Cytokine regulation of IL-13Ralpha2 and IL-13Ralpha1 in vivo and in vitro

BACKGROUND: IL-13 signals via a high-affinity receptor that includes IL-4Ralpha and IL-13Ralpha1 and binds to the decoy receptor IL-13Ralpha2. The processes that regulate the expression of these receptor subunits, however, are poorly defined. OBJECTIVE: These studies were designed to define the regulation of IL-13R components by T(H)2 and T(H)1 cytokines in vivo and in vitro. METHODS: Northern analysis, in situ hybridization, RT-PCR analysis, and immunoprecipitation were used to define the expression of IL-13Ralpha1 and IL-13Ralpha2 in lungs from lung targeted overexpression mice and lung fragments and cells in culture. RESULTS: IL-13Ralpha2 and IL-13Ralpha1 mRNA were detected at modest levels in lungs from control mice. In contrast, transgenic IL-13 caused a marked increase in IL-13Ralpha2 and IL-13Ralpha1 mRNA; this was most prominent in airway epithelial cells and macrophages. The effects of IL-13 on IL-13Ralpha2 were associated with comparable increases in protein production and were mediated by a blood leukocyte-independent and IL-4Ralpha-dependent mechanism. IL-13 stimulation of IL-13Ralpha1 was mediated via a blood leukocyte-dependent and partially IL-4Ralpha-dependent pathway. These effects were not specific for IL-13, because transgenic IL-4, IL-10, and IFN-gamma also stimulated IL-13Ralpha2 mRNA accumulation while stimulating-not altering and inhibiting-IL-13Ralpha1 mRNA accumulation, respectively. These regulatory events were mediated, at least in part, by direct effects of these cytokines, because IL-13, IL-4, and IFN-gamma had similar effects on IL-13Ralpha2 and/or IL-13Ralpha1 in epithelial cells and macrophages in in vitro culture. CONCLUSION: IL-13Ralpha2 and IL-13Ralpha1 are highly regulated in vivo and in vitro. These regulatory events might control IL-13 responses at sites of inflammation.     

Differential responses of human monocytes and macrophages to IL-4 and IL-13.

The primary interleukin-4 (IL-4) receptor complex on monocytes (type I IL-4 receptor) includes the 140-kDa alpha chain (IL-4R alpha) and the IL-2 receptor gamma chain, gamma(c), which heterodimerize for intracellular signaling, resulting in suppression of lipopolysaccharide (LPS)-inducible inflammatory mediator production. The activity of IL-13 on human monocytes is very similar to that of IL-4 because the predominant signaling chain (IL-4R alpha) is common to both receptors. In fact, IL-4R alpha with IL-13R alpha1 is designated both as an IL-13 receptor and the type II IL-4 receptor. When the anti-inflammatory activities of IL-4 and IL-13 were investigated on synovial fluid macrophages and compared with the responses by monocytes isolated from the patients at the same time as joint drainage, the response profiles differed with some responses similar in the two cell populations, others reduced on the inflammatory cells. Similar differences were recorded in the response profiles to IL-4 and IL-13 by monocytes and monocytes cultured for 7 days in macrophage colony-stimulating factor (M-CSF) or granulocyte-macrophage CSF (GM-CSF) (monocyte-derived macrophages, MDMac). MDMac have reduced gamma(c) mRNA levels and reduced expression of the functional 64-kDa gamma(c). There was a similar loss of IL-13R alpha1 mRNA on monocyte differentiation. In turn, there was a significant reduction in the ability of IL-4 and IL-13 to activate STAT6. These findings suggest that different functional responses to IL-4 and IL-13 by human monocytes and macrophages may result from reduced expression of gamma(c) and IL-13R alpha1.     

Characterization of IL-4 and IL-13 signals dependent on the human IL-13 receptor alpha chain 1: redundancy of requirement of tyrosine residue for STAT3 activation

IL-4 and IL-13 are pleiotropic cytokines whose biological activities overlap with each other. IL-13 receptor alpha chain 1 (IL-13R alpha 1) is necessary for binding to IL-13, and the heterodimer composed of IL-13R alpha 1 and IL-4R alpha chain transduces IL-13 and IL-4 signals; however, the functional mapping of the intracellular domain of IL-13R alpha 1 is not fully understood. In this study, we constructed wild and mutated types of human IL-13R alpha 1, and analyzed IL-4 and IL-13 signals using an IL-13R alpha 1-transfected human B cell line. Expression of IL-13R alpha 1 evoked STAT3 activation by IL-4 and IL-13, and in stimulated human B cells, on which IL-13R alpha 1 was highly expressed, IL-4 and IL-13 induced STAT3 activation. Replacement of the two tyrosine residues completely abolished STAT3 activation, although replacing either tyrosine residue alone retained it. Furthermore, we found that the Box1 region and the C-terminal tail of IL-13R alpha 1 were critical for binding to Tyk2, and activation of Jak1, Tyk2, the insulin receptor substrate-1 and STAT6 respectively. These results suggest that STAT3 activation is involved with IL-4 and IL-13 signals in human B cells along with the activation of STAT6, and that there is a unique sequence in IL-13R alpha 1 to activate STAT3.     

TNF and IL-1 generation by human monocytes in response to biomaterials.

The biocompatibility of surgically implanted materials may be compromised as a consequence of inflammatory reactions associated with phagocyte activation. Two important mediators of the inflammatory response are Interleukin-1 (IL-1) and tumor necrosis factor (TNF), both of which exert a wide range of biologic effects on many cells. This study was designed to evaluate the release of these cytokines by human monocytes (HM) brought into contact with four biomaterials utilized in clinical practice: polyurethane, expanded polytetrafluorethylene (ePTFE), Dacron velour, and woven Dacron. In vitro cultures for the generation of IL-1 and TNF by HM in the presence of the above biomaterials were established by exposing cells to each biomaterial in the presence and absence of lipopolysaccharide (LPS) with harvest of supernatants after 6 or 18 h. These studies showed that in the absence of LPS, IL-1 was released only by Dacron velour and woven Dacron associated monocytes while TNF was secreted in response to all of the materials. When LPS was present, however, monocytes associated with all of the materials released IL-1; and TNF release was greatly augmented. Further, the quantity of released cytokine was directly related to the duration of the association time. This study demonstrated that HM in association with various biomaterials were activated to produce both TNF and IL-1 and that the addition of nanogram quantities of LPS, such as would be produced if infection were present, greatly increased the amount of cytokines released.     

Distinct responses to histamine of STAT1 and STAT6 phosphoregulation in human T helper lymphocytes

Inflammation Research -     

FUT-175 inhibits the production of IL-6 and IL-8 in human monocytes.

We investigated the effect of FUT-175, a serine protease inhibitor, on the production of pro-inflammatory cytokines, interleukin-6 (IL-6) and interleukin-8 (IL-8), by monocytes stimulated with lipopolysaccharide (LPS). Monocytes isolated from healthy volunteers were incubated with LPS and various concentrations of FUT-175 for 12 hours. The productions of both IL-6 and IL-8, measured by enzyme-linked immunosorbent assay, were significantly inhibited by FUT at the concentration of 1 to 100 microg/ml in a dose-dependent manner. Furthermore, the expressions of IL-6 and IL-8 mRNAs were also inhibited by FUT-175. These data suggest that FUT-175 may reduce the pathological inflammatory conditions associated with enhanced production of proinflammatory cytokines including IL-6 and IL-8.     

IL-4 and IL-13 suppress prostaglandins production in human follicular dendritic cells by repressing COX-2 and mPGES-1 expression through JAK1 and STAT6

Originally discovered as a B cell growth and differentiation factor, IL-4 displays a variety of functions in many different cell types. Germinal center T cells are abundant producers of IL-4. In a recent report, we demonstrated that IL-4 inhibits prostaglandins (PGs) production in follicular dendritic cell (FDC)-like cells, HK. To understand the inhibitory mechanisms of IL-4, its effects on the biosynthesis of enzymes in charge of PG production were assessed in this study. Although IL-4 did not affect COX-1 expression, it specifically inhibited LPS-induced COX-2 biosynthesis at mRNA and protein levels. Protein expression of mPGES-1, a downstream enzyme of COX-2, was also markedly diminished by IL-4 but not by IL-10, maximizing the inhibitory activity. Next, we attempted to identify the early signaling molecules that led to this inhibition of COX-2 expression. Although IL-4 induced tyrosine phosphorylation of JAK1 and TYK2, RNA interference experiments revealed that only JAK1 was responsible for the IL-4-stimulated STAT6 phosphorylation. Knocking down JAK1 and STAT6 ablated the inhibitory effect of IL-4 on COX-2 expression and significantly reduced production of PGE(2) and prostacyclin. Similar results were obtained with IL-13. Pharmacologic inhibitors of ERK and p38 mitogen-activated protein kinases inhibited the COX-2 upregulation. However, IL-4 did not affect LPS-induced phosphorylation of ERK and p38. These results stress the essential roles of JAK1 and STAT6 in the early signaling pathway of IL-4 and IL-13 leading to suppression of COX-2 expression and repression of PG production by HK cells. Our results suggest that T cells via IL-4 play a regulatory role in PG generation in FDC. IL-4 therapeutics may be applied to immune disorders where normal and ectopic expression of germinal center reactions needs to be regulated.     

Effect of IL-18 on the release of IL-6 and its soluble receptors: sIL-6Ralpha and sgp130 by human neutrophils

The biological activities of IL-18 include its ability to induce the production of inflammatory cytokines such as IL-1 or IL-8 by immunocompetent cells. Our previous study demonstrated that rhIL-18 induces IL-1beta and, to a lesser exted, the secretion of IL-1beta regulatory proteins involving interleukin-1 receptor antagonist (IL-IRa) and soluble interleukin-1 receptor II (sIL-1RII) by neutrophils (PMN), suggesting a significant role of IL-18 in the reactions mediated by IL-1beta. In this study, we estimated the effect of rhIL-18 on the induction of IL-6 and its soluble receptors - sIL-6Ralpha and sgp130 by these cells. Results obtained indicate that IL-18 is a promising candidate for the enhanced secretion of IL-6 by human neutrophils. In contrast, we have not found a significant effect of IL-18 on the release of both soluble receptors of IL-6. The influence of IL-18 on the IL-6 production by PMN appears to indicate a potential role of IL-18 in the early steps of the inflammatory cascade and other immune reactions mediated by IL-6.     

Interleukin-15 enhances human neutrophil phagocytosis by a Syk-dependent mechanism: importance of the IL-15Ralpha chain

Interleukin-15 (IL-15) is a cytokine that possesses interesting, potential therapeutic properties. However, based on several parameters including activation of neutrophils, it is also recognized as a proinflammatory cytokine. The mechanisms by which IL-15 activates human neutrophil functions are not fully understood. Although these cells express a functional IL-15 receptor (IL-15R) composed of IL-15Ralpha, IL-2/15Rbeta (CD122), and gamma(c) (CD132) subunits, the role of each receptor component has not been investigated in IL-15-induced human neutrophil responses. In the present study, fluorescein-activated cell sorter analysis revealed that the ability of IL-15 to enhance neutrophil phagocytosis is not a result of increased expression of IL-15Ralpha, CD122, or CD132 on the neutrophil cell surface. Pretreatment of neutrophils with specific antibodies to IL-15Ralpha, CD122, or CD132 was found to inhibit phagocytosis of opsonized-sheep red blood cells by nearly 40%, 21%, and 27%, respectively. As expected, pretreatment of neutrophils with anti-IL-2Ralpha (CD25) had no effect. Pretreatment of cells with the Syk inhibitor piceatannol was found to significantly inhibit the ability of IL-15 to enhance phagocytosis. In addition, IL-15 was found to induce tyrosine phosphorylation of Syk that was largely inhibited by pretreating cells with piceatannol. Moreover, we found that Syk kinase is physically associated with IL-15Ralpha. We conclude that IL-15R enhances neutrophil phagocytosis by a Syk-dependent mechanism and that the IL-15Ralpha chain plays a key role in mediating this response, at least by interacting with Syk kinase.     

Leukotriene B4 up-regulates IL-6 rather than IL-1 synthesis in human monocytes

Leukotriene B₄ (LTB₄) preferentially induced IL-6 mRNA accumulation and IL-6 protein release as assessed by ELISA and the B9 cell bioassay. In contrast, minimal IL-1 mRNA or protein was induced by LTB₄ either in the absence or presence of muramyl dipeptide. Supernatants of LTB₄-treated monocytes consistently showed enhanced thymocyte costimulatory activity and this was abrogated by 75–80% by anti-IL-1 antibody. Baseline production of IL-1 appeared however to be sufficient for a synergistic stimulation of thymocytes in the presence of IL-6. Our results now help clarify that LTB₄ stimulated preferentially IL-6 production and that the observed LTB₄-induced augmentation in thymocyte responses to monocyte supernatants is due to augmented IL-6 contents in the presence of baseline minimal IL-1 production.     

Purified interleukin-1 (IL-1) from human monocytes stimulates acute-phase protein synthesis by rodent hepatocytes in vitro.

A universal component of inflammation is the increased synthesis of a series of plasma proteins (acute-phase proteins) by the liver. The postulated messenger of acute-phase protein induction is released by leucocytes at the site of inflammation and has been shown to co-purify with endogenous pyrogen or lymphocyte-activating factor. Interleukin-1, molecular weight 17,000, pI 6 X 8-7 X 2, was purified to homogeneity from adherent human blood monocytes by a combination of affinity chromatography, gel filtration and isoelectric focusing. We examined the direct effect of pure IL-1 on the induction of acute-phase protein synthesis in vitro using rat and mouse hepatocytes. IL-1 caused significant increased synthesis of alpha 1-acid glycoprotein and smaller increases in the synthesis of other acute-phase proteins, and significant decreased synthesis of albumin. The pattern of induction of acute-phase proteins differs from that seen with a separate 30,000 molecular weight hepatocyte-stimulating factor from human monocytes described previously. We conclude that human IL-1 is one of the mediators responsible for the acute-phase protein response of the liver in inflammation and can directly cause stimulation of specific gene expression in normal hepatocytes.     

IL-13 Activates STAT6 and Inhibits Liver Injury Induced by Ischemia/Reperfusion

Hepatic ischemia/reperfusion injury is initiated by the activation of Kupffer cells and their subsequent release of proinflammatory mediators, including tumor necrosis factor-alpha (TNFalpha). These mediators stimulate a cascade of events including up-regulation of CXC chemokines and vascular endothelial adhesion molecules, leading to hepatic neutrophil recruitment and tissue injury. Interleukin-13 (IL-13) is a cytokine that has been shown to suppress macrophage production of proinflammatory mediators. The objective of the current study was to determine whether IL-13 could regulate the liver inflammatory injury induced by ischemia and reperfusion. C57BL/6 mice underwent 90 minutes of partial hepatic ischemia followed by reperfusion with or without intravenous administration of recombinant murine IL-13. Hepatic ischemia/reperfusion increased expression of TNFalpha and macrophage inflammatory protein-2 (MIP-2), leading to hepatic neutrophil recruitment, hepatocellular injury, and liver edema. Administration of IL-13 reduced the production of TNFalpha and MIP-2 mRNA and protein. IL-13 suppressed liver neutrophil recruitment by up to 72% and hepatocellular injury and liver edema were each reduced by >60%. Administration of IL-13 had no effect on liver NFkappaB activation, but greatly increased the activation of STAT6. The data suggest that the hepatoprotective effects of IL-13 may be a result of STAT6 activation.     

HIV-1 does not alter in vitro and in vivo IL-10 production by human monocytes and macrophages

The present study analyses the ability of HIV-1 to modulate IL-10 production in cells of monocyte-macrophage lineage cultured in the presence of macrophage colony-stimulating factor (M-CSF). Both monocytes and macrophages spontaneously produced low amount of IL-10. Lipopolysaccharide (LPS) induced a strong IL-10 response in fresh monocytes and in M-CSF-treated macrophages. In contrast, macrophages cultured in the absence of M-CSF exhibited a marked decrease in their susceptibility to LPS stimulation. M-CSF increased the IL-10 response of macrophages to LPS by enhancing both the expression of membrane-bound CD14, the protein that serves as LPS receptor, and the sensibility of CD14-expressing cells to LPS stimulation. Neither spontaneous nor LPS-induced expression of IL-10 was modulated in monocytes and macrophages by infection with eight monocytotropic strains, as demonstrated by ELISA and cytofluorimetric analysis. In contrast, all the HIV-1 strains primed macrophages for an increased IL-6 response to LPS stimulation. To determine whether IL-10 production was associated with in vivo infection, monocytes from AIDS individuals were analysed for IL-10 production. We found that neither spontaneous nor LPS-induced IL-10 production were different between healthy controls and HIV-infected patients. Taken together, these data strongly suggest that HIV-1 infection of monocytes-macrophages does not play a significant role in the regulation of IL-10 in infected patients. This study also emphasizes the role of M-CSF activation in the regulation of the cytokine response in macrophages.     

IL-13Ralpha2 reverses the effects of IL-13 and IL-4 on bronchial reactivity and acetylcholine-induced Ca+ signaling

BACKGROUND: The interleukins IL-4 and IL-13 play a key role in the pathophysiology of asthma. The interleukin receptor IL-13Ralpha2 is believed to act as a decoy receptor, but until now, the functional significance of IL-13Ralpha2 remains vague. METHODS: Bronchial reactivity was quantified in murine lung slices by digital video microscopy and acetylcholine (ACH)-induced Ca(2+) signaling was measured in human airway smooth muscle cells (ASMC) using fluorescence microscopy. RESULTS: IL-4 or IL-13 up to 50 ng/ml induced bronchial hyperreactivity. But after incubation with 100 ng/ml this effect was lost and bronchial responsiveness was again comparable to the control level. The effects of IL-4 and IL-13 on bronchial reactivity were paralleled by the effects on ASMC proliferation. Fifty nanograms per milliliter of IL-4 and IL-13 increased the Ca(2+) response of human ASMC to ACH. At 100 ng/ml, however, the effects of the cytokines on the Ca(2+) response were no longer evident. The expression of IL-13Ralpha2 increased with increasing concentrations of IL-4 or IL-13, reaching its maximum at 100 ng/ml. Blocking IL-13Ralpha2, the loss of the effect of IL-4 and IL-13 at 100 ng/ml on human ASMC proliferation and the ACH-induced Ca(2+) response were no longer present. CONCLUSIONS: IL-4 and IL-13 induce bronchial hyperreactivity by changing the Ca(2+) homeostasis of ASMC. These effects are counteracted by IL-13Ralpha2. The biological significance of IL-13Ralpha2 might be a protective function by regulating IL-13- and IL-4-mediated signal transduction and thereby limiting pathological alterations in Th2-mediated inflammatory diseases.     

Ciprofloxacin treatment in vivo increases the ex vivo capacity of lipopolysaccharide-stimulated human monocytes to produce IL-1, IL-6 and tumour necrosis factor-alpha.

Because in vitro treatment with quinolones, at pharmacological concentrations, modifies lipopolysaccharide (LPS) induced production of cytokines by monocytes, we studied the effect of orally administered ciprofloxacin (25 mg/kg) on the capacity of peripheral blood monocytes of healthy volunteers to produce tumour necrosis factor-alpha (TNF-alpha), IL-1 activity, IL-1 alpha, IL-1 beta and IL-6 ex vivo in response to endotoxin stimulation. After 7 days of ciprofloxacin, the extracellular and cellular production of TNF-alpha, the cellular production of IL-1 activity, the extracellular and cellular production of IL-1 alpha, and the cellular production of IL-6 increased significantly. Seven days after the end of the treatment, values returned to basal levels or even lower. To our knowledge, this is the first demonstration that ciprofloxacin can modulate in vivo the capacity of human monocytes to react to an inflammatory stimulus such as endotoxin.     

Individual diversity of IL-6 generation by human monocytes with lentinan administration

Effect of in vivo administration of lentinan on IL-6 generation by human peripheral blood monocytes was examined using 5 healthy volunteers. Monocytes isolated from them before and 3 days after intravenous administration of 2 mg lentinan per person were cultured for 2 days and then the levels of IL-6 in the supernatants of cultured monocytes were determined. In vivo lentinan administration elicited an increase in IL-6 generation by monocytes in 3 of 5 cases. Failure to increase IL-6 generation by monocytes in two cases is characteristics of lentinan, since these monocytes generated an apparent level of IL-6 in response to lipopolysaccharide, a potent monocyte activator. Thus, it was demonstrated that lentinan was capable of augmenting IL-6 generation by human monocytes and that there was individual difference in their responsiveness to lentinan.     

The effect of gamma interferon on IL-1 secretion of in vitro differentiated human macrophages

After being cultured overnight, human monocytes lose their ability to secrete interleukin-1 (IL-1) when stimulated by lipopolysaccharide (LPS). However, when these monocytes were cultured for up to 9 days with various concentrations of interferon-gamma (IFN-gamma), these cells were found to retain their ability to secrete appreciable amounts of IL-1 on LPS stimulation. However, the effect was observed only if the monocytes were exposed to the IFN before LPS stimulation and simultaneous addition of IFN and LPS to macrophages was ineffective. This effect of IFN-gamma was related to the concentration of IFN added to the cultures and was completely neutralized by a monoclonal antibody to IFN-gamma. In addition to inducing IL-1 secretion, IFN-gamma also appeared to increase the overall production of IL-1, since reinduction of IL-1 secretion was not associated with a decrease in intracellular IL-1 content. When these macrophages were initially cultured with IFN-gamma, washed, and further cultured with IFN free medium, these macrophages were found to progressively lose their capacity to secrete IL-1 in response to LPS. Conversely, when monocytes were initially cultured in medium free of IFN, washed, and then further cultured in new medium, but now containing IFN-gamma, these macrophages were found to regain their capacity to secrete IL-1. However, the amount of reinduced IL-1 secretion decreased as the length of the initial culture period without IFN increased, with less than optimal IL-1 secretion occurring if monocytes were allowed to mature for 6 days before IFN-gamma pretreatment. In summary, these studies suggest that IFN-gamma may be important in enhancing IL-1 production and secretion by maturing macrophages and tissue macrophages and consequently may play a role in regulating the accessory cell activity of these cells for a variety of immune responses in vivo.