Eotaxin is specifically cleaved by hookworm metalloproteases preventing its action in vitro and in vivo.
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Eotaxin is a potent eosinophil chemoattractant that acts selectively through CCR3, which is expressed on eosinophils, basophils, mast cells, and Th2-type T cells. This arm of the immune system is believed to have evolved to control helminthic parasites. We hypothesized that helminths may employ mechanisms to inhibit eosinophil recruitment, to prolong worm survival in the host. We observed that the excretory/secretory products of the hookworm Necator americanus inhibited eosinophil recruitment in vivo in response to eotaxin, but not leukotriene B(4), a phenomenon that could be prevented by the addition of protease inhibitors. Using Western blotting, N. americanus supernatant was shown to cause rapid proteolysis of eotaxin, but not IL-8 or eotaxin-2. N. americanus homogenate was fractionated by gel filtration chromatography, and a FACS-based bioassay measured the ability of each fraction to inhibit the activity of a variety of chemokines. This resulted in two peaks of eotaxin-degrading activity, corresponding to approximately 15 and 50 kDa molecular mass. This activity was specific for eotaxin, as responses to other agonists tested were unaffected. Proteolysis of eotaxin was prevented by EDTA and phenanthroline, indicating that metalloprotease activity was involved. Production of enzymes inactivating eotaxin may be a strategy employed by helminths to prevent recruitment and activation of eosinophils at the site of infection. As such this represents a novel mechanism of regulation of chemokine function in vivo. The existence of CCR3 ligands other than eotaxin (e.g., eotaxin-2) may reflect the evolution of host counter measures to parasite defense systems. (+info)
Eosinophils in the human corpus luteum: the role of RANTES and eotaxin in eosinophil attraction into periovulatory structures.
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We evaluated the presence and number of eosinophils at varying stages in the human corpus luteum from 27 ovaries of women at reproductive age. Eosinophils preferentially accumulated in dilated microvessels of the thecal layer transforming into septa of the corpus luteum. The granulosa layer under luteinization, the thecal layer, and haemorrhages in the former antrum each contained low, moderate and high numbers of extravasated eosinophils respectively. Eosinophils decreased rapidly during the stages of secretion and regression. Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) systems were used to investigate the expression and regulation of the eosinophil-attracting chemokines RANTES (regulated on activation, normal T cell expressed and secreted) and eotaxin in granulosa cells obtained from follicular aspirates from women undergoing IVF. Contaminating leukocytes were determined by CD18 mRNA quantification. Granulosa cells expressed RANTES (n = 3; 43 +/- 14 pg/ml, mean +/- SEM). 4ss-phorbol-12-myristate-13-acetate (PMA; 211 +/- 53) and tumour necrosis factor alpha (TNFalpha) (238 +/- 59), but not interleukin (IL)-1 up-regulated RANTES at significant levels. In general, higher basal and stimulated RANTES mRNA and protein were found in cultures with higher CD18 mRNA levels than in those with lower levels. We found only traces of eotaxin mRNA and no eotaxin secretion, even in stimulated granulosa cell cultures, independently of leukocyte levels. Taken together, this is the first study demonstrating the selective presence of eosinophils in human periovulatory structures. RANTES, but not eotaxin, may play an active process in the accumulation of these cells. (+info)
Hyalinosis and Ym1/Ym2 gene expression in the stomach and respiratory tract of 129S4/SvJae and wild-type and CYP1A2-null B6, 129 mice.
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The C57BL/6, 129, and B6,129 mouse strains or stocks have been commonly used to generate targeted mutant mice. The pathology of these mice is not well characterized. In studies of these aging mice, we found high incidences of hyalinosis (eosinophilic cytoplasmic change) in the glandular stomach, respiratory tract, bile duct, and gall bladder of B6,129 CYP1A2-null and wild-type mice as well as in both sexes of the background 129S4/SvJae strain. The gastric lesions of the glandular stomach were found in 95.7% of female CYP1A2-null mice as well as in 45.7% of female 129S4/SvJae animals. The eosinophilic protein isolated from characteristic hyaline gastric lesions was identified as Ym2, a member of the chitinase family. Immunohistochemistry, using rabbit polyclonal antibodies to oligopeptides derived from the Ym1 sequence, detected focal to diffuse reactivity within both normal and abnormal nasal olfactory and respiratory epithelium, pulmonary alveolar macrophages, bone marrow myeloid cells, and the squamous epithelium of the forestomach and epithelium of the glandular stomach. Alveolar macrophages in acidophilic pneumonia, a major cause of death of aging 129 mice, and in mice with the me mutation also were highly immunoreactive. The possible cause of this protein excess in gastric and other lesions and its possible functions are discussed. (+info)
Bleomycin stimulates lung fibroblast and epithelial cell lines to release eosinophil chemotactic activity.
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The presence of eosinophils in the lungs of patients with pulmonary fibrosis correlates with poor prognosis or resistance to therapy. Furthermore, eosinophils localize to areas undergoing active fibrosis. It was hypothesized that a human lung fibroblast (HFL-1) and a human lung epithelial cell line (BEAS-2B) might release eosinophil chemotactic activity (ECA) in response to bleomycin, a chemotherapeutic agent associated with pulmonary fibrosis. HFL-1 and BEAS-2B cells were cultured in the presence of bleomycin and their supernatant fluids evaluated for ECA by means of a Boyden chamber method. HFL-1 and BEAS-2B cells released ECA in a dose- and time-dependent manner in response to bleomycin, and partial characterization revealed that the ECA was heterogeneous. ECA release from HFL-1 and BEAS-2B cells was significantly reduced by a leukotriene B4 (LTB4) receptor antagonist and an antibody directed against granulocyte-macrophage colony-stimulating factor. HFL-1 cells released LTB4, eotaxin, and GM-CSF constitutively, and BEAS-2B cells released LTB4, eotaxin, regulated on activation, normal T-cell expressed and presumably secreted, and GM-CSF constitutively. In both cases, the release of GM-CSF was significantly increased in response to bleomycin. These data suggest that lung fibroblasts and epithelial cells may modulate eosinophil recruitment into the lung in bleomycin-induced pulmonary fibrosis. (+info)
Erythromycin modulates eosinophil chemotactic cytokine production by human lung fibroblasts in vitro.
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Recent studies suggest that erythromycin can suppress the production of some cytokines and may be an effective treatment for asthma. Eosinophil chemotactic cytokines have been suggested to contribute to the pathogenesis of asthma by the recruitment of eosinophils. We hypothesized that erythromycin modulates eosinophil chemotactic cytokine production. To test the hypothesis, we evaluated the potential of erythromycin to modulate the release of eosinophil chemoattractants from the human lung fibroblast cell line HFL-1. HFL-1 released eotaxin, granulocyte-macrophage colony-stimulating factor, and regulated and normal T-cell expressed and presumably secreted (RANTES) in response to interleukin-1beta or tumor necrosis factor alpha. Erythromycin attenuated the release of these cytokines and eosinophil chemotactic activity by the HFL-1. The suppressive effect on eotaxin was the most marked of these cytokines. Erythromycin therapy also suppressed eotaxin mRNA significantly. These results suggest a mechanism that may account for the apparent beneficial action of macrolide antibiotics in the treatment of allergic airway disorders. (+info)
STAT6 mediates eotaxin-1 expression in IL-4 or TNF-alpha-induced fibroblasts.
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Eosinophils are attracted to sites of allergic inflammation by a number of chemoattractants including eotaxin-1. This chemokine can be secreted from epithelial cells and fibroblasts after IL-4 and TNF-alpha stimulation in a synergistic fashion. TNF-alpha activated gene expression at the transcriptional level in a STAT6-dependent manner, because: 1) eotaxin-1 promoter luciferase constructs were TNF-alpha inducible in STAT6-defective HEK293 cells only on cotransfection of STAT6 expression vector, an effect that was partially mediated by activation-induced binding of NF-kappa B proteins to a composite STAT6/NF-kappa B element; 2) reporter constructs defective in STAT6 DNA binding did not respond to TNF-alpha stimulation; 3) eotaxin-1 protein secretion was detected only in STAT6-transfected HEK293 cell supernatants on TNF-alpha treatment; and 4) a trans-dominant negative STAT6 protein inhibited TNF-alpha-induced eotaxin-1 secretion in primary fibroblasts. TNF-alpha inducibility of the IL-8 and monocyte chemoattractant protein-1 genes was not dependent on STAT6 expression in the same experimental systems. The inducing effect of IL-4 and IL-13 was also mediated by STAT6. The synergistic effect of IL-4 and TNF-alpha observed at the RNA and the protein level was not seen at the promoter level. The data demonstrate that both IL-4 and TNF-alpha induce eotaxin-1 expression at the level of transcription via a STAT6-mediated pathway. (+info)
Eotaxin promotes eosinophil transmigration via the activation of the plasminogen-plasmin system.
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The effect of eotaxin, a potent eosinophil chemotactic factor, on eosinophil transmigration through a reconstituted basal membrane (Matrigel) was evaluated. Eotaxin induced significant eosinophil transmigration in the presence of 10% fetal bovine serum (FBS) and interleukin-5. Its effect was optimal at 0.01 microM, and it plateaued at 18 h. Eotaxin's effect was greater with eosinophils from asthmatic subjects (61.1 +/- 3.4%) than with eosinophils from normal subjects (38.7 +/- 4.2%) (P < 0.001). Inhibition of metalloproteinases decreased eotaxin-induced transmigration by < or = 10.4%, whereas inhibition of the plasminogen-plasmin system decreased eotaxin's effect by < or = 44.4% (P = 0.0002). Moreover, eotaxin-induced transmigration was largely diminished in medium with low concentrations of serum [0.5% FBS: 6.1 +/- 2.4%; 10% FBS: 40.2 +/- 5.8% (P = 0.0001)] but returned to its initial level with the addition of plasminogen (2 U/mL) to 0.5% FBS (43.1 +/- 6.5%). These data show that eotaxin is an efficient promoter of eosinophil transmigration in vitro, that it is more potent with cells from asthmatics than with normal cells, and that its effect depends predominantly on the activation of the plasminogen-plasmin system. (+info)
Eotaxin (CCL11) induces in vivo angiogenic responses by human CCR3+ endothelial cells.
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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. (+info)