Functional characterization of IL-10 and its receptor subunits in a perciform fish, the mandarin fish, Siniperca chuatsi.

Interleukin (IL)-10 is an immune-regulatory cytokine with multiple functions. In the current study, IL-10 and its two receptors, IL-10R1 and IL-10R2 were identified in mandarin fish, Siniperca chuatsi. The inhibitory effect of mandarin fish IL-10 was investigated on pro-inflammatory cytokine expression and the ligand-receptor relationship. This IL-10 possesses conserved cysteine residues, predicted α-helices and a typical IL-10 family signature motif, similar to its mammalian orthologue, and IL-10R1 harbours predicted JAK1 and STAT3 binding sites in the intracellular region. The fish IL-10 and IL-10R1 exhibit high expression levels in several immune-related organs/tissues, such as spleen, trunk kidney and head kidney, and IL-10R2 possesses a constitutive expression pattern. The expression of IL-10 shows significant increase in spleen from infectious spleen and kidney necrosis virus (ISKNV) infected mandarin fish, where the two receptors also exhibit different levels of induced expression. Mandarin fish IL-10 also exhibits significant response to the stimulation of LPS, PHA and PMA, with the two receptors exhibiting an interesting decrease in expression following the treatment of PMA. The pro-inflammatory cytokines, IL-6, IL-1ß, IL-8, TNF-α, show diminished up-regulation in LPS-stimulated splenocytes pre-incubated with IL-10, indicating the anti-inflammatory roles of mandarin fish IL-10. In EPC cells transfected with different combinations of receptors, IL-10 can enhance the expression of suppressor of cytokine signalling 3 (SOCS3) only when IL-10R1 and IL-10R2 are both expressed, suggesting the participation of the two receptors in signal transduction of mandarin fish IL-10. Similar results are observed with the usage of chimeric receptors, IL-10R1/CRFB1 and IL-10R2/CRFB5. Overall, mandarin fish IL-10 shares conserved ligand-receptor system and the prototypical inhibitory activities on pro-inflammatory cytokine expression with mammalian IL-10, implying the evolutionary conservation of this cytokine.

Structure and function of interleukin-22 and other members of the interleukin-10 family.

The IL-10 family of cytokines is comprised of IL-10, IL-19, IL-20, IL-22, IL-24, IL-26, and IFN-lambdas (IL-28A, IL-28B, and IL-29). The IL-10 family members bind to shared class II cytokine receptor chains that associate in various combinations in heterodimeric complexes. Upon interleukin/receptor complex formation, these proteins switch on the Jak/STAT pathway and elicit pleiotropic biological responses whose variety sharply contrasts with their structural similarities. IL-10 family members are involved in several human diseases and health conditions and hence their structural analyses may provide valuable information to design specific therapeutic strategies. In this review, we describe the human interleukin-10 family of cytokines, focusing on their structures and functions, with particular attention given to IL-22 and IL-10. We report on the recently published structures of IL-10 cytokine family members and their complexes with cognate transmembrane and soluble receptors as well as on interleukin physiology and physiopathology.

Interleukin-10 does not affect IL-1-induced interleukin-6 and metalloproteinase production in human chondrosarcoma cells, SW1353.

Cartilage repair by transplantation of autologous chondrocytes is an option when restoring functional joints. Control of chondrocyte function is thus required. Interleukin-10 (IL-10) is a potent anti-inflammatory cytokine affecting the expression of a wide range of immune mediators in hematopoietic and non-hematopoietic cells. Previous studies indicated that IL-10 has therapeutic potential in the treatment of chronic inflammatory joint disorders such as rheumatoid arthritis and osteoarthritis. IL-10 has been found to be chondroprotective by down-regulating metalloproteinase expression and by inhibiting the synthesis of pro-inflammatory cytokines, such as IL-6, in immune cells. In contrast, the effects of IL-10 on chondrocytes are poorly understood and have to be identified with regard to their future clinical use. In this study, we investigated the effects of IL-10 on the expression of cartilage-degrading mediators in the human chondrosarcoma cell line, SW1353, after exposure to IL-1, a key mediator in cartilage and bone destruction. We found a strong induction of the pro-inflammatory cytokine, IL-6, in IL-1-exposed SW1353 cells. Surprisingly, IL-10 had no effect on IL-1-induced IL-6, pro-MMP1, and pro-MMP13 secretion. Although RT-PCR analyses demonstrated the expression of both receptor chains of the IL-10 receptor complex (IL-10R1 and IL-10R2), exposure of SW1353 to IL-10 did not lead to phosphorylation of STAT3, the major transcription factor induced by IL-10. This was not due to a defect in STAT3, because stimulation with IL-6 resulted in its phosphorylation. Failure of SW1353 cells to respond to IL-10 was consistent with a deficient surface expression of IL-10R1. From these results we conclude that IL-10 does not exert its chondroprotective character on chondrocytes directly. Furthermore, the unresponsiveness of chondrocytes towards IL-10 might explain the vulnerability of joint cartilage to inflammation.

Cloning, characterization and expression analysis of interleukin-10 from the zebrafish (Danio rerion).

Cytokines are proteins produced by many different cells of the immune system and play a significant role in initiating and regulating the inflammatory process. In this research, an important cytokine, interleukin-10 (IL-10) gene, has been identified and characterized from zebrafish (Danio rerio) genome database. Zebrafish IL-10 is located within a 2690 bp fragment and contains five exons and four introns, sharing the same organization with mammalian IL-10 genes. An open reading frame of 543 bp was found to encode a putative 180 amino acid protein with a signal peptide of 22 amino acids, which shares 29.7-80.9 % homology with amino acid sequences of other known IL-10. The signature motif of IL-10 is also conserved in zebrafish IL-10. The predicted transcript was finally confirmed by sequencing of cDNA clones. Multi-tissue reverse transcriptase PCR (RT-PCR) was performed to examine the tissue distribution and expression regulation of this gene in seven organs of normal and lipopolysaccharide (LPS) stimulation zebrafish. The results demonstrated that this gene was expressed slightly in normal kidney, gill and gut, no expression was detected in other four tissues. The expression was clearly upregulated after LPS stimulation. Using the ideal zebrafish model, further study of IL-10 characterization and function may provide insight on the understanding of the innate immune system.

Subtype & cytokine profiles of HIV infected individuals from south India.

BACKGROUND & OBJECTIVE: The global surveillance of human immunodeficiency virus (HIV) subtypes (clades) helps understand the global distribution and incidence of different HIV subtypes. As knowledge about subtypes circulating in an area is needed for developing a candidate vaccine, prevalence of the subtypes HIV-1 and HIV-2 were studied in south India. The profile of cytokines interleukin 10 (IL10) and interferon gamma (IFNgamma) in both types of infection were also analysed as these are considered indicators of disease progression. METHODS: Patients who belonged to the 4 south Indian States i.e. Tamil Nadu, Kerala, Karnataka and Andhra Pradesh were included. HIV-1 subtyping was carried out by the heteroduplex mobility analysis (HMA) while that of HIV-2 was done by direct sequencing. The quantitation of IFNgamma and IL-10 was carried out using commercial ELISA kits. RESULTS: Among the 82 HIV-1 infected individuals subtyped, 78 (95.1%) were subtype C while all 12 HIV-2 strains were subtype A. IL-10 concentration was significantly higher among HIV infected individuals compared to normal healthy controls. IFNgamma was significantly higher among symptomatic and AIDS groups compared to asymptomatic HIV-1 infected individuals. INTERPRETATION & CONCLUSION: HIV-1 subtype C and the HIV-2 subtype A are the major subtypes circulating in south India. The study showed a trend towards a shifting of the cytokine profile from Th1 to Th2/Th0 in HIV-1, HIV-2 infections, and HIV-1 and HIV-2 dual infected individuals as the disease progresses. This trend observed is not unlike that reported from the West, despite the difference in subtype profile.

Structural features of the interleukin-10 family of cytokines.

The interleukin-10 (IL-10) family of cytokines includes IL-10, a number of its viral gene homologs, and eight recently discovered cellular cytokines (IL-19, IL-20, IL-22, IL-24, IL-26, IFN-lambda1, IFN-lambda2, IFN-lambda3). IL-10 is an intercalated dimer consisting of two six-helix bundle domains. Signal transduction occurs when each domain of IL-10 binds to two receptor chains, IL-10R1 and IL-10R2. Viral homologs use the same IL-10 receptor system, while cellular homologs use their own receptors: three long receptor chains (IL-20R1, IL-22R1 and IFN-lambda1R1) and two short receptor chains (IL-20R2 and IL-10R2). Most of the cellular homologs belong to the IL-19 subfamily of cytokines including IL-19, IL-20, IL-22 and IL-24. It is likely that IFN-lambda1, IFN-lambda2, and IFN-lambda3 also belong to the same subfamily. All these proteins are monomers in solution. Crystal structures of IL-19 and IL-22 show that the molecules consist of seven helices (A-G) forming a seven-helix bundle with compact hydrophobic core inside. Structures of complexes of IL-10 and CMVIL-10 with an extracellular domain of high affinity receptor IL-10R1 (sIL-10R1) showed that ligand/receptor interactions are of mostly polar nature, with two hydrophobic patches around receptor residues Tyr43 and Phe143 at the top and bottom of the interface. The location and structure of the binding site for the second receptor chain are still unknown. It has also been shown that in the case of IL-19 and IL-20, IL-20R2 rather than IL-20R1 is a high-affinity receptor chain. This review summarizes all published three-dimensional structures of the cytokines representing the IL-10 family of homologs, including the IL-19 subfamily and their interaction with appropriate receptors.

Bystander activity of Ad-mda7: human MDA-7 protein kills melanoma cells via an IL-20 receptor-dependent but STAT3-independent mechanism.

The melanoma differentiation-associated gene-7 (mda-7/IL24) is a unique member of the IL-10 family of cytokines, with ubiquitous tumor cell proapoptotic activity. Transduction of tumor or normal cells with the mda-7 gene results in secretion of glycosylated MDA-7 protein. Recent data indicate that secreted MDA-7 protein functions as a pro-Th1 cytokine and as a potent antiangiogenic molecule. MDA-7 protein binds two distinct type II cytokine heterodimeric receptor complexes, IL-20R1/IL-20R2 (type 1 IL-20R) and IL-22R1/IL-20R2 (type 2 IL-20R). In this study we analyzed the activity of glycosylated secreted MDA-7 against human melanoma cells. MDA-7 protein induces phosphorylation and nuclear translocation of STAT3 in melanoma cells via both type 1 and type 2 IL-20R. MDA-7 induces dose-dependent cell death in melanoma tumor cells. MDA-7 receptor engagement results in up-regulation of BAX and subsequent apoptosis induction; this effect is mediated by STAT3-independent signaling. Additional IL-10 family members (IL-10, -19, -20, and -22) also activate STAT3; however, these ligands do not activate death pathways in melanoma. In normal cells, MDA-7 can bind to its cognate receptors and induce phosphorylation of STAT3, without cytotoxic sequelae. This study defines a tumor-selective cytotoxic bystander role for secreted MDA-7 protein and identifies a novel receptor-mediated, STAT3-independent, and PKR-independent death pathway.

Molecular modeling of the interleukin-19 receptor complex. Novel aspects of receptor recognition in the interleukin-10 cytokine family.

The interleukin-10 (IL-10) cytokine family consists of several viral and human homologs that exhibit distinct receptor binding specificities. In the present study, the complex between interleukin-19 (IL-19) and its physiological receptor-the interleukin-20 receptor alpha-chain (IL-20R1)-was modeled. The most prominent feature of this complex is an extended binding interface formed by a long loop of IL-20R1 and a bulge region of IL-19. The two regions exhibit complementary charges and have no structural counterparts in the IL-10/IL-10R1 complex but show some resemblance to the complex between interferon-gamma (IFN-gamma) and its receptor. Sequence comparison of the three cytokines (IL-19, IL-20, IL-24) that bind the IL-20R1 reveals a considerable conservation of the length of the interacting loops. One residue suggested to play a key role in receptor binding specificity is a conserved glutamate. The binding interface of IL-20R1 is rich in aromatic residues while the interfaces of its cytokine ligands are mainly formed by more flexible aliphatic amino acids. This structural feature might play an important role for the specific recognition of a single receptor chain by three different cytokines. [Figure: see text]. Comparison of the ligand/receptor interfaces in theA IL-10/IL-10R1,B IL-19/IL-20R1 andC IFN-gamma/receptor complexes. The translucent Connolly surfaces of the receptor and the ligand are shown in yellow and white, respectively. The backbone of the receptor and ligand are highlighted by a red and blue/green tube, respectively. For clarity, only one domain of the intertwined dimes is shown for IL-10 and IFN-gamma. Arrows denote the location of helix B and the corresponding structural elements in IL-19 and IFN-gamma as well as the location of receptor loop L2. As evident fromB andC an extended interaction surface is created in the IL-19/IL-20R1 and IFN-gamma/receptor complexes by the interaction of this structural element with a long loop of the respective receptor.

MDA-7/IL-24 is a unique cytokine--tumor suppressor in the IL-10 family.

The melanoma differentiation associated gene-7 (mda-7) cDNA was isolated by virtue of being induced during melanoma differentiation. Initial gene transfer studies convincingly demonstrated potent antitumor effects of mda-7. Further studies showed that the mechanism of antitumor activity was due to induction of apoptosis. Most striking was the tumor-selective killing by mda-7 gene transfer--normal cells were unaffected by Adenoviral delivery of mda-7 (Ad-mda7). A variety of molecules implicated in apoptosis and intracellular signaling are regulated by Ad-mda7 transduction. Different apoptosis effector proteins are regulated in different tumor types, suggesting that Ad-mda7 may regulate various signaling pathways. mda-7 encodes a secreted protein, MDA-7, which has now been designated as IL-24, and is a novel member of the IL-10 cytokine family. MDA-7/IL-24 protein is actively secreted from cells after mda-7 gene transfer. In human peripheral blood mononuclear cells (PBMC), STAT3 activation by MDA-7/IL-24 is followed by elaboration of secondary Th1 cytokines, demonstrating that MDA-7/IL-24 is a pro-Th1 cytokine. Furthermore, MDA-7/IL-24 is antagonized by the prototypic Th2 cytokine IL-10. MDA-7/IL-24 protein is endogenously expressed in cultured NK and B-cells and is also expressed in dendritic cells in tissues. MDA-7/IL-24 protein is expressed in nevi and melanoma primary tumors, to varying degrees, but is rarely expressed in malignant melanoma or other human tumors evaluated. Indeed, loss of MDA-7/IL-24 protein expression correlates strongly with melanoma tumor invasion and disease progression. The "bystander" effects proposed for MDA-7/IL-24 protein include immune stimulation, antiangiogenesis and receptor-mediated cytotoxicity. Thus, mda-7 is a unique multifunctional cytokine in the IL-10 family and may have potent antitumor utility in a clinical setting.

Genotyping interleukin-10 high and low producers with single-tube bidirectional allele-specific amplification.

A simple bidirectional allele-specific PCR method is described for determining the -1082 A and G alleles in the interleukin-10 (IL-10) promoter region. This polymorphism is associated with IL-10 production capacity, and it is thus interesting to see whether different infectious and autoimmune conditions are associated with it. With our method, the A and G alleles may be studied simultaneously in a single PCR reaction, as amplification of the different alleles is performed by using 3'-mismatched and partly overlapping allele-specific upstream and downstream primers around the -1082 site. The fast and simple method described here is especially suitable for large-scale association studies.