Decreased expression of membrane IL-5 receptor alpha on human eosinophils: II. IL-5 down-modulates its receptor via a proteinase-mediated process

In the accompanying study, we demonstrated that following Ag challenge, membrane (m)IL-5Ralpha expression is attenuated on bronchoalveolar lavage eosinophils, soluble (s)IL-5Ralpha is detectable in BAL fluid in the absence of increased steady state levels of sIL-5Ralpha mRNA, and BAL eosinophils become refractory to IL-5 for ex vivo degranulation. We hypothesized that IL-5 regulates its receptor through proteolytic release of mIL-5Ralpha, which in turn contributes to the presence of sIL-5Ralpha. Purified human peripheral blood eosinophils were incubated with IL-5 under various conditions and in the presence of different pharmacological agents. A dose-dependent decrease in mIL-5Ralpha was accompanied by an increase in sIL-5Ralpha in the supernatant. IL-5 had no ligand-specific effect on mIL-5Ralpha or sIL-5Ralpha mRNA levels. The matrix metalloproteinase-specific inhibitors BB-94 and GM6001 and tissue inhibitor of metalloproteinase-3 partially inhibited IL-5-mediated loss of mIL-5Ralpha, suggesting that sIL-5Ralpha may be produced by proteolytic cleavage of mIL-5Ralpha. IL-5 transiently reduced surface expression of beta-chain, but had no effect on the expression of GM-CSFRalpha. Pretreatment of eosinophils with a dose of IL-5 that down-modulated mIL-5Ralpha rendered these cells unable to degranulate in response to further IL-5 stimulation, but they were fully responsive to GM-CSF. These findings suggest that IL-5-activated eosinophils may lose mIL-5Ralpha and release sIL-5Ralpha in vivo, which may limit IL-5-dependent inflammatory events in diseases such as asthma.     

Potent neutralizing anti-CD1d antibody reduces lung cytokine release in primate asthma model

CD1d is a receptor on antigen-presenting cells involved in triggering cell populations, particularly natural killer T (NKT) cells, to release high levels of cytokines. NKT cells are implicated in asthma pathology and blockade of the CD1d/NKT cell pathway may have therapeutic potential. We developed a potent anti-human CD1d antibody (NIB.2) that possesses high affinity for human and cynomolgus macaque CD1d (K_D ∼100 pM) and strong neutralizing activity in human primary cell-based assays (IC₅₀ typically <100 pM). By epitope mapping experiments, we showed that NIB.2 binds to CD1d in close proximity to the interface of CD1d and the Type 1 NKT cell receptor β-chain. Together with data showing that NIB.2 inhibited stimulation via CD1d loaded with different glycolipids, this supports a mechanism whereby NIB.2 inhibits NKT cell activation by inhibiting Type 1 NKT cell receptor β-chain interactions with CD1d, independent of the lipid antigen in the CD1d antigen-binding cleft. The strong in vitro potency of NIB.2 was reflected in vivo in an Ascaris suum cynomolgus macaque asthma model. Compared with vehicle control, NIB.2 treatment significantly reduced bronchoalveolar lavage (BAL) levels of Ascaris-induced cytokines IL-5, IL-8 and IL-1 receptor antagonist, and significantly reduced baseline levels of GM-CSF, IL-6, IL-15, IL-12/23p40, MIP-1α, MIP-1β, and VEGF. At a cellular population level NIB.2 also reduced numbers of BAL lymphocytes and macrophages, and blood eosinophils and basophils. We demonstrate that anti-CD1d antibody blockade of the CD1d/NKT pathway modulates inflammatory parameters in vivo in a primate inflammation model, with therapeutic potential for diseases where the local cytokine milieu is critical.     

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.     

Human interleukin-3 inhibits the binding of granulocyte-macrophage colony-stimulating factor and interleukin-5 to basophils and strongly enhances their functional activity.

The human T cell-derived cytokines interleukin (IL)-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-5 were examined for their ability to bind specifically to human basophils and to regulate their function. Scatchard analysis of equilibrium binding studies showed that IL-3 and GM-CSF, bound to basophils with apparent dissociation constants (KD) = 8 x 10(-11) M and 3.9 x 10(-11) M, respectively. Specificity studies under conditions that prevent receptor internalization showed that the binding of IL-3, GM-CSF, and IL-5 was not inhibited by tumor necrosis factor (TNF)-alpha, IL-1 beta, interferon (IFN)-gamma, or G-CSF. However, receptors for IL-3, GM-CSF, and IL-5 interacted with each other on the basophil membrane, showing a unique spectrum of cross-reactivity, with IL-3 competing for GM-CSF and IL-5 binding, whereas GM-CSF and IL-5 showed little or no competition for IL-3 binding. In order to relate the binding properties of these cytokines to function, they were tested for their ability to influence basophil histamine release in an IgE/anti-IgE-dependent system. We found a hierarchy in the stimulation of basophil with the order of potency being IL-3 greater than GM-CSF greater than IL-5. In addition, IL-3 stimulated larger amounts of histamine release than GM-CSF or IL-5. The observation that IL-3 interacts with receptors for GM-CSF and IL-5 may have a bearing on its stronger functional effects and suggests a major role for IL-3 in the pathogenesis of hypersensitivity syndromes.     

Toll-like receptor (TLR)2 and TLR4 in human peripheral blood granulocytes: a critical role for monocytes in leukocyte lipopolysaccharide responses

Leukocyte responsiveness to LPS is dependent upon CD14 and receptors of the Toll-like receptor (TLR) family. Neutrophils respond to LPS, but conflicting data exist regarding LPS responses of eosinophils and basophils, and expression of TLRs at the protein level in these granulocyte lineages has not been fully described. We examined the expression of TLR2, TLR4, and CD14 and found that monocytes expressed relatively high levels of cell surface TLR2, TLR4, and CD14, while neutrophils also expressed all three molecules, but at low levels. In contrast, basophils expressed TLR2 and TLR4 but not CD14, while eosinophils expressed none of these proteins. Tested in a range of functional assays including L-selectin shedding, CD11b up-regulation, IL-8 mRNA generation, and cell survival, neutrophils responded to LPS, but eosinophils and basophils did not. In contrast to previous data, we found, using monocyte depletion by negative magnetic selection, that neutrophil responses to LPS were heavily dependent upon the presence of a very low level of monocytes, and neutrophil survival induced by LPS at 22 h was monocyte dependent. We conclude that LPS has little role in the regulation of peripheral blood eosinophil and basophil function, and that, even in neutrophils, monocytes orchestrate many previously observed leukocyte LPS response patterns.     

Decreased expression of membrane IL-5 receptor alpha on human eosinophils: I. Loss of membrane IL-5 receptor alpha on airway eosinophils and increased soluble IL-5 receptor alpha in the airway after allergen challenge

IL-5 is a key cytokine for eosinophil maturation, recruitment, activation, and possibly the development of inflammation in asthma. High concentrations of IL-5 are present in the airway after Ag challenge, but the responsiveness of airway eosinophils to IL-5 is not well characterized. The objectives of this study were to establish, following airway Ag challenge: 1) the expression of membrane (m)IL-5Ralpha on bronchoalveolar lavage (BAL) eosinophils; 2) the responsiveness of these cells to exogenous IL-5; and 3) the presence of soluble (s)IL-5Ralpha in BAL fluid. To accomplish these goals, blood and BAL eosinophils were obtained from atopic subjects 48 h after segmental bronchoprovocation with Ag. There was a striking reduction in mIL-5Ralpha on airway eosinophils compared with circulating cells. Furthermore, sIL-5Ralpha concentrations were elevated in BAL fluid, but steady state levels of sIL-5Ralpha mRNA were not increased in BAL compared with blood eosinophils. Finally, BAL eosinophils were refractory to IL-5 for ex vivo degranulation, suggesting that the reduction in mIL-5Ralpha on BAL eosinophils may regulate IL-5-mediated eosinophil functions. Together, the loss of mIL-5Ralpha, the presence of sIL-5Ralpha, and the blunted functional response (degranulation) of eosinophils to IL-5 suggest that when eosinophils are recruited to the airway, regulation of their functions becomes IL-5 independent. These observations provide a potential explanation for the inability of anti-IL-5 therapy to suppress airway hyperresponsiveness to inhaled Ag, despite a reduction in eosinophil recruitment.     

Potent neutralizing anti-CD1d antibody reduces lung cytokine release in primate asthma model

CD1d is a receptor on antigen-presenting cells involved in triggering cell populations, particularly natural killer T (NKT) cells, to release high levels of cytokines. NKT cells are implicated in asthma pathology and blockade of the CD1d/NKT cell pathway may have therapeutic potential. We developed a potent anti-human CD1d antibody (NIB.2) that possesses high affinity for human and cynomolgus macaque CD1d (K_D ～100 pM) and strong neutralizing activity in human primary cell-based assays (IC₅₀ typically <100 pM). By epitope mapping experiments, we showed that NIB.2 binds to CD1d in close proximity to the interface of CD1d and the Type 1 NKT cell receptor β-chain. Together with data showing that NIB.2 inhibited stimulation via CD1d loaded with different glycolipids, this supports a mechanism whereby NIB.2 inhibits NKT cell activation by inhibiting Type 1 NKT cell receptor β-chain interactions with CD1d, independent of the lipid antigen in the CD1d antigen-binding cleft. The strong in vitro potency of NIB.2 was reflected in vivo in an Ascaris suum cynomolgus macaque asthma model. Compared with vehicle control, NIB.2 treatment significantly reduced bronchoalveolar lavage (BAL) levels of Ascaris-induced cytokines IL-5, IL-8 and IL-1 receptor antagonist, and significantly reduced baseline levels of GM-CSF, IL-6, IL-15, IL-12/23p40, MIP-1α, MIP-1β, and VEGF. At a cellular population level NIB.2 also reduced numbers of BAL lymphocytes and macrophages, and blood eosinophils and basophils. We demonstrate that anti-CD1d antibody blockade of the CD1d/NKT pathway modulates inflammatory parameters in vivo in a primate inflammation model, with therapeutic potential for diseases where the local cytokine milieu is critical.     

The granulocyte macrophage colony stimulating factor (GM-CSF) regulates amyloid beta (Abeta) production

One of the hallmarks of Alzheimer’s disease (AD) is the accumulation of amyloid beta (Aβ) plaques in the brain parenchyma. An inflammatory component to AD has been suggested in association with increased cytokine release. We have previously shown that CD40L stimulation of microglia induces increases in pro-inflammatory cytokines such as interleukin-1β (IL-1β), IL-6, IL-8 and GM-CSF. We have also shown that CD40L stimulation increases Aβ levels in HEK-293 cells over-expressing both the amyloid precursor protein (APP) and CD40 (HEK/APPsw/CD40). In this study, we show that GM-CSF neutralizing antibodies mitigate the CD40L-induced production of Aβ in HEK/APPsw/CD40 cells. In addition, we demonstrate that treatment of these cells with recombinant GM-CSF significantly increases Aβ levels. Furthermore, we show that shRNA silencing of the GM-CSF receptor gene significantly reduces Aβ levels to below base line in non-stimulated HEK/APPsw/CD40 cells. Analysis of cell surface proteins revealed that silencing of the GM-CSF receptor also decreases APP endocytosis (therefore reducing the availability of APP to be cleaved in the endosomes). Taken together, our results suggest that GM-CSF operates downstream of CD40/CD40L interaction and that GM-CSF modulates Aβ production by influencing APP trafficking. GM-CSF signaling may be a suitable therapeutic target against Aβ production in AD.     

Induction of CD23, CD25 and CD4 expression on an eosinophilic cell line (EoL-3) by interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-5 (IL-5).

The effects of IL-3, GM-CSF and IL-5 on the expression of CD23 (Fc epsilon RII), CD25 (IL-2R/p55) and CD4 on an eosinophilic cell line (EoL-3) were investigated by flow cytometry. A separate incubation with IL-3, GM-CSF or IL-5 alone, did not induce the expression of CD23, CD25, or CD4. However, a sequential incubation with IL-3 for 6 days, then with IL-3 and GM-CSF for the following 6 days, induced a significant expression of CD23 and CD25. After a further incubation for 6 days with IL-3, GM-CSF and IL-5, CD4 was then expressed, while CD23 and CD25 expression still increased. The kinetics of expression of CR3/CD11b were parallel to that of CD23, but the expression of the transferrin receptor (CD71) remained negative. Northern blot analysis revealed the presence of mRNA encoding CD23, CD25 and CD4 in EoL-3 stimulated by IL-3, GM-CSF and IL-5. Culture with GM-CSF induced the binding of radiolabeled IL-5 to EoL-3 cells, with an increased affinity after incubation with IL-3, GM-CSF and IL-5. These data indicate that IL-3, GM-CSF and IL-5, might be involved in the expression of functional markers on eosinophil membrane.     

Antigen controls IL-7R alpha expression levels on CD8 T cells during full activation or tolerance induction

The high-affinity chain of the IL-7 receptor, IL-7Ralpha (CD127), is expressed by effector CD8 T cells that have the capacity to become memory cells. IL-7Ralpha expression is uniformly high on naive CD8 T cells, and the majority of these cells down-regulate expression upon antigenic challenge. At the peak of expansion, the fraction of effectors expressing high IL-7Ralpha varies depending on the response examined. The signals that a CD8 T cell receives during a response to Ag that lead to altered expression of IL-7Ralpha have not been fully defined. In vitro experiments demonstrated that Ag alone is sufficient to down-regulate IL-7Ralpha on all cells and most of the cells rapidly re-express the receptor upon removal from Ag. Expression was not altered by the B7.1 costimulatory ligand or when IL-12 was present to provide the signal needed for development of effector functions, indicating that TCR engagement is sufficient to regulate IL-7Ralpha expression. Consistent with this, in vivo priming with peptide Ag resulted in IL-7Ralpha expression that inversely correlated with Ag levels, and expression levels were not changed when IL-12 or adjuvant were administered with Ag. A large fraction of the cells present at the peak of expansion had re-expressed IL-7Ralpha, but most of these cells failed to survive; those that did survive expressed high IL-7Ralpha levels. Thus, Ag-dependent signals regulate IL-7Ralpha levels on responding CD8 T cells, and this occurs whether the responding cells become fully activated or are rendered tolerant by administration of peptide Ag alone.     

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.     

Novel technique for the direct flow cytofluorometric analysis of human basophils in unseparated blood and bone marrow, and the characterization of phenotype and peroxidase of human basophils

BACKGROUND: No technique has been reported to analyze directly the antigen expression on basophil leukocytes when using a flow cytometer; therefore, the exact phenotype of human basophils and the character of the peroxidase in basophils are not well understood. METHODS: Human blood basophils were purified by using an antibody against high-affinity Fc epsilon receptor (hFcepsilonR) and a MACS magnetic cell sorting system and then cytochemically stained. The phenotype and peroxidase of the human basophils were flow cytofluorometrically analyzed directly in unseparated blood and bone marrow samples as hFcepsilonR+/MBP+ (major basic protein)/Hist+ (histamine) light-density cells distributed in the high sidescatter area of lymphocytes on light scattergrams. RESULTS: The peroxidase granules of human basophils were stained by an anti-eosinophil peroxidase (EPO) antibody. The human blood basophils had common granulocyte markers plus CD25, i.e., they were CD11a/ CD11b/CD11c/CD25/CD38/CD13/CD33/hFcepsi lonR/MBP/Hist/ EPO positive, CD71 dim positive, CD14/CD15 partially positive, and CD2/CD3/CD7/CD122/CD16/CD56/CD57/ CD10/CD19/CD20/CD22/HLA-DR/MPO (myeloperoxidase)/CD23 negative. Further examination was done to analyze the expression of colony-stimulating factor receptors on three lineages of granulocytes, i.e., basophils, eosinophils, and neutrophils. The neutrophils were CD114 (G-CSFR)/CD116 (GM-CSFR)/CD124 [interleukin (IL)-4R]/CD126 (IL-6R) positive and CD123 (IL-3R)/CD125 (IL-5R) negative. In contrast, the eosinophils and basophils were CD116/CD123/CD125/CD126 positive and CD114/CD124 negative. CONCLUSIONS: This novel technique for directly characterizing human basophil leukocytes with flow cytometry may be a convenient way to screen the expression of surface antigens and the cytoplasmic expression of CD antigens and other proteins in human blood basophils and to analyze alterations of the character of basophils by cytokines and other biological substances in vivo and in vitro.     

Down-regulation of IL-7Ralpha expression in human T cells via DNA methylation

IL-7 is critical for the development and survival of T cells. Recently, we found two subsets of human CD8+ T cells expressing IL-7Ralpha(high) and IL-7Ralpha(low) with different cell survival responses to IL-7. Although these CD8+ T cell subsets have differential IL-7Ralpha gene expression, the mechanism for this is unknown. DNA methylation is an important gene regulatory mechanism and is associated with the inactivation of gene expression. Thus, we investigated a role for DNA methylation in differentially regulating IL-7Ralpha gene expression in human CD8+ T cells and Jurkat T cells. IL-7Ralpha(high)CD8+ T cells had decreased methylation in the IL-7Ralpha gene promoter compared with IL-7Ralpha(low)CD8+ T cells and Jurkat T cells with low levels of IL-7Ralpha. Treating Jurkat T cells with 5-aza-2'-deoxycytidine, which reduced DNA methylation, increased IL-7Ralpha expression. Plus, the unmethylated IL-7Ralpha gene promoter construct had higher levels of promoter activity than the methylated one as measured by a luciferase reporter assay. These findings suggest that DNA methylation is involved in regulating IL-7Ralpha expression in T cells via affecting IL-7Ralpha gene promoter activity, and that the methylation of this gene promoter could be a potential target for modifying IL-7-mediated T cell development and survival.     

CD25 T-lymphocytes induce CD11b on eosinophils in allergic nasal mucosa

In the allergic mucosa, there is a significant increase in numbers of CD25(+) cells and activated eosinophils. To determine whether a link exists between the activated T-lymphocytes and tissue eosinophils in nasal allergy, we studied CD25(+) cells in the nasal mucosa and compared the levels of soluble IL-2 receptor (sIL-2R) both in the serum and the nasal secretions, and further investigated expression of CD11b on eosinophils in the nasal lavage fluids and peripheral blood of patients with nasal allergy. We also examined the effects of the culture supernatant of Con A- and IL-2-activated T-lymphocytes on CD11b expression on eosinophils in the present study. The concentration of sIL-2R in the nasal secretions from patients with Japanese cedar pollinosis (JCP) was significantly higher than that from normal subjects (p < 0.01). The sIL-2R level was significantly higher in the nasal secretions than in the sera in patients (p < 0.01), and CD11b expression on eosinophils from nasal hvage fluid was significandy higher than that of eosinophils from peripheral blood of the same individuals (p < 0.01). The activated T-lymphocytes promoted eosinophil activation with upregulation of CD11b in vitro, and eosinophils in the nasal secretions from patients significantly expressed more CD11b in vivo. These results indicate that activation of T-lymphocytes is linked to eosinophil activation in nasal allergy.     

Cutting edge: human eosinophils regulate T cell subset selection through indoleamine 2,3-dioxygenase

Allergy involves eosinophilia and Th2 polarization. Indoleamine 2,3-dioxygenase (IDO)-catalyzed conversion of tryptophan to kynurenines (KYN) regulates T cell function. We show that human eosinophils constitutively express IDO. Eosinophils treated with IFN-gamma showed an 8-fold increase in IDO mRNA within 4 h; IL-3, IL-5, and GM-CSF had no effect on baseline IDO expression. IL-3 pretreatment of eosinophils reduced IFN-gamma-induced IDO mRNA expression below baseline. Conversely, GM-CSF, but not IL-5, resulted in a 2-fold increase in IFN-gamma-induced IDO. Treatment with IL-3, IL-5, GM-CSF, or IFN-gamma alone expressed IDO enzymatic activity (the presence of KYN in supernatants 48 h postculture). CD28 cross-linking resulted in measurable KYN in culture supernatants, inhibitable by a neutralizing anti-IFN-gamma. Coculture of eosinophils with an IFN-gamma-producing T cell line, but not IL-4-producing T cell clone, led to apoptosis and inhibition of CD3 or CD3/CD28-induced proliferation. Eosinophils infiltrating asthmatic lung and associated lymphoid tissue exhibited intracellular IDO immunoreactivity. Eosinophils may, therefore, maintain Th2 bias through IDO.     

Eotaxin potentiates antigen-dependent basophil IL-4 production.

Basophils are a major source of IL-4, which is a critical factor in the generation of allergic inflammation. Eotaxin induces chemotaxis mediated through the CC chemokine receptor 3 (CCR3) present on basophils as well as eosinophils and Th2 cells, thereby promoting cell recruitment. To determine whether eotaxin has other proinflammatory activity, we examined the effect of eotaxin on basophil IL-4 expression by flow cytometry. Eotaxin alone had no effect on basophil IL-4 production, but further increased allergen-stimulated IL-4 expression. Eotaxin also enhanced IL-4 release from purified basophils 2- to 4-fold, as determined by ELISA (p < 0.01). Addition of eotaxin to cultures resulted in a 40-fold left shift in the dose response to Ag. This effect was obtained with physiologic concentrations of eotaxin (10 ng/ml), was abrogated by an Ab to the CCR3 receptor, and was noted with other chemokine ligands of CCR3. Additionally, eotaxin augmented IL-3 priming of basophil IL-4 production in a synergistic manner (p < 0.01). In contrast, no priming was observed with either IL-5 or GM-CSF. These results establish a novel function for eotaxin and other chemokine ligands of CCR3: the potentiation of Ag-mediated IL-4 production in basophils, and suggest a potential nonchemotactic role for CC chemokines in the pathogenesis and amplification of inflammation.