant immunomodulatory potential. B. diffusa hexane, chloroform and ethanol extracts, and two pure compounds Bd-I (eupalitin-3- O-β- D-galactopyranoside) and Bd-II (eupalitin) were evaluated in vitro for their effect on T cell mitogen (phytohemagglutinin; PHA) stimulated proliferation of human peripheral blood mononuclear cell (PBMC), mixed lymphocyte culture, lipopolysaccharide (LPS) stimulated nitric oxide production by RAW 264.7, PHA and LPS induced IL-2 and TNF-α production, in human PBMCs, superoxide production in neutrophils, human natural killer (NK) cell cytotoxicity and nuclear translocation of nuclear factor-κB and AP-1 in PHA stimulated PBMCs. The chloroform and ethanol extracts inhibited PHA stimulated proliferation of peripheral blood mononuclear cells, two-way MLR, NK cell cytotoxicity as well as LPS induced NO production by RAW 264.7; the hexane extract showed no activity. Bd-I purified from the ethanolic extract at equivalent dose, inhibited PHA-stimulated proliferation of peripheral blood mononuclear cells, two-way MLR and NK cell cytotoxicity as well as LPS induced NO production by RAW 264.7 equally or more effectively than the parent ethanolic extract. Bd-I inhibited production of PHA stimulated IL-2 at the protein and mRNA transcript levels and LPS stimulated TNF-α production in human PBMCs; it also blocked the activation of DNA binding of nuclear factor-κB and AP-1, two major transcription factors centrally involved in expression of the IL-2 and IL-2R gene, which are necessary for T cell activation and proliferation. Our results report selective immunosuppressive activity of B. diffusa leaf extracts and that this activity lies in eupalitin-3- O-β- D-galactopyranoside (Bd-I) isolated and purified from the ethnaolic extract. Thus, Bd-I could be a candidate for development as an immunosuppressive agent.

HepG2 HUMAN HEPATOMA CELLS EXPRESS MULTIPLE CYTOKINE GENES

Although cytokines are known to be involved in the regulation of a variety of hepatocellular functions, hepatocytes themselves are generally considered only targets but not producers of these important mediators. In order to investigate whether cells of hepatocellular linages are a potential source of various regulatory cytokines we have estimated the multiple cytokine gene expression in the culture of well differentiated human HepG2 hepatoma cells using RT-PCR. Our findings demonstrate that HepG2 cells express mRNAs for interferon γ (IFN-γ), tumour necrosis factor α (TNF-α), transforming growth factor β (TGF-β), macrophage colony-stimulating factor (M-CSF), oncostatin-M (OSM), intercellular adhesion molecule (ICAM-1), interleukin 4 (IL-4), IL-5, IL-7, IL-10, IL-11, IL-12 and IL-6 receptor (IL-6R). At the same time the expression of IL-1, IL-2, IL-3, IL-6, CD40 ligand and IL-2R genes was not detected. It was concluded that hepatocytes are potential producers of a variety of cytokines, some of them being able to regulate hepatocellular functions directly, while others are important regulators of leukocyte activity. Thus, on the one hand, hepatocytes may express autoregulatory cytokines and on the other hand, influence the functions of other liver cells like Kupffer, Ito or endothelial cells. Due to their large amount, liver parenchymal cells could be an important source of sytemically acting pro- and anti-inflammatory and other regulatory cytokines.

The tumour associated cell surface antigen A6H is costimulatory for human CD4+ but not CD8+ T cells.

The A6H monoclonal antibody (mAb) recognizes a 120,000-140,000 MW antigen that is expressed at similar densities on 85-90% of human CD4+ and CD8+ T cells and on renal cell carcinomas. The binding of the A6H mAb induced a costimulatory signal in anti-CD3 activated T cells. In the present report, we show that A6H costimulated cell proliferation and cytokine production in purified CD4+ T cells. Unexpectedly, the CD8+ T-cell subpopulation failed to respond. CD4+ T cells costimulated with the A6H mAb upregulated CD80, CD86, CD71, interleukin-2 (IL-2)R alpha, IL-2R beta and IL-2R gamma, while no corresponding up-regulation of these cell surface molecules was seen in CD8+ T cells. In order to investigate the nature of the A6H mAb costimulus at the transcriptional level we have examined induction of the transcription factors OCT-1, AP-1 and NF-kappa B which are known to be transcriptional regulators of several cytokine and cytokine receptor genes, including the IL-2 and IL-2R genes. Co-ligation of the A6H antigen and the CD3 complex induced expression of the transcription factor AP-1 in CD4+ T cells, whereas no increase in NF-kappa B and octamer-binding (Oct) proteins was seen compared to T cells stimulated with anti-CD3 alone. Furthermore, no induction of AP-1 was seen in A6H costimulated CD8+ T cells. These results suggests that both proximal steps in CD8+ T-cell activation as well as the later phases are unresponsive to A6H ligation. Molecular differences of the A6H molecule or distinct regulation of the A6H transduced AP-1 activation pathway may exist in CD4+ and CD8+ T cell subpopulations.

Effect of 5-fluorouracil treatment on IL-2R gene expression in gastric carcinoma patients

Interleukin-2R gene expression was evaluated in peripheral blood T lymphocytes of patients with gastric carcinoma and normal healthy controls. The presence of mRNA for IL-2R alpha evaluated by Northern blot analysis revealed that unstimulated T cells expressed lower levels of IL-2R mRNA than PHA stimulated T cells. Expression of both IL-2R alpha transcripts (3.5 and 1.5 kb) were either not detectable or only weakly detectable on T lymphocytes from patients even after mitogenic stimulation. In contrast, a significant rise in the expression of both IL-2R alpha transcripts was observed on T cells from normal controls followed by mitogenic challenge. Enhanced expression of both IL-2R transcripts was also detected in a follow-up study of the patients, conducted one month following initial trial. of chemotherapy with 5-fluorouracil (5 Fu). Patients post chemotherapy were better responders of PHA stimulation as reflected by enhanced IL-2R gene expression following mitogenic challenge.

Interleukin-2 receptor (CD25) upregulation on human T-lymphocytes: sensitivity to immunosuppressants is defined by the mode of T-lymphocyte activation

We investigated the transcription and expression of the interleukin-2 receptor (IL-2R, CD25) in human T-lymphocytes after different modes of T-lymphocyte stimulation in the presence of the immunosuppressants cyclosporin (CsA) and tacrolimus (FK506) as well as the structurally related macrolide rapamycin. We demonstrate that CsA and FK506 inhibited IL-2R (CD25) gene transcription and protein expression after stimulation by anti-CD3 or ionomycin, but not by phorbol ester or IL-2. Rapamycin, which does not affect IL-2 transcription, surprisingly inhibited IL-2R upregulation after anti-CD3 or ionomycin-induced stimulation, but not by phorbol ester or IL-2. Interestingly, the protein synthesis inhibitor anisomycin inhibited anti-CD3- or ionomycin-, but not phorbol ester- and IL-2-induced CD25 gene transcription indicating the dependence on de novo protein synthesis of the former pathways. These data suggest that the pharmacological effect of the imunosuppressants tested on IL-2R gene expression may be predicted by the requirement of protein synthesis of a given activation pathway.

Cyclic AMP inhibits phosphatidylinositol-coupled and -uncoupled mitogenic signals in T lymphocytes. Evidence that cAMP alters PKC-induced transcription regulation of members of the jun and fos family of genes.

T lymphocyte stimulation via the Ag receptor results in activation of phospholipase C gamma 1 that catalyses the hydrolysis of phosphatidylinositol (PI). The hydrolysis generates inositol phosphate and diacylglycerol, which in turn, increase intracellular Ca2+ concentration and activates protein kinase C, respectively. Agonists operating via the adenylate cyclase pathway or cell permeable cAMP analogues inhibit T cell activation by interfering with the PI-turnover. We have shown that dbcAMP inhibits PI-independent mitogenic signals in T cells after stimulation with TPA plus ionomycin. dbcAMP inhibited the TPA plus ionomycin-induced transcription of IL-2 and IL-2R genes in EL4 cells, suggesting interference with biochemic events downstream to PI hydrolysis and upstream to transcription of early activation genes. Because many of the early genes operating in T cell mitogenesis possess a TPA-response element (TRE) in their promoter region, we tested the effect of cAMP on the TRE-binding protein, TPA-response element (TRE) in their promoter region, we tested the effect of cAMP on the TRE-binding protein, AP-1. dbcAMP increased the binding activity of nuclear proteins consisting of Fos:Jun heterodimers to a TRE-containing oligonucleotide, but altered the composition of Jun proteins in the AP-1. Furthermore, the TPA plus ionomycin-induced transcription program of members of the jun and fos family of genes was altered by dbcAMP, suggesting that inhibition of T cell proliferation by dbcAMP is a consequence of intervention in transcriptional regulation by TRE-binding proteins.

IL-2, IL-4, and IFN-gamma gene expression versus secretion in superantigen-activated T cells. Distinct requirement for costimulatory signals through adhesion molecules.

In the complete absence of APCs staphylococcal superantigens induced IL-2, IL-4, IL-5, IFN-gamma, and IL-2R gene transcripts in both highly purified human T cells and FACs sorted CD4+ memory (CD45RA-) T cells. Secretion of IL-2, IL-4, and IFN-gamma, as well as DNA synthesis, on the other hand, required the presence of monocytes. At cytokine gene transcript level, three patterns of expression were noted after superantigen activation of T cells in the presence vs the absence of APC. mRNA levels for IL-2 were markedly up-regulated in the presence of monocytes, IL-4 and IFN-gamma transcripts increased only modestly, and IL-5 and IL-2R mRNA levels were unaffected. Blocking mAbs against LFA-1 and LFA-3 added to staphylococcal enterotoxin B (SEB)-activated cultures of T cells and autologous monocytes, reproducibly decreased both T cell proliferation and genetic expression of IL-2, IL-4, IL-5, and IL-2R, although having little or no effect on IFN-gamma transcripts. Further, under those conditions of blocking, secretion of IL-2 and IL-4 was dramatically decreased, whereas IFN-gamma secretion remained essentially unchanged. In contrast, LFA-1 and LFA-3 mAbs completely abrogated IFN-gamma secretion from PHA-activated T cell-monocyte mixtures, although having no inhibitory effect on T cell proliferation. These results indicate a characteristic and differential involvement of adhesion molecule-mediated signals in superantigen-induced T cell proliferation, differential cytokine gene expression, and cytokine secretion.

Kinetics of IL-1β, IL-2, IL-2R, IL-4, IL-7 and IL-8 gene activity in human allograft biopsies after orthotopic liver transplantation: Elevated detection of IL-4 mRNA expression correlates with acute rejection

Kinetics of IL-1β, IL-2, IL-2R, IL-4, IL-7 and IL-8 gene activity in human allograft biopsies after orthotopic liver transplantation: Elevated detection of IL-4 mRNA expression correlates with acute rejection

Antibodies against major histocompatibility complex class II antigens directly inhibit the growth of T cells infected with Theileria parva without affecting their state of activation.

We have analyzed the effect of antibodies (Abs) directed against major histocompatibility complex (MHC) class II Abs on the proliferation of Theileria parva-infected (Tpi) T cells. Anti-MHC class II Abs exert a direct effect on Tpi T cells causing an acute block in their proliferation. The inhibition does not involve apoptosis and is also entirely reversible. The rapid arrest of DNA synthesis caused by anti-MHC class II Abs is not due to interference with the state of activation of the T cells since the transcriptional activator NF-kappa B remains activated in arrested cells. In addition, interleukin 2 (IL-2), IL-2R, and c-myc gene expression are also unaffected. By analyzing the cell-cycle phase distribution of inhibited cells, it could be shown that cells in all phases of the cell cycle are inhibited. The signal transduction pathway that results in inhibition was shown to be independent of protein kinase C and extracellular Ca2+. Tyrosine kinase inhibitors, however, partly reduced the level of inhibition and, conversely, phosphatase inhibitors enhanced it. The possible relevance of this phenomenon in other systems is discussed.

Rapid Il-2-induced adherence of human natural killer cells. Expression of mRNA for cytokines and IL-2 receptors in adherent NK cells.

Natural killer (NK) cells selected by IL-2-induced rapid adherence to plastic and called A-NK cells represent a phenotypically and functionally distinct subset of mature peripheral blood NK cells. To further characterize this subset of NK cells functionally, their potential to express mRNA for the IL-2R and various cytokines after IL-2 activation was examined. Highly purified normal human peripheral blood resting NK (R-NK) cells were obtained by negative immunoselection using OKT3 mAb and magnetic beads coated with goat anti-mouse Ig. By two-color flow cytometry, > 90% of these R-NK cells were either CD3-CD56+CD16+ or - or CD3-CD56-CD16+. R-NK cells were activated in the presence of 6000 IU/ml (22 nM) of IL-2 for different periods of time. After 1, 3, 5, or 24 h, plastic-adherent (A) and nonadherent (NA) NK cells were separated and compared for the expression of the IL-2R or cytokine mRNA by in situ hybridization, using 35[S]-cDNA probes. Only low proportions of R-NK cells expressed genes for IL-2Rp55 (16%) or cytokines IL-2 (20%), IFN-gamma (18%), TNF-alpha (16%), and TGF-beta (7%). Thus, the genes for the IL-2Rp55 and these cytokines were not constitutively expressed by most human R-NK cells, and there was no indication that the NK cells used in these experiments were activated in vivo or during the purification procedure. However, larger proportions of R-NK cells showed expression of mRNA for IL-1-beta (35%) and IL-6 (40%), which indicates that genes for these cytokines may be constitutively expressed in a substantial proportion of normal human circulating NK cells. When R-NK cells were incubated in the presence of 22 nM of IL-2 for 1 to 24 h and separated into A-NK cells and NA-NK cells, a large proportion of A-NK cells became positive for IL-2R and cytokine gene expression. In contrast, the proportion of mRNA-positive NA-NK cells was similar or lower than that observed for R-NK cells, with the exception of an increase in TGF-beta.(ABSTRACT TRUNCATED AT 400 WORDS)

Immune activation genes in inflammatory bowel disease

Background: Inflammatory bowel disease is associated with enhanced activation of T cells, but the genes responsible for this state are not well characterized. Methods: T-cell activation genes were studied in peripheral blood and intestinal mucosal mononuclear cells of control, Crohn's disease, and ulcerative colitis patients. Results: In all groups the expression of interleukin-2 (IL-2), IL-2 receptor α (IL-2Rα), and IL-2Rβ messenger RNA (mRNA) was significantly higher in intestinal than circulating cells, and it correlated well with protein levels. Both IL-2Rα and IL-2Rβ mRNA were abundant in mucosal cells, suggesting that a substantial number of them displays high affinity IL-2R. This would explain why intestinal cells proliferate more, express more IL-2 transcripts, and secrete more IL-2 than peripheral cells. Inflammatory bowel disease cells produced similar or higher IL-2Rα and IL-2Rβ mRNA than controls but generated significantly lower IL-2 mRNA. Thus, the reported defect of IL-2 activity in Crohn's disease and ulcerative colitis is probably related to decreased IL-2 transcription. Crohn's disease intestinal cells had the highest expression of IL-2R gene products. This provides a mechanism for their increased response to IL-2 and supports claims that elevated soluble IL-2Rα serum levels reflect gut T-cell hyperactivity in this disease. Conclusions: These findings underscore the importance of T cells in mucosal immunity and indicate that abnormal T-cell activation is intimately associated to the pathogenesis of inflammatory bowel disease.

Role of cytokines in the adoptive immunotherapy of an experimental model of human head and neck cancer by human IL-2-activated natural killer cells.

Peritumoral injection of human IL-2-activated natural killer cells into nude mice consistently induced regression of xenografts of human squamous cell carcinoma of the head and neck (SCCHN). To determine the mechanisms responsible for the tumor regression, the lymphoid cells infiltrating the tumor stroma at 24 to 48 h after adoptive immunotherapy were examined by in situ hybridization for the presence of mRNA for cytokines or IL-2R. Numerous lymphoid cells expressing cytokine or IL-2R genes were observed in these tumors, whereas the cultured IL-2-activated NK cells used for therapy were negative. Thus, it appeared that the transferred NK cells became activated in situ after coming into proximity with the tumor cells. To analyze this phenomenon, fresh or cultured human NK cells were coincubated in vitro with irradiated human SCCHN cell line, PCI-1, with or without the presence of IL-2. Expression of mRNA for IL-2R, perforin, and various cytokines was observed within 5 h. Contact with the tumor cells stimulated NK cells to proliferate, secrete IFN-gamma, TNF-alpha, and soluble IL-2R, up-regulate cell surface expression of IL2R p55 and p75 as well as CD16 Ag, and mediate higher levels of antitumor activity in 51Cr-release assays. In addition, supernatants of in vitro-activated NK cells significantly inhibited proliferation of SCCHN cell lines. By examining the effects of neutralizing mAb to various cytokines, this inhibitory activity was shown to be partially attributable to IFN-gamma. To determine the possible in vivo role of soluble factors produced by activated human NK cells, the supernatants (0.2 ml) or rIFN-gamma (10(5) U) were injected perilesionally each day for 2 wk into 3-day SCCHN established in immunosuppressed nude mice. These treatments caused significant (p less than 0.02) inhibition of tumor growth. The results of our studies indicate that human NK cells are strongly activated by SCCHN cells and that the consequent release of cytokines contribute to the regression of SCCHN growing in nude mice.