Kinetic analysis of high affinity forms of interleukin (IL)-13 receptors: suppression of IL-13 binding by IL-2 receptor gamma chain.

Interleukin-13 (IL-13) is a pleiotropic cytokine that controls growth, differentiation, and apoptosis of immune and tumor cells. To understand the mechanisms of interaction between IL-13 and IL-13 receptors (IL-13R), and the role of the IL-2 receptor common gamma chain (gammac) in IL-13 binding and processing, we have examined IL-13 binding kinetics, dissociation/shedding, and internalization in renal cell carcinoma (RCC) cell lines. We observed a new phenomena in that the apparent rate of association, but not the dissociation, was strongly related to IL-13 concentration. We also observed cooperativity phenomena in IL-13 and IL-13R interaction in control RCC (MLneo) cells, but not in cells transfected with gammac chain (MLgammac). The number of IL-13 binding sites, the effective rate of ligand association, and the dissociation rate constants were reduced in gammac-transfected cells compared to control RCC cells. Two forms of IL-13R were detected in these cell lines, which differed in the kinetics of endocytosis and dissociation/exocytosis. Only a small fraction of bound receptors (14-24%) was rapidly internalized and the same fraction of the ligand-receptor complexes was shed and/or dissociated. The expression of gammac chain did not change any of these processes. A two independent high-affinity and moderate-affinity receptor model fit the kinetic observations in gammac-transfected cells. However, in control cells, the binding kinetics were more complicated. A mathematical model that fit a set of kinetic and steady state data in control cells was selected from a set of possible models. This best-fit model predicts that 1) two different IL-13R are expressed on the cell membrane, 2) a minor fraction of IL-13R exist as microclusters (homodimers and/or heterodimers) without exogenous IL-13, 3) high morphological complexity of the gammac-negative control cell membrane affects the cooperativity phenomena of IL-13 binding, and 4) a large number of co-receptor molecules is present, which helps keep the ligand on the cell surface for a long period of time after fast IL-13 binding and provides a negative control for ligand binding via production of the high affinity inhibitor bound to IL-13. Our data demonstrate that gammac exerts dramatic changes in the kinetic mechanisms of IL-13 binding.     

Identification of distinct roles for a dileucine and a tyrosine internalization motif in the interleukin (IL)-13 binding component IL-13 receptor alpha 2 chain

Interleukin (IL)-13 receptor alpha2 (IL-13Ralpha2) chain is an essential binding component for IL-13-mediated ligand binding. Recently, we have demonstrated that this receptor chain also plays an important role in the internalization of IL-13. To study the mechanism of IL-13 internalization, we generated mutated IL-13Ralpha2 chains that targeted trileucine residues (Leu(335), Leu(336), and Leu(337)) in the transmembrane domain and a tyrosine motif (Tyr(343)) in the intracellular domain and transfected these cDNAs in COS-7 cells. Cells that expressed a C-terminally truncated IL-13Ralpha2 chain (Delta335) did not bind IL-13, suggesting that the trileucine region modulates IL-13 binding. Truncation of IL-13Ralpha2 chain with a mutation in the trileucine region resulted in significantly decreased internalization compared with wild type IL-13Ralpha2 chain transfected cells. COS-7 cells transfected with tyrosine motif mutants exhibited a similar internalization level compared with wild type IL-13Ralpha2 chain transfected cells; however, dissociation of cell surface IL-13 was faster compared with wild type IL-13Ralpha2 transfectants. These results were further confirmed by determining the cytotoxicity of a chimeric protein composed of IL-13 and a mutated form of Pseudomonas exotoxin (IL13-PE38QQR) to cells that expressed IL-13Ralpha2 chain mutants. We further demonstrate that the IL-13Ralpha2 chain is not ubiquitinated and that internalization of IL-13Ralpha2 did not depend on ubiquitination. Together, our findings suggest that the dileucine motif in the trileucine region and tyrosine motif participate in IL-13Ralpha2 internalization in distinct manners.     

Expression of the Ym2 lectin-binding protein is dependent on interleukin (IL)-4 and IL-13 signal transduction: identification of a novel allergy-associated protein.

Asthma pathophysiology is intimately regulated by CD4(+) Th2 lymphocytes and the cytokines interleukin (IL)-4 and IL-13. However, the mechanisms by which these cytokines promote disease have not been fully elucidated. In order to identify novel molecular mediators of allergy, a comparison was made of the bronchoalveolar lavage, which demonstrated that the Ym2 protein was abundantly up-regulated in the lung during the development of allergy. Low levels of the Ym1 isomer were also detected. Importantly, neither Ym1 nor Ym2 has been characterized previously in the context of allergic pulmonary inflammation. Western immunoblot showed that enhanced expression of these proteins was dependent on CD4(+) T cells and IL-4 or IL-13 signaling via the IL-4Ralpha subunit. In addition, intratracheal instillation of IL-13 into naive mice was sufficient to induce expression. Ym1 is homologous to eosinophil chemotactic factor L. However, only weak eosinophil chemotaxis was observed in response to Ym protein in both in vitro and in vivo assays. By contrast, the homology of Ym1 and Ym2 to proteins associated with tissue remodeling, together with the previous findings that Ym1 is homologous to chitinase and binds heparin sulfate and GlcN oligomers (chitobiose, chitotriose, and chitotetraose), strongly suggests these proteins play an important role in airway wall remodeling in the allergic lung.     

Production of interleukin (IL)-1beta, IL-1 receptor antagonist and IL-10 by blood mononuclear cells in chronic arthritis

Joint erosion is a prevalent feature of rheumatoid arthritis (RA) but not of many other chronic inflammatory arthritides (non-RA). Joint destruction is mediated by cytokines, primarily interleukin (IL)-1 and tumour necrosis factor. Less erosive activity in patients with non-RA compared to RA might be related to factors that inhibit production and/or function of IL-1. Release of IL-1beta, and the two antagonists, IL-1 receptor antagonist (IL-1ra) and IL-10 from blood mononuclear cells were therefore quantitated by ELISA in 22 patients with RA, 11 with non-RA and 15 healthy age-matched controls. Release of IL-1beta was comparable between the three groups but only detectable in cultures stimulated with lipopolysaccharide; it decreased in patients treated with prednisolone: 3.8 ng/10(6)monocytes (median) vs 11.7 (P=0.045). Release of IL-1ra was in all but IgG-stimulated cultures comparable between groups. The ratio of IL-1ra/IL-1beta was elevated in LPS-stimulated cells from RA patients only: 2.0 versus 1.3 (P=0.02). In contrast, IgG-induced IL-1ra release was significantly elevated only in non-RA patients: 95 ng/10(6)monocytes vs 40 (P=0.014), and the levels correlated positively to those of blood CRP (P=0.02). Though stimulated release of IL-10 was similar between the three groups, the levels were lower in non-erosive than erosive arthritis patients, and controls (P=0. 05). In conclusion, increased IgG-stimulated IL-1ra release and elevated IL-1ra/IL-1beta ratio may protect against actions of IL-1 in vivo, and decreased release of IL-10 might be related to features of non-erosive arthritis.     

Role of interleukin (IL)-1 type 1 receptor in mycobacterial infection.

It is important to gain a better understanding of IL-1-mediated signaling events in mycobacterial infection. In order to clarify the role of IL-1 receptor type 1 (IL-1 R1) in IL-1 R1, knockout (KO) mice were infected with either Mycobacterium tuberculosis H37Rv or Kurono strain by the respiratory route, and their ability to control mycobacterial growth, pulmonary granuloma formation, and cytokine mRNA expression was investigated. IL-1 R1 KO mice developed significantly larger (P< 0.01) granulomatous lesions with neutrophil infiltration in their lungs than wild-type mice did after infection with the M. tuberculosis Kurono strain. The number of mycobacterial colonies in lungs and spleen increased from five weeks post-infection. Interferon-y production by spleen cells was low in IL-1 R1 KO mice. It is concluded that the IL-1 R1 is essential for IL-1-mediated signaling events in mycobacterial infection.     

Interleukin (IL)-17 enhances prostaglandin F(2 alpha)-stimulated IL-6 synthesis in osteoblasts

We previously showed that prostaglandin F(2alpha) (PGF(2alpha)) and endothelin-1 (ET-1) induce interleukin (IL)-6 through the activation of protein kinase C-dependent p44/p42 mitogen-activated protein (MAP) kinase in osteoblast-like MC3T3-E1 cells. It has recently been reported that tumor necrosis factor-alpha-induced IL-6 synthesis is amplified by IL-17 in these cells. In the present study, we investigated the effect of IL-17 on the IL-6 synthesis stimulated by PGF(2alpha) in MC3T3-E1 cells. IL-17 significantly enhanced the PGF(2alpha)-induced IL-6 synthesis in a dose-dependent manner in the range between 0.1 and 10 ng/ml. IL-17 also enhanced the IL-6 synthesis stimulated by 12- O -tetradecanoylphorbol-13-acetate, a direct activator of protein kinase C. In addition, IL-17 amplified the IL-6 synthesis induced by ET-1. However, IL-17 hardly affected the phosphorylation of p44/p42 MAP kinase induced by PGF(2alpha) or ET-1. These results strongly suggest that IL-17 enhances the IL-6 synthesis stimulated by PGF(2alpha) as well as ET-1 in osteoblasts, and that the effect is exerted at a point downstream from p44/p42 MAP kinase.     

Up-regulation of interleukin (IL)-6 receptor gene expression in vitro and in vivo in IL-6 deprived myeloma cells.

Myeloma cells absolutely require interleukin-6 (IL-6) for growing in vivo in patients with multiple myeloma and exogenous IL-6-dependent myeloma cell lines have been reproducibly obtained. In this study we show a dramatic up-regulation of the IL-6 receptor (gp80 chain) gene expression in myeloma cell lines following the removal of exogenous IL-6. Such a regulation was also known to occur in IL-6-deprived myeloma cells in vivo in three patients who were treated with optimal doses of anti-IL-6 monoclonal antibodies. The direct effect of IL-6 on IL-6 receptor gene expression in myeloma cells was further confirmed by adding IL-6 to an autonomously growing myeloma cell line.     

Interleukin (IL)-7 induces rapid activation of Pyk2, which is bound to Janus kinase 1 and IL-7Ralpha

Interleukin-7 (IL-7) receptor signaling begins with activation of the Janus tyrosine kinases Jak1 and Jak3, which are associated with the receptor complex. To identify potential targets of these kinases, we examined Pyk2 (a member of the focal adhesion kinase family) using an IL-7-dependent murine thymocyte line, D1. We demonstrate that stimulation of D1 (or normal pro-T) cells by IL-7 rapidly increased tyrosine phosphorylation and enzymatic activity of Pyk2, with kinetics slightly lagging that of Jak1 and Jak3 phosphorylation. Conversely, IL-7 withdrawal resulted in a marked decrease of Pyk2 phosphorylation. Pyk2 was found to be physically associated with Jak1 prior to IL-7 stimulation and to increase its association with IL-7Ralpha chain following IL-7 stimulation. Pyk2 appeared to be involved in cell survival, because antisense Pyk2 accelerated the cell death process. Activation of Pyk2 via the muscarinic and nicotinic receptors using carbachol or via intracellular Ca(2+) rise using ionomycin/phorbol myristate acetate promoted survival in the absence of IL-7. These data support a role for Pyk2 in coupling Jak signaling to the trophic response.     

Allergen rush immunotherapy increases interleukin (IL)-12 production and IL-12 receptor beta2 chain expression in patients with allergic asthma

Interleukin (IL)-12 production and IL-12 receptor (IL-12R) beta2 chain expression were investigated in patients with allergic asthma successfully treated with rush immunotherapy (RIT) and control patients with mild allergic asthma. Peripheral blood mononuclear cells (PBMCs) were stimulated with Dermatophagoides farinae (Der f), and production of cytokines was measured. Furthermore, the effects of cytokines on IL-12R beta2 chain expression on CD4(+) T cells were investigated. Production by PBMCs of IL-12 and IFN-gamma was significantly higher and production of IL-4 was significantly lower after stimulation with Der f allergen in RIT-treated patients than in control patients. Significant increases in the expression of IL-12R beta2 chain before and after stimulation of CD4(+) T cells with IL-12 or IFN-gamma were observed in RIT-treated patients compared with that in control patients. Allergen RIT increases IL-12 production and IL-12R beta2 chain expression and thus may convert cytokine production from Th2 to Th1 or Th0 type in allergic asthma.     

Chimeric cytokine receptor can transduce expansion signals in interleukin 6 receptor alpha (IL-6Ralpha)-, IL-11Ralpha-, and gp130-low to -negative primitive hematopoietic progenitors

We generated transgenic mice expressing chimeric receptors, which comprise extracellular domains of the human granulocyte-macrophage colony-stimulating factor (hGM-CSF) receptor and transmembrane and cytoplasmic domains of the mouse leukemia inhibitory factor receptor. In suspension cultures of lineage-negative (Lin(-)), 5-fluorouracil-resistant bone marrow cells of the transgenic mice, a combination of hGM-CSF and stem cell factor (SCF) induced exponential expansions of mixed colony-forming unit. The combination of hGM-CSF and SCF was effective on enriched, Lin(-)Sca-1(+)c-kit(+) progenitors and increased either mixed colony-forming unit or cobblestone area-forming cells. In case of stimulation with hGM-CSF and SCF, interleukin-6 (IL-6) and SCF, or IL-11 and SCF, the most efficient expansion was achieved with hGM-CSF and SCF. When Lin(-)Sca-1(+)c-kit(+)CD34(-) further enriched progenitors were clone sorted and individually incubated in the presence of SCF, hGM-CSF stimulated a larger number of cells than did IL-6, IL-6 and soluble IL-6 receptor (IL-6R), or IL-11. These data suggest the presence of IL-6Ralpha-, IL-11Ralpha-, and gp130-low to -negative primitive hematopoietic progenitors. Such primitive progenitors are equipped with signal transduction molecules and can expand when these chimeric receptors are genetically introduced into the cells and stimulated with hGM-CSF in the presence of SCF.     

Lysophosphatidic acid induces interleukin-13 (IL-13) receptor alpha2 expression and inhibits IL-13 signaling in primary human bronchial epithelial cells

Interleukin-13 (IL-13), a Th2 cytokine, plays a pivotal role in pathogenesis of bronchial asthma via IL-13 receptor alpha1 (IL-13Ralpha1) and IL-4 receptor alpha (IL-4Ralpha). Recent studies show that a decoy receptor for IL-13, namely IL-13Ralpha2, mitigates IL-13 signaling and function. This study provides evidence for regulation of IL-13Ralpha2 production and release and IL-13-dependent signaling by lysophosphatidic acid (LPA) in primary cultures of human bronchial epithelial cells (HBEpCs). LPA treatment of HBEpCs in at imedependent fashion increased IL-13Ralpha2 gene expression without altering the mRNA levels of IL-13Ralpha1 and IL-4Ralpha. Pretreatment with pertussis toxin (100 ng/ml, 4 h) or transfection of c-Jun small interference RNA or an inhibitor of JNK attenuated LPA-induced IL-13Ralpha2 gene expression and secretion of soluble IL-13Ralpha2. Overexpression of catalytically inactive mutants of phospholipase D (PLD) 1 or 2 attenuated LPA-induced IL-13Ralpha2 gene expression and protein secretion as well as phosphorylation of JNK. Pretreatment of HBEpCs with 1 microM LPA for 6 h attenuated IL-13-but not IL-4-induced phosphorylation of STAT6. Transfection of HBEpCs with IL-13Ralpha2 small interference RNA blocked the effect of LPA on IL-13-induced phosphorylation of STAT6. Furthermore, pretreatment with LPA (1 microM, 6 h) attenuated IL-13-induced eotaxin-1 and SOCS-1 gene expression. These results demonstrate that LPA induces IL-13Ralpha2 expression and release via PLD and JNK/AP-1 signal transduction and that pretreatment with LPA down-regulates IL-13 signaling in HBEpCs. Our data suggest a novel mechanism of regulation of IL-13Ralpha2 and IL-13 signaling that may be of physiological relevance to airway inflammation and remodeling.     

Circulating levels of the interleukin (IL)-4 receptor and of IL-18 in patients with Plasmodium falciparum malaria

Circulating levels of sIL-4R, IL-18 and IFN-gamma were studied by ELISA in 36 Gabonese patients with Plasmodium falciparum malaria (29 children, 7 adults). Drug induced clearance of parasitemia, studied in 22 patients with mild disease, was accompanied by a rapid decrease of sIL-4R and IFN-gamma to normal values and an increase of circulating IL-18, suggesting the downregulation of a type 2 biased immune response and a dissociated type 1 responsiveness while resolving parasitemia. Comparing subgroups with hyperparasitemia/severe anemia and mild malaria, children with severe malaria had significant higher levels of sIL-4R and IFN-gamma, whereas IL-18 levels were not statistically different. Furthermore, among those children, higher levels of circulating IL-18 correlated with a lower degree of parasitemia.     

Evidence for a critical role for the cytoplasmic region of the interleukin 2 (IL-2) receptor gamma chain in IL-2, IL-4, and IL-7 signalling.

The high-affinity interleukin 2 receptor (IL-2R) consists of at least three distinct subunits: the IL-2R alpha chain (IL-2R alpha), beta chain (IL-2R beta), and gamma chain (IL-2R gamma). It has been shown that the cytoplasmic region of IL-2R beta, but not of IL-2R alpha, is essential for IL-2 signalling to the cell interior. In the present study, we examined the functional role of the IL-2R gamma cytoplasmic region in the IL-3-dependent mouse hematopoietic cell line BAF-B03, which expresses the endogenous IL-2R alpha and IL-2R gamma, or its subline F7, which additionally expresses human IL-2R beta cDNA. We show that overexpression of a mutant IL-2R gamma, lacking all but 7 amino acids of its cytoplasmic region, results in the selective inhibition of IL-2-induced c-fos gene activation and cellular proliferation in F7 cells. When two chimeric receptor molecules in which the cytoplasmic regions of IL-2R beta and IL-2R gamma had been swapped with each other (IL-2R beta/gamma and IL-2R gamma/beta) were coexpressed in BAF-B03, the cells responded to IL-2. These results indicate the critical importance of the IL-2-induced functional cooperation of the two cytoplasmic regions. Finally, we provide evidence that the IL-2R gamma cytoplasmic region is also critical for the IL-4 and IL-7-induced growth signal transduction in BAF-B03.