Citations

Abstract Glucocorticoids characteristically induce eosinopenia in vivo and are effective for treating allergic and other eosinophilic disorders. We studied the effect of glucocorticoids on cytokine-induced survival of human eosinophils in vitro. Eosinophils were purified from normal or mildly atopic volunteers by Percoll density gradient and incubated for 4 days in the presence of cytokine plus steroid. Cell viabilities were determined by staining cells with fluorescein diacetate and propidium iodide. In the absence of glucocorticoids, human rIL-5 enhanced eosinophil survival in a dose-dependent manner, from 22 fM for a minimal effect to 2200 fM for maximal effect. When eosinophils were cultured with a submaximal concentration of rIL-5 (220 fM), dexamethasone, methylprednisolone, and hydrocortisone inhibited eosinophil survival in a dose-dependent manner. Inhibition was time-dependent and required at least 2 days’ exposure of eosinophils to dexamethasone. Dexamethasone, methylprednisolone, and hydrocortisone at 1000 nM inhibited survival by 88 +/- 2, 66 +/- 9 and 37 +/- 7%. In contrast, estradiol and testosterone (1000 nM) had no effect on eosinophil survival. When eosinophils were incubated with varying concentrations of human rIL-5 and 1000 nM dexamethasone, survival inhibition was reduced at higher concentrations of human rIL-5, and completely abolished by human rIL-5 23,000 fM. Human recombinant granulocyte-macrophage CSF, human rIL-3, and human rIFN-γ also enhanced eosinophil survival in a dose-dependent manner and dexamethasone (1000 nM) strongly inhibited cell survival when submaximal concentrations of these cytokines were used. The effects of dexamethasone were reversed by higher concentrations of granulocyte-macrophage CSF (10 U/ml) and IL-3 (3 ng/ml). However, even 1000 U/ml IFN-γ did not overcome dexamethasone inhibition, indicating a difference between the mechanism of eosinophil survival induced by IFN-γ and other cytokines. These results suggest that glucocorticoids exert a direct, inhibitory effect on eosinophil survival, which may be important in the treatment of allergic and other eosinophilic disorders. Antagonism of this effect by higher amounts of cytokine may be a mechanism for glucocorticoid resistance.

Abstract Eosinophil granule proteins, such as major basic protein (MBP), eosinophil peroxidase (EPO), and eosinophil cationic protein (ECP), possess a wide range of biologic activities including the ability to activate other cells, such as basophils, neutrophils, and platelets. Here we have analyzed the effects of these proteins on eosinophils themselves. MBP and EPO, at concentrations as low as 0.1 micrograms/ml, induced eosinophil degranulation as measured by release of eosinophil-derived neurotoxin (EDN); in contrast, ECP, at 1 micrograms/ml, was inactive. MBP (10 micrograms/ml) and EPO (0.1 micrograms/ml) induced EDN release comparable with one of the strongest agonists for eosinophils, secretory IgA. Pretreatment of cells with dibutyryl cAMP or cytochalasin B completely abolished the EDN release induced by MBP and EPO, suggesting that the effects of MBP and EPO are not due to cytotoxic lysis of the cells. Degranulation induced by MBP was only partially dependent on calcium, and no elevation of intracellular Ca2+ concentration ([Ca2+]i) was observed in eosinophils stimulated with MBP. MBP stimulated the production, up to eightfold, of IL-8 by eosinophils in a dose-dependent manner. The MBP-stimulated expression of IL-8 mRNA by eosinophils was confirmed by reverse transcription-PCR. The MBP-stimulated production of IL-8 was inhibited by actinomycin D, but not by cyclosporin A. Furthermore, MBP and calcium ionophore ionomycin synergistically induced production of leukotriene C4 from eosinophils. Thus, MBP and EPO may act as autocrine mediators in the pathogenesis of eosinophil-associated diseases, such as bronchial asthma.

Abstract Eosinophil major basic protein (MBP), a highly charged polycation, forms the core of the eosinophil granule and mediates tissue damage in allergic disease. Purified MBP was studied for capacity to regulate the generation of classical and alternative-amplification pathway C3 convertases because previous studies have shown that other polycations (protamine, poly-L-lysine) and polyanions (heparin) may play important roles in regulating C activation. MBP inhibited the generation of EAC1,4b,2a and EAC4b,3b,Bb,P but appeared to inhibit the generation of classical pathway convertase more than the alternative amplification pathway convertase at a given dose. Dose-response curves with MBP were steeper than curves seen with polyanion (heparin). MBP did not lyse cellular intermediates at concentrations that caused almost total inhibition of convertase generation. One mechanism of inhibition of convertase generation may have been through an action on C3b, because preincubation of MBP with an EAC4b,3b cellular intermediate interfered with the ability of this cellular intermediate to be lysed. Furthermore, MBP prevented consumption of B in a reaction mixture that contained factors B, D, and C3b, also suggesting an action on C3b. Reduced and alkylated MBP (A-MBP) was compared with native MBP, which possesses two reactive sulfhydryl groups, to determine whether charge alone is responsible for blocking convertase generation; native MBP rapidly associates and is relatively insoluble at neutral and alkaline pH whereas A-MBP remains soluble. A-MBP impaired convertase generation, did not appear to remain bound to cellular intermediates and did not suppress B consumption in the fluid phase assay. This suggests that the ability of MBP to regulate C activation is complex and not entirely through its net charge. Finally, although heparin or MBP alone may prevent C activation, when these substances were present at the same time there was no effect on C activation suggesting that charge neutralization may abrogate the effects of these charged substances on C activation. Taken together, these studies suggest that MBP at physiologic concentrations may regulate in vivo C activation at the tissue level.

Abstract B10.Ek alpha transgenic mice were mated with H2-E B10.Q and SWR mice. F1 and F1 x parental strain backcross progeny were tested for arthritis and autoimmune reactivity to mouse type II collagen (MII) after immunization with bovine, chick, deer, or human type II collagen. The results were correlated with the H-2 haplotype (b/q vs q/q) and the TCR V beta profile of peripheral blood T cells in each mouse. Hybrid progeny expressed TCR profiles different from either parent because of the TCR V beta genomic deletions of SWR mice (V beta a), the wild-type TCR allele of C57Bl/10 (B10) mice (V beta b), and the intrathymic negative selection processes resulting from cell surface expression of Ek alpha-A q beta or Eb beta-Ek alpha, together with the integrated retroviral genes Mtv-9 originating in B10 mice and Mtv-7 (Mls-1a) from SWR mice. (B10.Ek alpha x SWR)F1 mice developed higher IgG anti-MII Ab titers, but much milder arthritis than (B10.E x B10.Q)F1 mice. Expression of Ek alpha did not change the level of IgG anti-MII Ab nor the degree of susceptibility to collagen-induced arthritis (CIA) in the H-2q/q and H-2b/q progeny of (B10.Ek alpha x B10.Q)F1 x B10.Q matings, indicating that the Mtv-9-reactive, TCR V beta 5+, and V beta 11+ T cells are not critical to CIA. Among bovine type II collagen-immunized (B10.Ek alpha x SWR)F1 x SWR backcross mice: 1) arthritis severity is associated with the presence of V beta b (p < or = 0.01) and expression of Ek alpha (p < or = 0.05), but not with the MHC haplotype (b/q vs q/q); 2) regression analysis showed a significant association (R = 0.99) between IgG anti-MII Ab titers and the level of Mtv-7-reactive V beta 6+ T cells that was detectable in the IgG1, but not the IgG2a subclass. The data prompt the speculation that Mtv-7-reactive V beta 6+ (or V beta 7+) T cells in (B10.EK alpha x SWR)F1 x SWR mice express Th2-type properties, and thus contribute to the combination of mild arthritis but high anti-MII Ab titers that characterize mice of SWR heritage.

Abstract By using microscopic, fluorometric, and immunologic methods, we have assessed the effects of eosinophil granule proteins on the microcirculation of the hamster cheek pouch. The plasma clearance of FITC-dextran 150 (FITC-dx 150) was used to quantify macromolecular transport. Topical application of major basic protein (MaBP) at 0.1 and 0.5 nM increased the clearance of FITC-dx 150 from a base line of 591 to 1283 and 1966 nl/60 min/g, respectively. Numerous muscle fasciculations were also observed with the 0.5 nM dose of MaBP. Eosinophil cationic protein (ECP) was as potent as MaBP and caused an increase in the clearance of FITC-dx 150, 0.5 nM eliciting 2156 nl/60 min/g. In contrast, topical application of 0.5 nM eosinophil peroxidase (EPO) increased clearance of FITC-dx 150 to a significantly lower level, 1113 nl/60 min/g. Supplementing 0.5 nM EPO with 1 nM H2O2 enhanced the clearance of FITC-dx 150 to 2404 nl/60 min/g, suggesting separate cationic charge and enzymatic activity-related effects. Compared with these eosinophil granule proteins, eosinophil-derived neurotoxin (EDN) required a 2000-fold higher concentration (1 microM) to elicit a significant increase in the clearance of FITC-dx 150 (1505 nl/60 min/g). Neither EPO, EPO+H2O2, ECP, nor EDN at 1 mM caused muscle fasciculations. Quantitative analysis of the suffusates from the preparations exposed to these eosinophil proteins did not contain histamine. Our results demonstrate that MaBP, ECP, EPO, and EDN increase microvascular transport in the hamster cheek pouch and that this increase is independent of endogenous histamine release. The concentrations of eosinophil granule proteins causing increased vascular permeability are achieved in many pathologic conditions suggesting that the granule proteins play an important role in disease.

Abstract We examined the bactericidal activity of two proteins that are abundant in the cytoplasmic granules of human eosinophils, major basic protein (MBP) and eosinophil cationic protein (ECP). Unlike the human neutrophil's peptide defensins, both MBP and ECP killed stationary phase Staphylococcus aureus 502A in a simple nutrient-free buffer solution. Although MBP also killed Escherichia coli ML-35 with considerable efficacy under these experimental conditions, the in vitro activity of ECP against E. coli was considerably enhanced if mid-logarithmic phase bacteria replaced stationary phase organisms or if the assay medium was enriched with trypticase soy broth. The antibacterial activity of both eosinophil proteins was modulated by incubation time, protein concentration, temperature and pH. A pBR322-transformed derivative of E. coli ML-35 was used to examine the effects of ECP and MBP on integrity of the bacterial inner membrane (IM) and outer membrane. Although both MBP and ECP caused outer and inner membrane permeabilization when nutrients were present, only MBP was effective under nutrient-free conditions. Two proton ionophores (DNP and carbonyl cyanide m-chlorophenyl hydrazone) protected E. coli from the bactericidal effects of ECP but not from MBP. These findings establish that MBP and ECP have bactericidal properties and suggest that these proteins kill E. coli by similar but nonidentical mechanisms marked by an attack on the target cell's membranes. In view of evidence that high concentrations of ECP and MBP exist in cytoplasmic granules whose contents are translocated to phagocytic vacuoles, we suggest that MBP and ECP contribute to the eosinophil's ability to kill ingested bacteria.

Abstract Major basic protein (MBP), an arginine-rich basic polypeptide that constitutes the crystalloid core of the large specific eosinophil granule, has previously been shown to stimulate noncytolytic histamine release from human basophils and rat mast cells by an IgE-independent mechanism. Two additional basic polypeptides present in eosinophil granules, eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin (EDN), were examined for similar activity in the present study. Acid-solubilized eosinophil granules were fractionated by chromatography on a Sephadex G-50 column. Incubation of basophil-containing human mononuclear cells with the individual column fractions demonstrated that histamine release occurred only with the fractions that contained MBP. The selectivity of the basophil response for MBP was confirmed by using equimolar concentrations of purified MBP, ECP, and EDN. In contrast, both MBP and ECP, but not EDN, stimulated histamine release from purified rat peritoneal mast cells. Reduction and alkylation of the MBP molecule diminished the response of human basophils to MBP but enhanced the potency of the molecule with rat mast cells. The distinct potency of MBP as a stimulus for histamine secretion from human basophils suggests that eosinophil release of MBP may be a specific event in the augmentation of immediate hypersensitivity reactions and other disorders characterized by eosinophilia.

Abstract The eosinophil granule contains a series of basic proteins, including major basic protein, eosinophil peroxidase, eosinophil-derived neurotoxin (EDN), and eosinophil cationic protein (ECP). Both EDN and ECP are neurotoxins and helminthotoxins. Comparison of the partial N-terminal amino acid sequences of EDN and ECP showed 67% identity; surprisingly, they also showed structural homology to pancreatic ribonuclease (RNase). Therefore, we determined whether EDN and ECP possess RNase enzymatic activity. By spectrophotometric assay of acid soluble nucleotides formed from yeast RNA, purified EDN showed RNase activity similar to bovine pancreatic RNase, whereas ECP was 50 to 100 times less active. The RNase activity associated with ECP was not significantly inhibited after exposure of ECP to polyclonal or monoclonal antibody to EDN. These results indicate that EDN and ECP both possess RNase activity, the RNase activity of EDN and ECP is specific, and EDN and ECP have maintained not only structural but also functional homology to pancreatic RNase.

Abstract To study the cytotoxic reactions responsible for mediating eosinophil damage to schistosomula of Schistosoma mansoni, we have used cytoplasts (eosinophil or neutrophil vesicles devoid of granules and nuclei, with an intact oxidase in their plasma membrane) in combination with purified eosinophil cationic protein (ECP) or major basic protein (MBP) in a cytotoxicity test toward schistosomula. Suboptimal concentrations of ECP (10(-6) M) or MBP (10(-6) M) resulting in less than 10% killing were used in combination with cytoplasts. Cytoplasts alone in the presence of immune serum tested over a wide range of cytoplast:schistosomula ratios generated superoxide and hydrogen peroxide, but were unable to damage schistosomula. However, when a suboptimal ECP concentration (10(-6) M) was combined with neutroplasts or eosinoplasts, 43.9% +/- 8.5 (n = 7) and 24.7% +/- 9.8 (n = 3), respectively, of the schistosomula were killed. Oxygen metabolites were responsible for the synergism, because cytoplasts from a patient with chronic granulomatous disease were unable to act in synergy with ECP. In contrast to ECP, no synergism was found between cytoplasts and MBP (10(-6) to 2 X 10(-5)M). These results show that oxygen metabolites are important for the killing of schistosomula by lowering the concentration of ECP needed to inflict damage.

Abstract Human eosinophils contain several distinctive proteins including eosinophil granule MBP and the membrane-associated CLC protein (lysophospholipase). Human basophils also contain these proteins, indicating biochemical similarities between eosinophils and basophils. To determine whether MBP or CLC protein is present in connective tissue mast cells, we studied human lung and cutaneous mast cells by immunofluorescence by utilizing specific antibodies to CLC and MBP. Cytocentrifuge slides of enriched lung mast cells and mast cells in sections of formalin-fixed, paraffin-embedded cutaneous tissue from urticaria pigmentosa lesions were stained for CLC and MBP. Neither pulmonary nor cutaneous mast cells stained for CLC protein or MBP. In contrast, lung and cutaneous eosinophils in the same preparations showed bright staining for both proteins. The failure to find CLC protein and MBP in mast cells provides additional evidence of dissimilarity between mast cells and basophils, and an immunochemical means to distinguish between them.