Role of CCR5 in the pathogenesis of IL-13-induced inflammation and remodeling

IL-13 is a major effector at sites of Th2 inflammation and tissue remodeling. In these locations, it frequently coexists with the CCR5 chemokine receptor and its ligands MIP-1alpha/CCL3 and MIP-1beta/CCL4. We hypothesized that CCR5 induction and activation play important roles in the pathogenesis of IL-13-induced tissue responses. To test this hypothesis, we evaluated the effects of IL-13 on the expression of CCR5 in the murine lung. We also compared the effects of lung-targeted transgenic IL-13 in mice treated with anti-CCR5 or an Ab control and mice with wild-type or null CCR5 loci. These studies demonstrate that IL-13 is a potent stimulator of epithelial cell CCR5 expression. They also demonstrate that CCR5 neutralization or a deficiency of CCR5 significantly decreases IL-13-induced inflammation, alveolar remodeling, structural and inflammatory cell apoptosis, and respiratory failure and death. Lastly, these studies provide mechanistic insights by demonstrating that CCR5 is required for optimal IL-13 stimulation of select chemokines (MIP-1alpha/CCL3, MIP-1beta/CCL4, MCP-1/CCL-2), matrix metalloproteinase-9 and cell death regulators (Fas, TNF, TNFR1, TNFR2, Bid), optimal IL-13 inhibition of alpha1-antitrypsin, and IL-13-induction of and activation of caspases-3, -8, and-9. Collectively, these studies demonstrate that CCR5 plays a critical role in the pathogenesis of IL-13-induced inflammation and tissue remodeling.     

Granulocyte-macrophage colony-stimulating factor is required for bronchial eosinophilia in a murine model of allergic airway inflammation

GM-CSF plays an important role in inflammation by promoting the production, activation, and survival of granulocytes and macrophages. In this study, GM-CSF knockout (GM-CSF(-/-)) mice were used to investigate the role of GM-CSF in a model of allergic airway inflammation. In allergic GM-CSF(-/-) mice, eosinophil recruitment to the airways showed a striking pattern, with eosinophils present in perivascular areas, but almost completely absent in peribronchial areas, whereas in wild-type mice, eosinophil infiltration appeared in both areas. In the GM-CSF(-/-) mice, mucus production in the airways was also reduced, and eosinophil numbers were markedly reduced in the bronchoalveolar lavage (BAL)(3) fluid. IL-5 production was reduced in the lung tissue and BAL fluid of GM-CSF(-/-) mice, but IL-4 and IL-13 production, airway hyperresponsiveness, and serum IgE levels were not affected. The presence of eosinophils in perivascular but not peribronchial regions was suggestive of a cell migration defect in the airways of GM-CSF(-/-) mice. The CCR3 agonists CCL5 (RANTES) and CCL11 (eotaxin-1) were expressed at similar levels in GM-CSF(-/-) and wild-type mice. However, IFN-gamma mRNA and protein were increased in the lung tissue and BAL fluid in GM-CSF(-/-) mice, as were mRNA levels of the IFN-gamma-inducible chemokines CXCL9 (Mig), CXCL10 (IP-10), and CXCL11 (I-Tac). Interestingly, these IFN-gamma-inducible chemokines are natural antagonists of CCR3, suggesting that their overproduction in GM-CSF(-/-) mice contributes to the lack of airway eosinophils. These findings demonstrate distinctive abnormalities to a model of allergic asthma in the absence of GM-CSF.     

Chitin Elicits CCL2 from Airway Epithelial Cells and Induces CCR2-Dependent Innate Allergic Inflammation in the Lung

Chitin exposure in the lung induces eosinophilia and alternative activation of macrophages and is correlated with allergic airway disease. However, the mechanism underlying chitin-induced polarization of macrophages is poorly understood. In this paper, we show that chitin induces alternative activation of macrophages in vivo but does not do so directly in vitro. We further show that airway epithelial cells bind chitin in vitro and produce CCL2 in response to chitin both in vitro and in vivo. Supernatants of chitin-exposed epithelial cells promoted alternative activation of macrophages in vitro, whereas Ab neutralization of CCL2 in the supernate abolished the alternative activation of macrophages. CCL2 acted redundantly in vivo, but mice lacking the CCL2 receptor, CCR2, showed impaired alternative activation of macrophages in response to chitin, as measured by arginase I, CCL17, and CCL22 expression. Furthermore, CCR2 knockout mice exposed to chitin had diminished reactive oxygen species products in the lung, blunted eosinophil and monocyte recruitment, and impaired eosinophil functions as measured by expression of CCL5, IL-13, and CCL11. Thus, airway epithelial cells secrete CCL2 in response to chitin and CCR2 signaling mediates chitin-induced alternative activation of macrophages and allergic inflammation in vivo.     

Migration of eosinophils across endothelial cell monolayers: interactions among IL-5, endothelial-activating cytokines, and C-C chemokines

Eosinophils are the predominant cell type recruited in inflammatory reactions in response to allergen challenge. The mechanisms of selective eosinophil recruitment in allergic reactions are not fully elucidated. In this study, the ability of several C-C chemokines to induce transendothelial migration (TEM) of eosinophils in vitro was assessed. Eotaxin, eotaxin-2, monocyte chemotactic protein (MCP)-4, and RANTES induced eosinophil TEM across unstimulated human umbilical vein endothelial cells (HUVEC) in a concentration-dependent manner with the following rank order of potency: eotaxin approximately eotaxin-2 > MCP-4 approximately RANTES. The maximal response induced by eotaxin or eotaxin-2 exceeded that of RANTES or MCP-4. Preincubation of eosinophils with anti-CCR3 Ab (7B11) completely blocked eosinophil TEM induced by eotaxin, MCP-4, and RANTES. Activation of endothelial cells with IL-1beta or TNF-alpha induced concentration-dependent migration of eosinophils, which was enhanced synergistically in the presence of eotaxin and RANTES. Anti-CCR3 also inhibited eotaxin-induced eosinophil TEM across TNF-alpha-stimulated HUVEC. The ability of eosinophil-active cytokines to potentiate eosinophil TEM was assessed by investigating eotaxin or RANTES-induced eosinophil TEM across resting and IL-1beta-stimulated HUVEC in the presence or absence of IL-5. The results showed synergy between IL-5 and the chemokines but not between IL-5 and the endothelial activator IL-1beta. Our data suggest that eotaxin, eotaxin-2, MCP-4, and RANTES induce eosinophil TEM via CCR3 with varied potency and efficacy. Activation of HUVEC by IL-1beta or TNF-alpha or priming of eosinophils by IL-5 both promote CCR3-dependent migration of eosinophils from the vasculature in conjunction with CCR3-active chemokines.     

Ligation of Fc gamma RII (CD32) pivotally regulates survival of human eosinophils

The low-affinity IgG Fc receptor, FcgammaRII (CD32), mediates various effector functions of lymphoid and myeloid cells and is the major IgG Fc receptor expressed by human eosinophils. We investigated whether FcgammaRII regulates both cell survival and death of human eosinophils. When cultured in vitro without growth factors, most eosinophils undergo apoptosis within 96 h. Ligation of FcgammaRII by anti-CD32 mAb in solution inhibited eosinophil apoptosis and prolonged survival in the absence of growth factors. Cross-linking of human IgG bound to FcgammaRII by anti-human IgG Ab or of unoccupied FcgammaRII by aggregated human IgG also prolonged eosinophil survival. The enhanced survival with anti-CD32 mAb was inhibited by anti-granulocyte-macrophage-CSF (GM-CSF) mAb, suggesting that autocrine production of GM-CSF by eosinophils mediated survival. In fact, mRNA for GM-CSF was detected in eosinophils cultured with anti-CD32 mAb. In contrast to mAb or ligands in solution, anti-CD32 mAb or human IgG, when immobilized onto tissue culture plates, facilitated eosinophil cell death even in the presence of IL-5. Cell death induced by these immobilized ligands was accompanied by DNA fragmentation and was inhibited when eosinophil beta2 integrin was blocked by anti-CD18 mAb, suggesting that beta2 integrins play a key role in initiating eosinophil apoptosis. Thus, FcgammaRII may pivotally regulate both survival and death of eosinophils, depending on the manner of receptor ligation and beta2 integrin involvement. Moreover, the FcgammaRII could provide a novel mechanism to control the number of eosinophils at inflammation sites in human diseases.     

Inhibition of airway inflammation by amino-terminally modified RANTES/CC chemokine ligand 5 analogues is not mediated through CCR3

Chemokines play a key role in the recruitment of activated CD4(+) T cells and eosinophils into the lungs in animal models of airway inflammation. Inhibition of inflammation by N-terminally modified chemokines is well-documented in several models but is often reported with limited dose regimens. We have evaluated the effects of doses ranging from 10 ng to 100 micro g of two CC chemokine receptor antagonists, Met-RANTES/CC chemokine ligand 5 (CCL5) and aminooxypentane-RANTES/CCL5, in preventing inflammation in the OVA-sensitized murine model of human asthma. In the human system, aminooxypentane-RANTES/CCL5 is a full agonist of CCR5, but in the murine system neither variant is able to induce cellular recruitment. Both antagonists showed an inverse bell-shaped inhibition of cellular infiltration into the airways and mucus production in the lungs following allergen provocation. The loss of inhibition at higher doses did not appear to be due to partial agonist activity because neither variant showed activity in recruiting cells into the peritoneal cavity at these doses. Surprisingly, neither was able to bind to the major CCR expressed on eosinophils, CCR3. However, significant inhibition of eosinophil recruitment was observed. Both analogues retained high affinity binding for murine CCR1 and murine CCR5. Their ability to antagonize CCR1 and CCR5 but not CCR3 was confirmed by their ability to prevent RANTES/CCL5 and macrophage inflammatory protein-1beta/CCL4 recruitment in vitro and in vivo, while they had no effect on that induced by eotaxin/CCL11. These results suggest that CCR1 and/or CCR5 may be potential targets for asthma therapy.     

Differential roles of CC chemokine ligand 2/monocyte chemotactic protein-1 and CCR2 in the development of T1 immunity

CCR2 and its major ligand, chemokine ligand 2 (CCL2)/monocyte chemotactic protein-1, have been found to influence T1/T2 immune response polarization. Our objective was to directly compare the roles of CCR2 and CCL2 in T1/T2 immune response polarization using a model of pulmonary Cryptococcus neoformans infection. Either deletion of CCR2 or treatment of wild-type mice with CCL2 neutralizing Ab produced significant and comparable reductions in macrophage and T cell recruitment into the lungs following infection. Both CCL2 neutralization and CCR2 deficiency resulted in significantly diminished IFN-gamma production, and increased IL-4 and IL-5 production by lung leukocytes (T1 to T2 switch), but only CCR2 deficiency promoted pulmonary eotaxin production and eosinophilia. In the lung-associated lymph nodes (LALN), CCL2-neutralized mice developed Ag-specific IFN-gamma-producing cells, while CCR2 knockout mice did not. LALN from CCR2 knockout mice also had fewer MHCII(+)CD11c(+) and MHCII(+)CD11b(+) cells, and produced significantly less IL-12p70 and TNF-alpha when stimulated with heat-killed yeast than LALN from wild-type or CCL2-neutralized mice, consistent with a defect in APC trafficking in CCR2 knockout mice. Neutralization of CCL2 in CCR2 knockout mice did not alter immune response development, demonstrating that the high levels of CCL2 in these mice did not play a role in T2 polarization. Therefore, CCR2 (but not CCL2) is required for afferent T1 development in the lymph nodes. In the absence of CCL2, T1 cells polarize in the LALN, but do not traffic from the lymph nodes to the lungs, resulting in a pulmonary T2 response.     

Eotaxin/CCL11 suppresses IL-8/CXCL8 secretion from human dermal microvascular endothelial cells

The CC chemokine eotaxin/CCL11 is known to bind to the receptor CCR3 on eosinophils and Th2-type lymphocytes. In this study, we demonstrate that CCR3 is expressed on a subpopulation of primary human dermal microvascular endothelial cells and is up-regulated by TNF-alpha. We found that incubation of human dermal microvascular endothelial cells with recombinant eotaxin/CCL11 suppresses TNF-alpha-induced production of the neutrophil-specific chemokine IL-8/CXCL8. The eotaxin/CCL11-suppressive effect on endothelial cells was not seen on IL-1beta-induced IL-8/CXCL8 release. Eotaxin/CCL11 showed no effect on TNF-alpha-induced up-regulation of growth-related oncogene-alpha or IFN-gamma-inducible protein-10, two other CXC chemokines tested, and did not affect production of the CC chemokines monocyte chemoattractant protein-1/CCL2 and RANTES/CCL5, or the adhesion molecules ICAM-1 and E-selectin. These results suggest that eotaxin/CXCL11 is not effecting a general suppression of TNF-alphaR levels or signal transduction. Suppression of IL-8/CXCL8 was abrogated in the presence of anti-CCR3 mAb, pertussis toxin, and wortmannin, indicating it was mediated by the CCR3 receptor, G(i) proteins, and phosphatidylinositol 3-kinase signaling. Eotaxin/CCL11 decreased steady state levels of IL-8/CXCL8 mRNA in TNF-alpha-stimulated cells, an effect mediated in part by an acceleration of IL-8 mRNA decay. Eotaxin/CCL11 may down-regulate production of the neutrophil chemoattractant IL-8/CXCL8 by endothelial cells in vivo, acting as a negative regulator of neutrophil recruitment. This may play an important biological role in the prevention of overzealous inflammatory responses, aiding in the resolution of acute inflammation or transition from neutrophilic to mononuclear/eosinophilic inflammation.     

Cross-talk between ICAM-1 and granulocyte-macrophage colony-stimulating factor receptor signaling modulates eosinophil survival and activation

Reversal of eosinophilic inflammation has been an elusive therapeutic goal in the management of asthma pathogenesis. In this regard, GM-CSF is a primary candidate cytokine regulating eosinophil activation and survival in the lung; however, its molecular mechanism of propagation and maintenance of stimulated eosinophil activation is not well understood. In this study, we elucidate those late interactions occurring between the GM-CSF receptor and activated eosinophil signaling molecules. Using coimmunoprecipitation with GM-CSF-stimulated eosinophils, we have identified that the GM-CSF receptor beta-chain (GMRbeta) interacted with ICAM-1 and Shp2 phosphatase, as well as Slp76 and ADAP adaptor proteins. Separate experiments using affinity binding with a tyrosine-phosphorylated peptide containing an ITIM (ICAM-1 residues 480-488) showed binding to Shp2 phosphatase and GMRbeta. However, the interaction of GMRbeta with the phosphorylated ICAM-1-derived peptide was observed only with stimulated eosinophil lysates, suggesting that the interaction of GMRbeta with ICAM-1 required phosphorylated Shp2 and/or phosphorylated GMRbeta. Importantly, we found that inhibition of ICAM-1 in activated eosinophils blocked GM-CSF-induced expression of c-fos, c-myc, IL-8, and TNF-alpha. Moreover, inhibition of ICAM-1 expression with either antisense oligonucleotide or an ICAM-1-blocking Ab effectively inhibited ERK activation and eosinophil survival. We concluded that the interaction between ICAM-1 and the GM-CSF receptor was essential for GM-CSF-induced eosinophil activation and survival. Taken together, these results provide novel mechanistic insights defining the interaction between ICAM-1 and the GM-CSF receptor and highlight the importance of targeting ICAM-1 and GM-CSF/IL-5/IL-3 receptor systems as a therapeutic strategy to counter eosinophilia in asthma.     

CCL3 (MIP-1alpha) induces in vitro migration of GM-CSF-primed human neutrophils via CCR5-dependent activation of ERK 1/2

CCL3 (MIP-1alpha), a prototype of CC chemokines, is a potent chemoattractant toward human neutrophils pre-treated with GM-CSF for 15 min. GM-CSF-treated neutrophils migrate also to the selective CCR5 agonist CCL4 (MIP-1beta). CCL3- and CCL4-triggered migration of GM-CSF-primed neutrophils was inhibited by the CCR5 antagonist TAK-779. Accordingly, freshly isolated neutrophils express CCR5. Extracellular signal-regulated kinases (ERK)-1/2 and p38 mitogen-activated protein kinase (MAPK) inhibitors blocked CCL3-induced migration of GM-CSF-primed neutrophils. When the activation of ERK-1/2 and p38 MAPK by CCL3 and the classical neutrophilic chemokine CXCL8 (IL-8) were compared, both the chemokines were capable of activating p38 MAPK. On the contrary, whereas both ERK-1 and ERK-2 were activated by CXCL8, no ERK-1 band was detectable after CCL3 triggering. Finally, neutrophil pre-treatment with GM-CSF activated both ERK-1 and ERK-2. This suggests that by activating ERK-1, GM-CSF renders neutrophils rapidly responsive to CCL3 stimulation throughout CCR5 which is constitutively expressed on the cell surface.     

Eotaxin (CCL11) induces in vivo angiogenic responses by human CCR3+ endothelial cells

Chemokines are attractants and regulators of cell activation. Several CXC family chemokine members induce angiogenesis and promote tumor growth. In contrast, the only CC chemokine, reported to play a direct role in angiogenesis is monocyte-chemotactic protein-1. Here we report that another CC chemokine, eotaxin (also known as CCL11), also induced chemotaxis of human microvascular endothelial cells. CCL11-induced chemotactic responses were comparable with those induced by monocyte-chemotactic protein-1 (CCL2), but lower than those induced by stroma-derived factor-1alpha (CXCL12) and IL-8 (CXCL8). The chemotactic activity was consistent with the expression of CCR3, the receptor for CCL11, on human microvascular endothelial cells and was inhibited by mAbs to either human CCL11 or human CCR3. CCL11 also induced the formation of blood vessels in vivo as assessed by the chick chorioallantoic membrane and Matrigel plug assays. The angiogenic response induced by CCL11 was about one-half of that induced by basic fibroblast factor, and it was accompanied by an inflammatory infiltrate, which consisted predominantly of eosinophils. Because the rat aortic sprouting assay, which is not infiltrated by eosinophils, yielded a positive response to CCL11, this angiogenic response appears to be direct and is not mediated by eosinophil products. This suggests that CCL11 may contribute to angiogenesis in conditions characterized by increased CCL11 production and eosinophil infiltration such as Hodgkin's lymphoma, nasal polyposis, endometriosis, and allergic diathesis.     

Extremely rapid and intense induction of apoptosis in human eosinophils by anti-CD30 antibody treatment in vitro

Apoptosis is an important cellular mechanism for controlling cell viability and proliferation. With respect to eosinophils, cytokines prolong their survival, whereas corticosteroids reduce their survival in vitro. CD30, a member of the TNFR family, is expressed on the surface of many cell types, including Hodgkin's lymphoma cells. CD30 is capable of inducing apoptosis after Ab treatment in some cell lines. To determine whether this surface structure is involved in apoptosis of human eosinophils, we examined its expression and the effect of anti-CD30 Ab treatment on the viability of eosinophils. Purified human eosinophils expressed low, but consistently detectable, levels of CD30. Immobilized, but not soluble, forms of anti-CD30 Abs (HRS-4 and Ber-H8) or recombinant mouse CD30 ligand exhibited an extremely rapid and intense survival-reducing effect on the eosinophils in the presence of exogenous IL-5; this effect was both concentration and time dependent. Furthermore, high concentrations of IL-5 could not reverse the reduced survival rates. After treatment with anti-CD30 Ab, gel electrophoresis of DNA extracted from the eosinophils demonstrated changes consistent with apoptosis. The immobilized F(ab')(2) of the anti-CD30 Ab failed to induce eosinophil apoptosis. The addition of anti-CD18 Ab also completely abrogated the induction of eosinophil apoptosis. Further examination using specific signal transduction inhibitors suggested the involvement of p38, mitogen-activated protein kinase kinase 1/2, and specific tyrosine kinase, but not NF-kappaB, in the induction of CD30-mediated eosinophil apoptosis. These data demonstrate that CD30 can modify eosinophil survival by causing an extremely rapid and intense induction of apoptosis through a tightly regulated intracellular signaling pathway.     

TLR3-mediated synthesis and release of eotaxin-1/CCL11 from human bronchial smooth muscle cells stimulated with double-stranded RNA

Respiratory infections with RNA viruses, such as rhinovirus or respiratory syncytial virus, are a major cause of asthma exacerbation, accompanied by enhanced neutrophilic and/or eosinophilic inflammation of the airways. We studied the effects of dsRNA synthesized during RNA virus replication, and of its receptor, TLR3, on the synthesis of eosinophilic chemokines in bronchial smooth muscle cells (BSMC). Synthetic dsRNA, polyinosinic-cystidic acid (poly(I:C)), induced the synthesis of eosinophilic chemokines, eotaxin-1/CCL11 and RANTES/CCL5, from primary cultures of human BSMC, and IL-4 increased synergistically the synthesis of poly(I:C)-induced CCL11. A robust eosinophil chemotactic activity was released from BSMC stimulated with poly(I:C) and IL-4, which was mostly inhibited by preincubation with an anti-CCL11, but not with an anti-CCL5 Ab. Although the immunoreactivity of TLR3 was detectable on the cellular surface of BSMC by flow cytometric analysis, pretreatment with an anti-TLR3-neutralizing Ab failed to block the poly(I:C)-induced synthesis of CCL11. We have determined by confocal laser-scanning microscopy that the immunoreactivity of TLR3 was aggregated intracellularly in poly(I:C)-stimulated BSMC, colocalizing with fluorescein-labeled poly(I:C). The synthesis of CCL11 was prominently inhibited by the transfection of TLR3-specific small interfering RNA or by bafilomycin A1, an endosomal acidification inhibitor, further supporting the essential role played by intracellular TLR3 in the synthesis of poly(I:C)-induced CCL11 in BSMC. In conclusion, these observations suggest that, by activating intracellular TLR3 in BSMC, respiratory RNA virus infections stimulate the production of CCL11 and enhance eosinophilic inflammation of the airways in the Th2-dominant microenvironment.     

Differential regulation of human eosinophil IL-3, IL-5, and GM-CSF receptor alpha-chain expression by cytokines: IL-3, IL-5, and GM-CSF down-regulate IL-5 receptor alpha expression with loss of IL-5 responsiveness,but up-regulate IL-3 receptor alpha expression

Our recent data suggested that tissue eosinophils may be relatively insensitive to anti-IL-5 treatment. We examined cross-regulation and functional consequences of modulation of eosinophil cytokine receptor expression by IL-3, IL-5 GM-CSF, and eotaxin. Incubation of eosinophils with IL-3, IL-5, or GM-CSF led to reduced expression of IL-5R alpha, which was sustained for up to 5 days. Eosinophils incubated with IL-5 or IL-3 showed diminished respiratory burst and mitogen-activated protein kinase kinase phosphorylation in response to further IL-5 stimulation. In contrast to these findings, eosinophil expression of IL-3R alpha was increased by IL-3, IL-5, and GM-CSF, whereas GM-CSF receptor alpha was down-regulated by GM-CSF, but was not affected by IL-3 or IL-5. CCR3 expression was down-regulated by IL-3 and was transiently reduced by IL-5 and GM-CSF, but rapidly returned toward baseline. Eotaxin had no effect on receptor expression for IL-3, IL-5, or GM-CSF. Up-regulation of IL-3R alpha by cytokines was prevented by a phosphoinositol 3-kinase inhibitor, whereas this and other signaling inhibitors had no effect on IL-5R alpha down-regulation. These data suggest dynamic and differential regulation of eosinophil receptors for IL-3, IL-5, and GM-CSF by the cytokine ligands. Since these cytokines are thought to be involved in eosinophil development and mobilization from the bone marrow and are present at sites of allergic inflammation, tissue eosinophils may have reduced IL-5R expression and responsiveness, and this may explain the disappointing effect of anti-IL-5 therapy in reducing airway eosinophilia in asthma.     

A phosphosite screen identifies autocrine TGF-beta-driven activation of protein kinase R as a survival-limiting factor for eosinophils

The differential usage of signaling pathways by chemokines and cytokines in eosinophils is largely unresolved. In this study, we investigate signaling similarities and differences between CCL11 (eotaxin) and IL-5 in a phosphosite screen of human eosinophils. We confirm many previously known pathways of cytokine and chemokine signaling and elucidate novel phosphoregulation in eosinophils. The signaling molecules that were stimulated by both agents were members of the ERK1/2 and p38 MAPK pathways and their downstream effectors such as RSK and MSK1/2. Both agents inhibited S6 kinase, protein kinase Cepsilon, and glycogen synthase kinase 3 alpha and beta. The molecules that were differentially regulated include STATs and protein kinase R (PKR). One of the chief findings in this investigation was that PKR and eukaryotic initiation factor 2alpha are phosphorylated under basal conditions in eosinophils and neutrophils. This basal phosphorylation was linked to autocrine secretion of TGF-beta in eosinophils. TGF-beta directly activates PKR in eosinophils. Basal phosphorylation of PKR was inhibited by incubation of eosinophils with a neutralizing anti-TGF-beta Ab suggesting its physiological importance. We show that inhibition of PKR activity prolongs eosinophil survival. The eosinophil survival factor IL-5 strongly suppresses phosphorylation of PKR. The biological relevance of IL-5 inhibition of phospho-PKR was established by the observation that ex vivo bone marrow-derived eosinophils from OVA-immunized mice had no PKR phosphorylation in contrast to the high level of phosphorylation in sham-immunized mice. Together, our findings suggest that survival of eosinophils is in part controlled by basal activation of PKR through autocrine TGF-beta and that this could be modulated by a Th2 microenvironment in vivo.     

IL-5 and granulocyte-macrophage colony-stimulating factor activate STAT3 and STAT5 and promote Pim-1 and cyclin D3 protein expression in human eosinophils

Allergic inflammation is characterized by elevated eosinophil numbers and by the increased production of the cytokines IL-5 and GM-CSF, which control several eosinophil functions, including the suppression of apoptosis. The JAK/STAT pathway is important for several functions in hemopoietic cells, including the suppression of apoptosis. We report in this study that STAT3, STAT5a, and STAT5b are expressed in human eosinophils and that their signaling pathways are active following IL-5 or GM-CSF treatment. However, in airway eosinophils, the phosphorylation of STAT5 by IL-5 is reduced, an event that may be related to the reduced expression of the IL-5Ralpha on airway eosinophils. Furthermore, IL-5 and GM-CSF induced the protein expression of cyclin D3 and the kinase Pim-1, both of which are regulated by STAT-dependent processes in some cell systems. Pim-1 is more abundantly expressed in airway eosinophils than in blood eosinophils. Because Pim-1 reportedly has a role in the modulation of apoptosis, these results suggest that Pim-1 action is linked to the suppression of eosinophil apoptosis by these cytokines. Although cyclin D3 is known to be critical for cell cycle progression, eosinophils are terminally differentiated cells that do not proceed through the cell cycle. Thus, this apparent cytokine regulation of cyclin D3 suggests that there is an alternative role(s) for cyclin D3 in eosinophil biology.     

Chemokine receptor expression and function in CD4+ T lymphocytes with regulatory activity

We have investigated the chemokine receptor expression and migratory behavior of a new subset of nickel-specific skin-homing regulatory CD4(+) T cells (Th(IL-10)) releasing high levels of IL-10, low IFN-gamma, and undetectable IL-4. These cells inhibit in a IL-10-dependent manner the capacity of dendritic cells to activate nickel-specific Tc1 and Th1 lymphocytes. RNase protection assay and FACS analysis revealed the expression of a vast repertoire of chemokine receptors on resting Th(IL-10), including the Th1-associated CXCR3 and CCR5, and the Th2-associated CCR3, CCR4, and CCR8, the latter at higher levels compared with Th2 cells. The most active chemokines for resting Th(IL-10), in terms of calcium mobilization and in vitro migration, were in order of potency: CCL2 (monocyte chemoattractant protein-1, CCR2 ligand), CCL4 (macrophage-inflammatory protein-1beta, CCR5 ligand), CCL3 (macrophage-inflammatory protein-1alpha, CCR1/5 ligand), CCL17 (thymus and activation-regulated chemokine, CCR4 ligand), CCL1 (I-309, CCR8 ligand), CXCL12 (stromal-derived factor-1, CXCR4), and CCL11 (eotaxin, CCR3 ligand). Consistent with receptor expression down-regulation, activated Th(IL-10) exhibited a reduced or absent response to most chemokines, but retained a significant migratory capacity to I-309, monocyte chemoattractant protein-1, and thymus and activation-regulated chemokine. I-309, which was ineffective on Th1 lymphocytes, attracted more efficiently Th(IL-10) than Th2 cells. I-309 and CCR8 mRNAs were not detected in unaffected skin and were up-regulated at the skin site of nickel-allergic reaction, with an earlier expression kinetics compared with IL-10 and IL-4. Results indicate that skin-homing regulatory Th(IL-10) lymphocytes coexpress functional Th1- and Th2-associated chemokine receptors, and that CCR8/I-309-driven recruitment of both resting and activated Th(IL-10) cells may be critically involved in the regulation of Th1-mediated skin allergic disorders.     

The C10/CCL6 chemokine and CCR1 play critical roles in the pathogenesis of IL-13-induced inflammation and remodeling

IL-13 is a potent stimulator of inflammation and tissue remodeling that plays a key role in the pathogenesis of a wide variety of human disorders. To further understand these responses, studies were undertaken to define the role(s) of the chemokine C10/CCL6 in the pathogenesis of IL-13-induced alterations in the murine lung. IL-13 was a very potent stimulator of C10/CCL6 mRNA and protein, and IL-13-induced inflammation, alveolar remodeling, and compliance alterations were markedly ameliorated after C10/CCL6 neutralization. Treatment with anti-C10/CCL6 decreased the levels of mRNA encoding matrix metalloproteinase-2 (MMP-2), MMP-9, and tissue inhibitor of metalloproteinase-4 (TIMP-4) in lungs from wild-type mice. C10/CCL6 neutralization also decreased the ability of IL-13 to stimulate the production of monocyte chemoattractant protein-1, macrophage inflammatory protein-1alpha, MMP-2, MMP-9, and cathepsins-K, -L, and -S and the ability of IL-13 to inhibit alpha1-antitrypsin. In accord with these findings, a targeted null mutation of CCR1, a putative C10/CCL6 receptor, also decreased IL-13-induced inflammation and alveolar remodeling and caused alterations in chemokines, proteases, and antiproteases comparable to those seen after C10/CCL6 neutralization. These C10/CCL6 and CCR1 manipulations did not alter the production of transgenic IL-13. These studies demonstrate that IL-13 is a potent stimulator of C10/CCL6 and highlight the importance of C10/CCL6 and signaling via CCR1 in the pathogenesis of the IL-13-induced pulmonary phenotype. They also describe a C10/CCL6 target gene cascade in which C10/CCL6 induction is required for optimal IL-13 stimulation of selected chemokines (monocyte chemoattractant protein-1 and MIP-1alpha) and proteases (MMP-2, MMP-9, and cathepsins-K, -L, and -S) and the inhibition of alpha1-antitrypsin.     

CCR3 expression induced by IL-2 and IL-4 functioning as a death receptor for B cells

We report that CCR3 is not expressed on freshly isolated peripheral and germinal B cells, but is up-regulated after stimulation with IL-2 and IL-4 (approximately 98% CCR3(+)). Ligation of CCR3 by eotaxin/chemokine ligand (CCL) 11 induces apoptosis in IL-2- and IL-4-stimulated primary CD19(+) (approximately 40% apoptotic cells) B cell cultures as well as B cell lines, but has no effect on chemotaxis or cell adhesion. Freshly isolated B cells express low levels of CD95 and CD95 ligand (CD95L) (19 and 21%, respectively). Expression is up-regulated on culture in the presence of a combination of IL-2, IL-4, and eotaxin/CCL11 (88% CD95 and 84% CD95L). We therefore propose that ligation of such newly induced CCR3 on peripheral and germinal B cells by eotaxin/CCL11 leads to the enhanced levels of CD95 and CD95L expression. Ligation of CD95 by its CD95L expressed on neigboring B cells triggers relevant death signaling pathways, which include an increase in levels of Bcl-2 expression, its functional activity, and the release of cytochrome c from the mitochondria into the cytosol. These events initiate a cascade of enzymatic processes of the caspase family, culminating in programmed cell death. Interaction between CCR3 and eotaxin/CCL11 may, besides promoting allergic reactions, drive activated B cells to apoptosis, thereby reducing levels of Ig production, including IgE, and consequently limit the development of the humoral immune response. The apoptotic action of eotaxin/CCL11 suggests a therapeutic modality in the treatment of B cell lymphoma.     

New approaches in the treatment of asthma

Asthma is a common and complex inflammatory disease of the airways that remains incurable. Current forms of therapy are long term and may exhibit associated side-effect problems. Major participants in the development of an asthma phenotype include the triggering stimuli such as the allergens themselves, cells such as T cells, epithelial cells and mast cells that produce a variety of cytokines including IL-5, GM-CSF, IL-3, IL-4 and IL-13 and chemokines such as eotaxin. Significantly, the eosinophil, a specialized blood cell type, is invariably associated with this disease. The eosinophil has long been incriminated in the pathology of asthma due to its ability to release preformed and unique toxic substances as well as newly formed pro-inflammatory mediators. The regulation of eosinophil production and function is carried out by soluble peptides or factors. Of these IL-5, GM-CSF and IL-3 are of paramount importance as they control eosinophil functional activity and are the only known eosinophilopoietic factors. In addition they regulate the eosinophil life span by inhibiting apoptosis. While one therapeutic approach in asthma is directed at inhibiting single eosinophil products such as leukotrienes or single eosinophil regulators such as IL-5, we believe that the simultaneous inhibition of more than one component is preferable. This may be particularly important with eosinophil regulators in that not only IL-5, but also GM-CSF has been repeatedly implicated in clinical studies of asthma. The fact that GM-CSF is produced by many cells in the body and in copious amounts by lung epithelial cells highlights this need further. Our approach takes advantage of the fact that the IL-5 and GM-CSF receptors (as well as IL-3 receptors) utilize a shared subunit to bind, with high affinity, to these cytokines and the same common subunit mediates signal transduction culminating in all the biological activities mentioned. By generating the monoclonal antibody BION-1 to the cytokine binding region of the common subunit (betac) we have shown that the approach of inhibiting IL-5, GM-CSF and IL-3 binding and the resulting stimulation of eosinophil production and function with a single agent is feasible. Furthermore we have used BION-1 as a tool to crystallize and define the structure of the cytokine binding domain of betac. This knowledge and this approach may lead to the generation of novel therapeutics for the treatment of asthma.