Cutting edge: immune cells as sources and targets of the IL-10 family members?

This study investigated the expression of five novel human IL-10-related molecules and their receptors in blood mononuclear cells. IL-19 and IL-20 were found to be preferentially expressed in monocytes. IL-22 and IL-26 (AK155) expression was exclusively detected in T cells, especially upon type 1 polarization, and in NK cells. IL-24 (melanoma differentiation-associated gene 7) expression was restricted to monocytes and T cells. Detection of these molecules in lymphocytes was predominantly linked to cellular activation. Regarding T cells, IL-26 was primarily produced by memory cells, and its expression was independent on costimulation. In contrast to the high expression of receptors for IL-10 homologs in different tissues and cell lines, monocytes and NK, B, and T cells showed clear expression only of IL-10R1, IL-10R2, and IL-20R2. In these cells, IL-20R2 might be part of a still-unknown receptor complex. Therefore, immune cells may represent a major source but a minor target of the novel IL-10 family members.     

Human interleukin 24 (MDA-7/IL-24) protein kills breast cancer cells via the IL-20 receptor and is antagonized by IL-10

The melanoma differentiation-associated gene-7 (mda-7/IL-24) is a unique member of the interleukin 10 (IL-10) family of cytokines, with ubiquitous tumor cell pro-apoptotic activity. Recent data have shown that IL-24 is secreted as a glycosylated protein and functions as a pro-Th1 cytokine and as a potent anti-angiogenic molecule. In this study, we analyzed the activity of Ad-mda7 and its protein product, secreted IL-24, against human breast cancer cells. We show that Ad-mda7 transduction of human breast cancer cells results in G₂/M phase cell cycle arrest and apoptotic cell death, which correlates with secretion of IL-24 protein. Neutralizing antibody against IL-24 significantly inhibited Ad-mda7 cytotoxicity. IL-24 and IL-10 both engage their cognate receptors on breast cancer cells resulting in phosphorylation and activation of STAT3, however, IL-10 receptor binding failed to induce cell killing, indicating that tumor cell killing by IL-24 is independent of STAT3 phosphorylation. Treatment with exogenous IL-24 induced apoptosis in breast cancer cells and this effect was abolished by addition of anti-IL-24 antibody or anti-IL-20R1, indicating that bystander cell killing is mediated via IL-24 binding to the IL-20R1/IL-20R2 heterodimeric receptor complex. Co-administration of the related cytokine IL-10 inhibited killing mediated by IL-24 and concomitantly inhibited IL-24 mediated up-regulation of the tumor suppressor proteins, p53 and p27^Kip1. In summary, we have defined a tumor-selective cytotoxic bystander role for secreted IL-24 protein and identified a novel receptor-mediated death pathway in breast cancer cells, wherein the related cytokines IL-24 and IL-10 exhibit antagonistic activity.     

Mda-7/IL-24 induces apoptosis of diverse cancer cell lines through JAK/STAT-independent pathways

Experimental evidence documents that the MDA-7/IL-24 protein (an IL-10 family cytokine) binds to IL-20 and IL-22 receptor complexes resulting in the activation of JAK/STAT signaling pathways. Recent published reports utilizing human blood derived primary lymphocytes have provided additional confirmatory evidence relating to the cytokine properties of this molecule. A notable attribute of mda-7/IL-24 is its cancer cell-specific apoptosis inducing capacity, which currently remains incompletely understood. Treatment with distinctive tyrosine kinase inhibitors (Genistein and AG18) or a JAK-selective inhibitor (AG490) did not prevent Ad.mda-7 induced apoptosis in diverse cell lines. In addition, there is no apparent correlation between patterns of expression of IL-20R1, IL-20R2, and IL-22R mRNA and susceptibility to Ad.mda-7 in different cell lines. Furthermore, Ad.mda-7 is able to induce killing in STAT/JAK deficient cells. In contrast, treatment with the p38(MAPK) selective inhibitor SB203580, partially inhibited apoptosis induced by Ad.mda-7 in different cell lines. These results demonstrate for the first time that signaling events leading to susceptibility to Ad.mda-7 induced apoptosis, might be tyrosine kinase independent and can thus be distinguished from its cytokine function related properties mediated by the IL-20/IL-22 receptor complexes that require JAK/STAT kinase activity.     

Interleukins 19, 20, and 24 signal through two distinct receptor complexes. Differences in receptor-ligand interactions mediate unique biological functions

Cytokines that signal through Class II receptors form a distinct family that includes the interferons and interleukin 10 (IL-10). Recent identification of several IL-10 homologs has defined a cytokine subfamily that includes AK155, IL-19, IL-20, IL-22, and IL-24. Within this subfamily, IL-19, IL-20, and IL-24 exhibit substantial sharing of receptor complexes; all three are capable of signaling through IL-20RA/IL-20RB, and IL-20 and IL-24 both can also use IL-22R/IL-20RB. However, the biological effects of these three cytokines appear quite distinct: immune activity with IL-19, skin biology with IL-20, and tumor apoptosis with IL-24. To more fully elucidate their interactions with the receptor complexes, we have performed a series of in vitro assays. Reporter, proliferation, and direct STAT activation assays using cell lines expressing transfected receptors revealed differences between the receptor complexes. IL-19 and IL-24 also exhibited growth inhibition on a cell line endogenously expressing all three receptor subunits, an effect that was seen at cytokine levels two orders of magnitude above those required for STAT activation or proliferation. These results demonstrate that, although this subclass exhibits receptor complex redundancy, there are differences in ligand/receptor interactions and in signal transduction that may lead to specificity and a distinct biology for each cytokine.     

IL22/IL-22R Pathway Induces Cell Survival in Human Glioblastoma Cells

Interleukin-22 (IL-22) is a member of the IL-10 cytokine family that binds to a heterodimeric receptor consisting of IL-22 receptor 1 (IL-22R1) and IL-10R2. IL-22R expression was initially characterized on epithelial cells, and plays an essential role in a number of inflammatory diseases. Recently, a functional receptor was detected on cancer cells such as hepatocarcinoma and lung carcinoma, but its presence was not reported in glioblastoma (GBM). Two GBM cell lines and 10 primary cell lines established from patients undergoing surgery for malignant GBM were used to investigate the expression of IL-22 and IL-22R by using quantitative RT-PCR, western blotting and confocal microscopy studies. The role of IL-22 in proliferation and survival of GBM cell lines was investigated in vitro by BrdU and ELISA cell death assays. We report herein that the two subunits of the IL-22R complex are expressed on human GBM cells. Their activation, depending on exogenous IL-22, induced antiapoptotic effect and cell proliferation. IL-22 treatment of GBM cells resulted in increased levels of phosphorylated Akt, STAT3 signaling protein and its downstream antiapoptotic protein Bcl-xL and decreased level of phosphorylated ERK1/2. In addition, IL-22R subunits were expressed in all the 10 tested primary cell lines established from GBM tumors. Our results showed that IL-22R is expressed on GBM established and primary cell lines. Depending on STAT3, ERK1/2 and PI3K/Akt pathways, IL-22 induced GBM cell survival. These data are consistent with a potential role of IL-22R in tumorigenesis of GBM. Since endogenous IL-22 was not detected in all studied GBM cells, we hypothesize that IL-22R could be activated by immune microenvironmental IL-22 producing cells.     

Interleukin-2 enhances the growth of human melanoma cells derived form primary but not from metastatic tumours

Previously, we demonstrated that in vitro treatment of B16F10 murine melanoma cells with interleukin-2 (IL-2) enhances proliferation and metastasis. To further investigate the role played by IL-2 in human melanomas, we studied the expression of IL-2/IL-2 receptor and the effect of IL-2 on the proliferation of melanoma cell lines derived from primary (A375 and RMS cell lines) and metastatic (Hs294T cell line) tumours. We found a constitutive expression of cytoplasmic IL-2 and alpha, beta and gamma-subunits of the IL-2R on the surface of the three melanoma cell lines. The presence of IL-2 in the culture increased the proliferation rate in A375 and RMS cell lines, but no effect was observed in Hs294T metastatic cells. Biologically active IL-2 could be found in the supernatant of the three melanoma cell lines, particularly in A375 and RMS cells, in which an inhibition of the proliferation rate was observed when IL-2 was blocked. Moreover, the combination of anti-IL-2R beta and anti-IL-2R gamma blocking antibodies induced a significant down-regulation of cell proliferation in the three melanoma cell lines, and the combination of anti-IL-2R alpha, anti-IL-2R beta and anti-IL-2R gamma blocking antibodies inhibited IL-2-mediated growth stimulation in A375 and Hs294T cell lines. In RMS cells, a more significant effect was observed when only IL-2R gamma was blocked. Finally, exogenous IL-2 modulated the IL-2 endogenously produced by melanoma cells. These data show that IL-2 may modulate the growth of melanoma cells through autocrine or/and paracrine mechanisms.     

Loss of IL-6 receptor expression in cervical carcinoma cells inhibits autocrine IL-6 stimulation: abrogation of constitutive monocyte chemoattractant protein-1 production

IL-6 is synthesized in human papilloma virus (HPV)-transformed cervical carcinoma cell lines and is supposed to stimulate these cells in an autocrine manner. We studied IL-6 production and responsiveness in nonmalignant HPV-transformed keratinocytes and cervical carcinoma cells in detail. IL-6 was detected in cervical carcinomas in situ. Correspondingly, HPV-positive carcinoma cell lines expressed high IL-6 levels. However, these carcinoma cell lines showed low responsiveness to IL-6 as revealed by low constitutive STAT3 binding activity, which was not further enhanced by exogenous IL-6. In contrast, in vitro-transformed nonmalignant keratinocytes without endogenous IL-6 production strongly responded to exogenous IL-6 with activation of STAT3. STAT3 protein expression levels were comparable in both responsive and nonresponsive cell lines. Also, gp130, the upstream signal-transducing receptor subunit conveying IL-6 signals into the cell, was expressed in all tested cell lines. However, the IL-6 binding subunit gp80 was lost in the malignant cells. Addition of soluble gp80 was sufficient to restore IL-6 responsiveness in carcinoma cells as shown by enhanced activation of STAT3 binding activity. As a consequence of the restored IL-6 responsiveness, carcinoma cells strongly produced the chemokine monocyte chemoattractant protein-1 (MCP-1). Our data demonstrate that cervical carcinoma cells producing high amounts of IL-6 only weakly respond to IL-6 in an autocrine manner due to limited gp80 expression. While production of IL-6 might contribute to a local immunosuppressive effect, silencing an autocrine IL-6 response prevents constitutive production of the mononuclear cell-attracting chemokine MCP-1. Both mechanisms might help the tumor to escape the immune system.     

Role of the interleukin (IL)-28 receptor tyrosine residues for antiviral and antiproliferative activity of IL-29/interferon-lambda 1: similarities with type I interferon signaling

Interferon (IFN)-lambda 1, -lambda 2, and -lambda 3 are the latest members of the class II cytokine family and were shown to have antiviral activity. Their receptor is composed of two chains, interleukin-28R/likely interleukin or cytokine or receptor 2 (IL-28R/LICR2) and IL-10R beta, and mediates the tyrosine phosphorylation of STAT1, STAT2, STAT3, and STAT5. Here, we show that activation of this receptor by IFN-lambda 1 can also inhibit cell proliferation and induce STAT4 phosphorylation, further extending functional similarities with type I IFNs. We used IL-28R/LICR2-mutated receptors to identify the tyrosines required for STAT activation, as well as antiproliferative and antiviral activities. We found that IFN-lambda 1-induced STAT2 tyrosine phosphorylation is mediated through tyrosines 343 and 517 of the receptor, which showed some similarities with tyrosines from type I IFN receptors involved in STAT2 activation. These two tyrosines were also responsible for antiviral and antiproliferative activities of IFN-lambda 1. By contrast, STAT4 phosphorylation (and to some extent STAT3 activation) was independent from IL-28R/LICR2 tyrosine residues. Taken together, these observations extend the functional similarities between IFN-lambdas and type I IFNs and shed some new light on the mechanisms of activation of STAT2 and STAT4 by these cytokines.     

Cutting edge: STAT activation by IL-19, IL-20 and mda-7 through IL-20 receptor complexes of two types

IL-10-related cytokines include IL-20 and IL-22, which induce, respectively, keratinocyte proliferation and acute phase production by hepatocytes, as well as IL-19, melanoma differentiation-associated gene 7, and AK155, three cytokines for which no activity nor receptor complex has been described thus far. Here, we show that mda-7 and IL-19 bind to the previously described IL-20R complex, composed by cytokine receptor family 2-8/IL-20Ralpha and DIRS1/IL-20Rbeta (type I IL-20R). In addition, mda-7 and IL-20, but not IL-19, bind to another receptor complex, composed by IL-22R and DIRS1/IL20Rbeta (type II IL-20R). In both cases, binding of the ligands results in STAT3 phosphorylation and activation of a minimal promoter including STAT-binding sites. Taken together, these results demonstrate that: 1) IL-20 induces STAT activation through IL-20R complexes of two types; 2) mda-7 and IL-20 redundantly signal through both complexes; and 3) IL-19 signals only through the type I IL-20R complex.     

Predominant expression of the long isoform of GP130-like (GPL) receptor is required for interleukin-31 signaling

Gp130-like receptor (GPL) is a newly identified cytokine receptor. A recent study reported the involvement of GPL, together with OSMR, in the formation of the receptor complex for IL-31, a novel immune cytokine with a skin tropism. In the present work, we analyzed the signaling properties of IL-31 in glioblastoma and melanoma tumor cells. We demonstrate that in response to IL-31, its receptor complex recruits Jak1, Jak2, STAT1, -3, -5 signaling pathways, as well as the Pi3 kinase / AKT cascade. SHP-2 and Shc adapter molecules are also recruited and contribute to an increased activation of the MAP kinase pathway in response to IL-31. Different responses were observed depending on the expression of short or long GPL receptor isoform within the studied cell lines. We show that the short form of GPL receptor exerts a profound inhibitory effect on the signaling of IL-31 and behaves as a dominant negative receptor.     

Interleukin-26: an IL-10-related cytokine produced by Th17 cells

IL-26 is classified as a member of the IL-10 cytokine family because it has limited sequence homology to IL-10 and the IL-10-related cytokines. The human IL-26 gene, IL26, is located on chromosome 12q15 between the genes for two other important class-2 cytokines, IFNG (IFN-γ) and IL22 (IL-22). IL-26 is often co-expressed with IL-22 by activated T cells, especially Th17 cells. It signals through a heterodimeric receptor complex composed of the IL-20R1 and IL-10R2 chains. IL-26 receptors are primarily expressed on non-hematopoietic cell types, particularly epithelial cells. Signaling through IL-26 receptor complexes results in the activation of STAT1 and STAT3 with subsequent induction of IL-26-responsive genes. The biological functions of IL-26 have only begun to be defined.     

Cyclooxygenase regulates human oropharyngeal carcinomas via the proinflammatory cytokine IL-6: a general role for inflammation?

High levels of prostaglandins are produced in human oropharyngeal carcinoma (OPC). Five human OPC cell lines tested expressed both isoforms of cyclooxygenases (COX). The pan-COX inhibitor ketorolac continuously and significantly decreased PGE(2) production and IL-6 and IL-8 levels in all OPC cell lines tested, but did not affect IL-1alpha, GM-CSF levels, or in vitro tumor cell growth. In contrast, ketorolac reduced OPC growth in vivo. The OPC cell lines used express the IL-6 receptor, and IL-6 stimulation of these cells causes transduction to occur via STAT3 pathway activation. Coincubation with OPC cell lines with conditioned medium from a TPA-exposed HL-60 cells stimulated growth proportional to the IL-6 levels measured in the conditioned medium. This growth effect was specifically inhibited by anti-IL-6 antibody. These results are consistent with cytokine products of inflammatory cells having paracrine growth effects on OPC. If chronic inflammation plays a role in promoting the development of OPC, this mechanism may also apply to other epithelial tumor systems modulated by COX activity.     

Cutting edge: IL-26 signals through a novel receptor complex composed of IL-20 receptor 1 and IL-10 receptor 2

The receptor for IL-26 (AK155), a cytokine of the IL-10 family, has not previously been defined. We demonstrate that the active receptor complex for IL-26 is a heterodimer composed of two receptor proteins: IL-20R1 and IL-10R2. Signaling through the IL-26R results in activation of STAT1 and STAT3 which can be blocked by neutralizing Abs against IL-20R1 or IL-10R2. IL-10R2 is broadly expressed on a wide variety of tissues, whereas only a limited number of tissues express IL-20R1. Therefore, the ability to respond to IL-26 is restricted by the expression of IL-20R1. IL-10, IL-19, IL-20, IL-22, and IL-24 fail to signal through the combination of IL-10R2 and IL-20R1 proteins, demonstrating that this receptor combination is unique and specific for IL-26.     

Multiple reasons for an inefficient STAT1 response upon IL-6-type cytokine stimulation

IL-6-type cytokines play an important role during inflammation and the immune response. In addition, they are involved in haematopoiesis, liver and neuronal regeneration, embryonic development and fertility. We found that IL-6-type cytokine stimulation of cell lines and primary human macrophages results in a different distribution of the DNA-binding competent STAT dimer species in the cytosol and nucleus as demonstrated by electrophoretic mobility shift assays. In the absence of detergent, STAT3/STAT3, STAT1/STAT3 were the predominant species in the cytoplasm while STAT3/STAT3 was predominant in the nucleus. However, in detergent containing total cellular lysates and nuclear fractions prepared with detergent containing buffers, the STAT1/STAT1 homodimer was as prominent or even more prominent than STAT3/STAT3 and STAT1/STAT3. We were interested in the cause of this discrepancy since STAT1-regulated genes have not been described to be expressed upon IL-6-type cytokine stimulation. In addition to the more transient STAT1 activation, IL-6-type cytokines such as IL-6 and OSM lead to a much less efficient STAT1 activation compared to the potent STAT1 activators IFNγ and IFNα. Studies with STAT1-deficient cells revealed that STAT1 activation does not seem to be an important competitive process to STAT3 activation arguing again for a very inefficient STAT1 activation upon IL-6-type cytokine stimulation. We also describe that pY-STAT3 is much more efficiently shuttled into the nucleus than pY-STAT1.