Molecular modeling on some human interleukins sharing gamma c of interleukin-2 receptor, and structure-function relationships

Five models for human interleukin-7 (HIL-7), HIL-9, HIL-13, HIL-15 and HIL-17 have been generated by SYBYL software package. The primary models were optimized using molecular dynamics and molecular mechanics methods. The final models were optimized using a steepest descent algorithm and a subsequent conjugate gradient method. The complexes with these interleukins and the common gamma chain of interleukin-2 receptor (IL-2R) were constructed and subjected to energy minimization. We found residues, such as Gln127 and Tyr103, of the common gamma chain of IL-2R are very important. Other residues, e.g. Lys70, Asn128 and Glu162, are also significant. Four hydrophobic grooves and two hydrophilic sites converge at the active site triad of the gamma chain. The binding sites of these interleukins interaction with the common gamma chain exist in the first helical and/or the fourth helical domains. Therefore, we conclude that these interleukins binds to the common gamma chain of IL-2R by the first and the fourth helix domain. Especially at the binding sites of some residues (lysine, arginine, asparagine, glutamic acid and aspartic acid), with a discontinuous region of the common gamma chain of IL-2R, termed the interleukins binding sites (103-210). The study of these sites can be important for the development of new drugs. (C) 2000 Elsevier Science B.V. All rights reserved.

Identification of a gene for an ancient cytokine, interleukin 15-like, in mammals; interleukins 2 and 15 co-evolved with this third family member, all sharing binding motifs for IL-15Rα

Interleukins 2 and 15 (IL-2 and IL-15) are highly differentiated but related cytokines with overlapping, yet also distinct functions, and established benefits for medical drug use. The present study identified a gene for an ancient third IL-2/15 family member in reptiles and mammals, interleukin 15-like (IL-15L), which hitherto was only reported in fish. IL-15L genes with intact open reading frames (ORFs) and evidence of transcription, and a recent past of purifying selection, were found for cattle, horse, sheep, pig and rabbit. In human and mouse the IL-15L ORF is incapacitated. Although deduced IL-15L proteins share only ~21 % overall amino acid identity with IL-15, they share many of the IL-15 residues important for binding to receptor chain IL-15Rα, and recombinant bovine IL-15L was shown to interact with IL-15Rα indeed. Comparison of sequence motifs indicates that capacity for binding IL-15Rα is an ancestral characteristic of the IL-2/15/15L family, in accordance with a recent study which showed that in fish both IL-2 and IL-15 can bind IL-15Rα. Evidence reveals that the species lineage leading to mammals started out with three similar cytokines IL-2, IL-15 and IL-15L, and that later in evolution (1) IL-2 and IL-2Rα receptor chain acquired a new and specific binding mode and (2) IL-15L was lost in several but not all groups of mammals. The present study forms an important step forward in understanding this potent family of cytokines, and may help to improve future strategies for their application in veterinarian and human medicine.

Serum Levels of Interleukins 6, 10, and 13 Before and After Treatment of Classic Hodgkin Lymphoma

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Role of interleukins in the regulation of basophil development and secretion

It is well appreciated that differentiation, growth, and function of basophils are regulated by a network of cytokines, and that these cells express a unique composition of surface receptors including interleukin-binding sites. In the current article, most recent discoveries around cytokine regulation of basophils are discussed and compared with previous data.

Expression and regulation of interleukins in human head and neck squamous cell carcinoma

Patients with head and neck squamous cell carcinoma (HNSCC) demonstrate abnormal cell-mediated immunity which is most pronounced at the primary tumor site. Therefore, we tested whether this aberrant immunity could be due to tumor-derived cytokines. We investigated the presence of cytokine mRNA and protein in 8 HNSCC-derived cell lines; RT-PCR results indicated mRNA's for IL-1$\alpha$ and TGF-$\alpha$ (8/8), TGF-$\beta$ (7/8), IL-1$\beta$ (7/8), IL-4 and IL-6 (4/8). IL-2, IFN-$\gamma,$ and TNF-$\alpha$ mRNA was not detected. Supernatants from 6 of these cell lines were analyzed by ELISA and IL-1$\alpha,$ IL-1$\beta,$ and IL-6 were markedly increased compared to HPV-16 immortalized human oral keratinocytes. IL-1$\alpha$ was found in the highest concentration $>$IL-6 $>$ IL-1$\beta.$^ To approach the mechanisms of cytokine regulation, 4 cell lines were compared for HPV DNA presence, p53 status, and cytokine expression. An association between HPV DNA and cytokine expression was not found. However, cell lines secreting the most IL-6 had mutant p53 and/or HPV 16 E6/E7 expression. Further regulatory investigations revealed that exogenous IL-1$\alpha$ and/or IL-1$\beta$ minimally stimulated the proliferation of 2/3 cell lines, as well as strongly induced IL-6 production in 3/3; this effect was completely abrogated by IL-1Ra. IL-1Ra also inhibited the secretion of IL-1$\alpha$ and IL-1$\beta$ in 2/3 cell lines. These data suggest an IL-1 autocrine loop in certain HNSCC cell lines. Because IL-2 induces IL-1 and is used in therapy of HNSCC, the expression of IL-2 receptor was also investigated; IL-2 $\alpha$ and $\beta$ subunits were detected in 3/3 cell lines and $\gamma$ subunits was detected in one. Exogenous IL-2 inhibited the proliferation, but stimulated the secretion of IL-1$\alpha$ in 2/3, and IL-1$\beta$ and IL-6 in 1/3 cell lines.^ To determine if our cell line findings were applicable to patients, immunohistochemistry was performed on biopsies from 12 invasive tumors. Unexpectedly, universal intracellular production of IL-1$\alpha,$ IL-1$\beta,$ and IL-6 protein was detected. Therefore, the aberrant elaboration of biologically active IL-1 and IL-6 may contribute to altered immune status in HNSCC patients. ^

Hematopoietic and lung abnormalities in mice with a null mutation of the common beta subunit of the receptors for granulocyte-macrophage colony-stimulating factor and interleukins 3 and 5.

Gene targeting was used to create mice with a null mutation of the gene encoding the common beta subunit (beta C) of the granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 3 (IL-3; multi-CSF), and interleukin 5 (IL-5) receptor complexes (beta C-/- mice). High-affinity binding of GM-CSF was abolished in beta C-/- bone marrow cells, while cells from heterozygous animals (beta C+/- mice) showed an intermediate number of high-affinity receptors. Binding of IL-3 was unaffected, confirming that the IL-3-specific beta chain remained intact. Eosinophil numbers in peripheral blood and bone marrow of beta C-/- animals were reduced, while other hematological parameters were normal. In clonal cultures of beta C-/- bone marrow cells, even high concentrations of GM-CSF and IL-5 failed to stimulate colony formation, but the cells exhibited normal quantitative responsiveness to stimulation by IL-3 and other growth factors. beta C-/- mice exhibited normal development and survived to young adult life, although they developed pulmonary peribronchovascular lymphoid infiltrates and areas resembling alveolar proteinosis. There was no detectable difference in the systemic clearance and distribution of GM-CSF between beta C-/- and wild-type littermates. The data establish that beta C is normally limiting for high-affinity binding of GM-CSF and demonstrate that systemic clearance of GM-CSF is not mediated via such high-affinity receptor complexes.

Tyrosine phosphorylation and activation of STAT5, STAT3, and Janus kinases by interleukins 2 and 15.

The cytokines interleukin 2 (IL-2) and IL-15 have similar biological effects on T cells and bind common hematopoietin receptor subunits. Pathways that involve Janus kinases (JAKs) and signal transducers and activators of transcription (STATs) have been shown to be important for hematopoietin receptor signaling. In this study we identify the STAT proteins activated by IL-2 and IL-15 in human T cells. IL-2 and IL-15 rapidly induced the tyrosine phosphorylation of STAT3 and STAT5, and DNA-binding complexes containing STAT3 and STAT5 were rapidly activated by these cytokines in T cells. IL-4 induced tyrosine phosphorylation and activation of STAT3 but not STAT5. JAK1 and JAK3 were tyrosine-phosphorylated in response to IL-2 and IL-15. Hence, the JAK and STAT molecules that are activated in response to IL-2 and IL-15 are similar but differ from those induced by IL-4. These observations identify the STAT proteins activated by IL-2 and IL-15 and therefore define signaling pathways by which these T-cell growth factors may regulate gene transcription.

Blockade of T- and B-lymphocyte development by antibody to the gamma c subunit of the receptors for interleukins 2, 4, and 7.

Cytokines are important regulators of hematopoesis. Mutations in gamma c, which is a subunit shared by the receptors for interleukin (IL) 2, IL-4, and IL-7, have been causally associated with human X chromosome-linked severe combined immunodeficiency disease. This finding indicates a mandatory role for cytokine receptor signaling at one or more stages of lymphocyte development. To evaluate the cellular level at which gamma c is critical for lymphopoiesis, the effect of monoclonal antibodies to gamma c on the capacity of syngeneic bone marrow cells to reconstitute the hematopoietic compartment of lethally irradiated recipient mice was examined. We show that monoclonal antibody to gamma c blocked lymphocyte development at or before the appearance of pro-B cells and prior to or at the seeding of the thymus by precursor cells while erythromyeloid cell development was normal. These results suggest that one level of lymphocyte development that requires gamma c is a point in hematopoietic cell differentiation near the divergence of lymphopoiesis and erythromyelopoesis.

Pluripotent hematopoietic stem cells contain high levels of mRNA for c-kit, GATA-2, p45 NF-E2, and c-myb and low levels or no mRNA for c-fms and the receptors for granulocyte colony-stimulating factor and interleukins 5 and 7.

Pluripotent hematopoietic stem cells (PHSCs) were highly enriched from mouse bone marrow by counterflow centrifugal elutriation, lineage subtraction, and fluorescence-activated cell sorting based on high c-kit receptor expression (c-kitBR). We used reverse transcriptase polymerase chain reaction to assay the c-kitBR subset and the subsets expressing low (c-kitDULL) and no (c-kitNEG) c-kit receptor for expression of mRNA encoding hematopoietic growth factor receptors and transcription factors. The c-kitBR cells had approximately 3.5-fold more c-kit mRNA than unfractionated bone marrow cells. The c-kitDULL cells had 47-58% of the c-kit mRNA found in c-kitBR cells and the c-kitNEG cells had 4-9% of the c-kit mRNA present in c-kitBR cells. By comparing mRNA levels in c-kitBR cells (enriched for PHSCs) with those of unfractionated bone marrow, we demonstrated that c-kitBR cells contained low or undetectable levels of mRNA for c-fms, granulocyte colony-stimulating factor receptor, interleukin 5 receptor (IL-5R), and IL-7R. These same cells had moderate levels of mRNA for erythropoietin receptor, IL-3R subunits IL-3R alpha (SUT-1), AIC-2A, and AIC-2B, IL-6R and its partner gp-130, and the transcription factor GATA-1 and high levels of mRNA for transcription factors GATA-2, p45 NF-E2, and c-myb. We conclude from these findings that PHSCs are programmed to interact with stem cell factor, IL-3, and IL-6 but not with granulocyte or macrophage colony-stimulating factor. These findings also indicate that GATA-2, p45 NF-E2, and c-myb activities may be involved in PHSC maintenance or proliferation.

IL-20 is epigenetically regulated in NSCLC and down regulates the expression of VEGF

Background IL-20 is a pleiotrophic member of the IL-10 family and plays a role in skin biology and the development of haematopoietic cells. Recently, IL-20 has been demonstrated to have potential anti-angiogenic effects in non-small cell lung cancer (NSCLC) by down regulating COX-2. Methods The expression of IL-20 and its cognate receptors (IL-20RA/B and IL-22R1) was examined in a series of resected fresh frozen NSCLC tumours. Additionally, the expression and epigenetic regulation of this family was examined in normal bronchial epithelial and NSCLC cell lines. Furthermore, the effect of IL-20 on VEGF family members was examined. Results The expression of IL-20 and its receptors are frequently dysregulated in NSCLC. IL-20RB mRNA was significantly elevated in NSCLC tumours (p < 0.01). Protein levels of the receptors, IL-20RB and IL-22R1, were significantly increased (p < 0.01) in the tumours of NSCLC patients. IL-20 and its receptors were found to be epigenetically regulated through histone post-translational modifications and DNA CpG residue methylation. In addition, treatment with recombinant IL-20 resulted in decreased expression of the VEGF family members at the mRNA level. Conclusions This family of genes are dysregulated in NSCLC and are subject to epigenetic regulation. Whilst the anti-angiogenic properties of IL-20 require further clarification, targeting this family via epigenetic means may be a viable therapeutic option in lung cancer treatment. © 2011 Elsevier Ltd. All rights reserved.

Interleukin-23 mediates the intestinal response to microbial β-1,3-glucan and the development of spondyloarthritis pathology in SKG mice

Objective Spondyloarthritides (SpA) occur in 1% of the population and include ankylosing spondylitis (AS) and arthropathy of inflammatory bowel disease (IBD), with characteristic spondylitis, arthritis, enthesitis, and IBD. Genetic studies implicate interleukin-23 (IL-23) receptor signaling in the development of SpA and IBD, and IL-23 overexpression in mice is sufficient for enthesitis, driven by entheseal-resident T cells. However, in genetically prone individuals, it is not clear where IL-23 is produced and how it drives the SpA syndrome, including IBD or subclinical gut inflammation of AS. Moreover, it is unclear why specific tissue involvement varies between patients with SpA. We undertook this study to determine the location of IL-23 production and its role in SpA pathogenesis in BALB/c ZAP-70W163C-mutant (SKG) mice injected intraperitoneally with β-1,3-glucan (curdlan). Methods Eight weeks after curdlan injection in wild-type or IL-17A-/- SKG or BALB/c mice, pathology was scored in tissue sections. Mice were treated with anti-IL-23 or anti-IL-22. Cytokine production and endoplasmic reticulum (ER) stress were determined in affected organs. Results In curdlan-treated SKG mice, arthritis, enthesitis, and ileitis were IL-23 dependent. Enthesitis was specifically dependent on IL-17A and IL-22. IL-23 was induced in the ileum, where it amplified ER stress, goblet cell dysfunction, and proinflammatory cytokine production. IL-17A was pathogenic, while IL-22 was protective against ileitis. IL-22+CD3- innate-like cells were increased in lamina propria mononuclear cells of ileitis-resistant BALB/c mice, which developed ileitis after curdlan injection and anti-IL-22. Conclusion In response to systemic β-1,3-glucan, intestinal IL-23 provokes local mucosal dysregulation and cytokines driving the SpA syndrome, including IL-17/IL-22-dependent enthesitis. Innate IL-22 production promotes ileal tolerance.

Fine mapping and replication of genetic risk Loci in primary sclerosing cholangitis

Background and aims. Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease characterized by progressive inflammation and fibrosis of the bile ducts eventually leading to biliary cirrhosis. Recent genetic studies in PSC have identified associations at 2q13, 2q35, 3p21, 4q27, 13q31 and suggestive association at 10p15. The aim of this study was to further characterize and refine the genetic architecture of PSC. Methods. We analyzed previously reported associated SNPs at four of these non-HLA loci and 59 SNPs tagging the IL-2/IL-21 (4q27) and IL2RA (10p15) loci in 992 UK PSC cases and 5162 healthy UK controls. Results. The most associated SNPs identified were rs3197999 (3p21 (MST1), p = 1.9 × 10 -6, OR A vs G = 1.28, 95% CI (1.16-1.42)); rs4147359 (10p15 (IL2RA), p = 2.6 × 10 -4, OR A vs G = 1.20, 95% CI (1.09-1.33)) and rs12511287 (4q27 (IL-2/IL-21), p = 3.0 × 10 -4, OR A vs T = 1.21, 95% CI (1.09-1.35)). In addition, we performed a meta-analysis for selected SNPs using published summary statistics from recent studies. We observed genome-wide significance for rs3197999 (3p21 (MST1), P combined = 3.8 × 10 -12) and rs4147359 (10p15 (IL2RA), P combined = 1.5 × 10 -8). Conclusion. We have for the first time confirmed the association of PSC with genetic variants at 10p15 (IL2RA) locus at genome-wide significance and replicated the associations at MST1 and IL-2/IL-21 loci in a large homogeneous UK population. These results strongly implicate the role of IL-2/IL2RA pathway in PSC and provide further confirmation of MST1 association. © Informa Healthcare.

ERAP2 functional knockout in humans does not alter surface heavy chains or HLA-B27, inflammatory cytokines or endoplasmic reticulum stress markers

Introduction Single nucleotide polymorphisms in ERAP2 are strongly associated with ankylosing spondylitis (AS). One AS-associated single nucleotide polymorphism, rs2248374, causes a truncated ERAP2 protein that is degraded by nonsense-mediated decay. Approximately 25% of the populations of European ancestry are therefore natural ERAP2 knockouts. We investigated the effect of this associated variant on HLA class I allele presentation, surface heavy chains, endoplasmic reticulum (ER) stress markers and cytokine gene transcription in AS. Methods Patients with AS and healthy controls with either AA or GG homozygous status for rs2248374 were studied. Antibodies to CD14, CD19-ECD, HLA-A-B-C, Valpha7.2, CD161, anti-HC10 and anti-HLA-B27 were used to analyse peripheral blood mononuclear cells. Expression levels of ER stress markers (GRP78 and CHOP) and proinflammatory genes (tumour necrosis factor (TNF), IL6, IL17 and IL22) were assessed by qPCR. Results There was no significant difference in HLAclass I allele presentation or major histocompatibility class I heavy chains or ER stress markers GRP78 and CHOP or proinflammatory gene expression between genotypes for rs2248374 either between cases, between cases and controls, and between controls. Discussion Large differences were not seen in HLAB27 expression or cytokine levels between subjects with and without ERAP2 in AS cases and controls. This suggests that ERAP2 is more likely to influence AS risk through other mechanisms.

Prognostic importance of the tumour microenvironment in follicular lymphoma patients treated with immunochemotherapy

Follicular lymphoma (FL) is the second most common non-Hodgkin lymphoma. It is an indolent and clinically heterogeneous disease, which is generally considered incurable. Currently, immunochemotherapy has significantly improved the outcome of FL patients. This is based on the combination of rituximab, a monoclonal anti-CD20 antibody, with chemotherapy, and is used at present as a standard first-line therapy in FL. Thus far, however, patients have been selected for treatment based on clinical risk factors and indices that were developed before the rituximab era. Therefore, there is a growing need to understand the molecular mechanisms underlying the disease, which would not only provide information to predict survival in the rituximab era, but also enable the design of more targeted therapeutic strategies. In this study, our aim was to identify genes predicting the outcome in FL patients treated with immunochemotherapy. Thus, we performed a cDNA microarray with 24 FL patients. When gene expression differences from diagnostic tumour samples were related to the clinical outcome, we identified novel genes with a prognostic impact on survival. The expression of selected genes was further characterized with quantitative PCR and immunohistochemistry (IHC). Interestingly, the prognostic influence of these genes was often associated with their expression in non-malignant cells instead of tumour cells. Based on the observed gene expression patterns, we analyzed the abundance and prognostic value of non-malignant immune cells in 95-98 FL patients treated with immunochemotherapy. We observed that a high content of tumour-associated macrophages was a marker of a favourable prognosis. In contrast, the accumulation of mast cells correlated with a poor outcome and was further associated with tumour vascularity. Increased microvessel density also correlated with an inferior outcome. In addition, we used the same microarray data with a systems biology approach to identify signalling pathways or groups of genes capable of separating patients with favourable or adverse outcomes. Among the transcripts, there were many genes associated with signal transducers and activators of the transcription (STAT5a) pathway. When IHC was used as validation, STAT5a expression was mostly observed in T-cells and follicular dendritic cells, and expression was found to predict a favourable outcome. In cell cultures, rituximab was observed to induce the expression of STAT5a-associated interleukins in human lymphoma cell lines, which might provide a possible link for the cross-talk between rituximab-induced FL cells and their microenvironment. In conclusion, we have demonstrated that the microenvironment has a prognostic role in FL patients treated with immunochemotherapy. The results also address the importance of re-evaluating the prognostic markers in the rituximab era of lymphoma therapies.