Localization of human eosinophil granule major basic protein, eosinophil cationic protein, and eosinophil-derived neurotoxin by immunoelectron microscopy

By utilizing the colloidal gold particle technique, we localized eosinophil granule major basic protein, eosinophil cationic protein (ECP), and eosinophil-derived neurotoxin (EDN) in human nasal polyp sections by immunoelectron microscopy. Sections stained with affinity chromatography purified rabbit anti-human major basic protein, and subsequently with gold colloidal particle-goat anti-rabbit IgG, showed gold particles predominantly within granule cores, and not within other eosinophil organelles, plasma cells, mast cells, lymphocytes, or neutrophils. Sections stained with anti-ECP or anti-EDN showed gold particles concentrated over the granule matrix with fewer particles centrally. Control sections treated with preimmunization sera showed no staining of cells or organelles. These results verify the localization of major basic protein to the crystalloid core of the human eosinophil granule and show that ECP and EDN reside in the granule matrix. This technique provides a means of accurately locating the sites of major basic protein, ECP, and EDN deposition and thus of identifying eosinophil degranulation patterns in human disease.     

Two eosinophil granule proteins, eosinophil peroxidase and major basic protein, inhibit mucin release from hamster tracheal surface epithelial cells in primary culture

OBJECTIVES AND DESIGN: Effects of eosinophil proteins on airway goblet cell mucin release were investigated using primary hamster tracheal surface epithelial (HTSE) cell cultures. MATERIALS AND METHODS: HTSE cells were metabolically labeled using 3H-glucosamine and chased in the presence of varying concentrations of eosinophil proteins. The amount of 3H-mucin in the spent media was measured by Sepharose CL-4B gel-filtration column chromatography. Possible cytotoxicity by the eosinophil proteins was assessed by measurements of both lactic dehydrogenase and mucin release during as well as after the treatment period. RESULTS: (1) Neither eosinophil cationic protein nor eosinophil-derived neurotoxin affected mucin release at concentrations up to 10(-6) M; (2) both major basic protein (MBP) and eosinophil peroxidase (EPO) inhibited mucin release in a dose-dependent fashion, and the inhibitory effect by these proteins appeared to be reversible; (3) neither MBP nor EPO caused any apparent cytotoxicity at concentrations up to 10(-6) M. CONCLUSION: Eosinophil proteins such as MBP and EPO inhibit mucin release from primary HTSE cells without causing any apparent cytotoxicity.     

Eosinophil cationic protein stimulates and major basic protein inhibits airway mucus secretion

Possible roles of eosinophil (EO) products in modulating the release of mucus from airway explants were investigated. Cell- and membrane-free lysates from purified human EOs (1 to 20 x 10(5)) caused a dose-dependent release of respiratory glycoconjugates (RGC) from cultured feline tracheal explants. Crude extracts from isolated EO granules also stimulated RGC release, suggesting that a granular protein might be responsible. Three proteins derived from EO granules, EO-derived neurotoxin, EO cationic protein (ECP), and major basic protein (MBP) were separated by sequential sizing and affinity chromatography. ECP (0.025 to 25 micrograms/ml) caused a dose-dependent increase in RGC release from both feline and human airway explants and also stimulated the release of the serous cell-marker, lactoferrin, from human bronchial explants. EO-derived neurotoxin (0.025 to 50 micrograms/ml) failed to affect RGC release, whereas MBP (50 micrograms/ml) significantly inhibited RGC release from feline explants. Thus, ECP stimulates RGC and lactoferrin release from airway explants, whereas MBP inhibits RGC release.     

Cytotoxic properties of eosinophil granule major basic protein for tumor cells

BACKGROUND: Eosinophil granule major basic protein (MBP) mediates many eosinophil-associated immune functions and it adheres eosinophils to parasite targets. METHODS: We compared the toxicities of MBP and melittin to K562 and HL-60 cells using five cytotoxicity methods. RESULTS: Trypan blue staining, propidium iodide/ CellTrackertrade markGreen staining and incorporation of 14C-leucine assays indicated that MBP damages most cells by 1 h. In contrast, 51Cr and lactic dehydrogenase (LDH) release assays indicated that MBP damages most cells only at 20 h. All five methods indicated that melittin damages nearly all cells by 1 h. To resolve these discrepancies, the procedures were modified. Without cell transfer, dye staining methods showed that MBP produces very little cytotoxicity at 4 h. 51Cr and LDH assays, modified to mimic cell transfer, showed increased cytotoxicities at 4 h. The 14C-leucine assay modified by solubilization of cells with SDS and by trichloroacetic acid precipitation showed increased recovery of labeled protein and, thus, lower cytotoxicity, about 50%, at 4 h. CONCLUSION: Overall, MBP's ability to cause molecular and cellular adhesion confounds cytotoxicity measurements. A modified 14C-leucine assay overcame MBP's adhesiveness and provided an accurate measure of cytotoxicity.     

Donor sensitivity to basophil activation by eosinophil granule major basic protein

Leukocytes of 22 individuals were incubated with a suboptimal concentration of eosinophil granule major basic protein and the amount of histamine release was determined. Cells from 20 of the 22 donors had a histamine release of greater than or equal to 15%. The use of selected donors on repeated occasions revealed significant variability in individual donor responsiveness. The findings support a role for major basic protein activation of human basophils in allergic reactions.     

An immunofluorescent method for specific staining of eosinophil granule major basic protein

We have developed an indirect immunofluorescent method for detection of the guinea pig eosinophil granule major basic protein (MBP) in formalin-fixed, paraffinembedded tissues. The method is specific for MBP, permits MBP localization in cells and tissues, and eliminates eosinophil non-specific fluorescent staining.     

Cytophilic and cytotoxic properties of human eosinophil peroxidase plus major basic protein.

The cytophilic and cytotoxic properties of an acetate-buffered solution of human eosinophil peroxidase (EPO) plus major basic protein (MBP) were studied to determine the cytotoxic potential of localized eosinophil degranulation in human tissues. When incubated with EPO + MBP for 5 minutes, viable cells of six unrelated types (Sp 2/0; HeLa; human gastric adenocarcinoma; acute lymphocytic leukemia; IM-9; benign lymphoid hyperplasia) developed varying degrees of cytochemically detectable deposits of EPO on the cell membranes. A single-step propidium iodide exclusion assay was then used to show that EPO + MBP in the absence of hydrogen peroxide is substantially cytotoxic only to the acute lymphocytic leukemia and IM-9 cells. In the presence of 0.003% hydrogen peroxide, EPO + MBP was cytotoxic to five types of cells. It is concluded that human EPO in the presence of MBP has an affinity for the membrane of diverse cell types. The toxicity of EPO + MBP is markedly enhanced by the presence of hydrogen peroxide.     

Eosinophil major basic protein: first identified natural heparanase-inhibiting protein

BACKGROUND: Heparanase and eosinophils are involved in several diseases, including inflammation, cancer, and angiogenesis. OBJECTIVE: We sought to determine whether eosinophils produce active heparanase. METHODS: Human peripheral blood eosinophils were isolated by immunoselection and tested for heparanase protein (immunocytochemistry, Western blot), mRNA (RT-PCR) and activity (Na(2)[(35)S]O(4)-labeled extracellular matrix degradation) before and after activation. Heparanase intracellular localization (confocal laser microscopy) and ability to bind to eosinophil major basic protein (MBP) were also evaluated (immunoprecipitation). A model of allergic peritonitis resulting in eosinophilia was induced in TNF knockout and wild-type mice for in vivo studies. RESULTS: Eosinophils synthesized heparanase mRNA and contained heparanase in the active (50-kd) and latent (65-kd) forms. Heparanase partially co-localized with and was bound to MBP. No heparanase enzymatic activity was detected in eosinophils resting or activated with various agonists, including GM-CSF/C5a. Eosinophil lysates and MBP inhibited recombinant heparanase activity in a concentration-dependent manner (100%, 2 x 10(-7) mol/L). Eosinophil peroxidase and eosinophil cationic protein, but not myelin basic protein or compound 48/80, partially inhibited heparanase activity. Poly-l-arginine at very high concentrations caused an almost complete inhibition. In allergic peritonitis, heparanase activity in the peritoneal fluid inversely correlated with eosinophil number. CONCLUSIONS: MBP is the first identified natural heparanase-inhibiting protein. Its presence in the eosinophil granules might indicate a protective function of these cells in diseases associated with inflammation and cancer progression.     

Lack of eosinophil peroxidase or major basic protein impairs defense against murine filarial infection

Eosinophils are a hallmark of allergic diseases and helminth infection, yet direct evidence for killing of helminth parasites by their toxic granule products exists only in vitro. We investigated the in vivo roles of the eosinophil granule proteins eosinophil peroxidase (EPO) and major basic protein 1 (MBP) during infection with the rodent filaria Litomosoides sigmodontis. Mice deficient for either EPO or MBP on the 129/SvJ background developed significantly higher worm burdens than wild-type mice. Furthermore, the data indicate that EPO or MBP is involved in modulating the immune response leading to altered cytokine production during infection. Thus, in the absence of MBP, mice showed increased interleukin-10 (IL-10) production after stimulation of macrophages from the thoracic cavity where the worms reside. In addition to elevated IL-10 levels, EPO(-/-) mice displayed strongly increased amounts of the Th2 cytokine IL-5 by CD4 T cells as well as a significantly higher eosinophilia. Interestingly, a reduced ability to produce IL-4 in the knockout strains could even be seen in noninfected mice, arguing for different innate propensities to react with a Th2 response in the absence of either EPO or MBP. In conclusion, both of the eosinophil granule products MBP and EPO are part of the defense mechanism against filarial parasites. These data suggest a hitherto unknown interaction between eosinophil granule proteins, defense against filarial nematodes, and cytokine responses of macrophages and CD4 T cells.     

Pharmacological control of human basophil histamine release stimulated by eosinophil granule major basic protein.

Eosinophil granule major basic protein (MBP) is a 13,800 MW arginine-rich polypeptide that is unique among basic molecules in its ability to stimulate human basophil histamine release. We examined the Ca2+ requirements and pharmacological regulation of MBP-stimulated histamine release. Minimal MBP-induced histamine release occurred in the absence of extracellular Ca2+, whereas addition of 0.1 mM Ca2+ resulted in 70% of the maximum histamine release response. Maximum histamine release required 0.5 to 1 mM extracellular Ca2+. The MBP-induced histamine release was blocked by a calmodulin antagonist and by theophylline and was partially inhibited by an inhibitor of phospholipase A2. Release was unaffected by inhibition of protein kinase C. Basophil pretreatment with pertussis toxin also resulted in a concentration-dependent inhibition of release, suggesting involvement of a GTP regulatory protein in the activation mechanism. Histamine release stimulated by a 13,900 MW poly-L-arginine exhibited a dissimilar pharmacological profile from that of MBP. These results support the non-cytolytic nature of the MBP activation mechanism and identify pharmacological approaches for control of MBP-induced mediator release.     

Cytotoxicity of human eosinophil granule major basic protein to human nasal sinus mucosa in vitro

The toxic effects of the human eosinophil granule major basic protein (MBP), reduced and alkylated, were studied on human nasal mucosa in vitro. With a microscope coupled with a television monitor (magnification x 2500) and videotape recorder, we investigated the effects of MBP on the mucosa and the ciliary activity of single cells. In nasal mucosal specimens from normal individuals, MBP, 5 mumol/L and 10 mumol/L, significantly inhibited (p less than 0.01) ciliary activity by 4 and 1 hours of exposure, respectively. At these same MBP concentrations, the mucosal surface profiles were altered by 4 hours of exposure, and ciliostasis was 75% to 100% complete by 9 and 6 hours, respectively. In a mucosal specimen from a patient with nasal allergy, 1 mumol/L of MBP significantly inhibited (p less than 0.01) ciliary activity by 1 hour; alteration of the mucosal surface profile appeared by 3 hours of exposure, and ciliostasis was 75% to 100% by 13 hours. Similar alterations of the mucosal surface profile were observed with specimens from a second patient with allergies; in contrast, 1 mumol/L of MPB had no effect on specimens from a nonallergic patient. These results indicate that MBP damages human upper respiratory epithelium, causing ciliostasis and alteration of the epithelial surface at concentrations likely achieved in vivo. Furthermore, the mucosal specimens from two allergic patients were damaged by concentrations of MBP that had no effect on mucosal specimens from a normal individual.     

Peptide-based analysis of amino acid sequences important to the biological activity of eosinophil granule major basic protein

Synthetic peptides corresponding to amino acid sequences in eosinophil granule major basic protein (MBP) were evaluated for cytotoxic activity toward K562 cells and for ability to stimulate basophil mediator release. Results obtained using 14 peptides spanning the 117-amino acid sequence of MBP in overlapping fashion indicated that the activities mapped to peptide sequences near the amino and carboxy termini of MBP. The activity of these regions was confirmed using two peptides corresponding to MBP residues 18-45 and 89-117. A 20-h incubation with 5 microM peptide 18-45 or peptide 89-117 caused approximately the same levels (>60%) of cytotoxicity in K562 cells as 5 microM MBP. Similarly, a 30-min incubation with peptides 18-44 and 89-117 stimulated basophil histamine release in a concentration-dependent manner over the range of 5-20 microM. The level of release stimulated by 20 microM peptide 89-117 approached that stimulated by 2 microM MBP. A 20 microM concentration of peptide 89-117 also stimulated leukotriene C4 (LTC4) production by the basophils. Neither peptide 18-45 nor peptide 89-117 was cytotoxic for basophils under the experimental conditions for histamine and LTC4 release, as determined by 51Cr release. These results indicate that two MBP peptide sequences, including one (89-117) that contains a unique carbohydrate-binding region, share the biologic activities of MBP.     

Major basic protein, but not eosinophil cationic protein or eosinophil protein X, is related to atopy in cystic fibrosis.

Increased eosinophil granule proteins have been described in serum and sputum samples of patients with cystic fibrosis (CF). It has been assumed that eosinophil degranulation is enhanced in atopic subjects - as in asthmatics. Since in CF no differences in eosinophil cationic protein (ECP), eosinophil protein X (EPX), and eosinophil peroxidase between atopic and nonatopic subjects have been detected, we investigated whether major basic protein (MBP) is increased in serum and sputum samples derived from atopic (n = 14) compared with nonatopic CF subjects (n = 26). In CF patients, high mean serum (sputum) levels of ECP 29.7 microg/l (2.7 mg/l), EPX 53.7 microg/l (7.9 mg/l), and MBP 984.6 microg/l but low sputum MBP levels (57.4 microg/l) were measured. In addition, in serum and in sputum samples, a significant correlation between MBP and ECP (P<0.03 and P<0.0001, respectively) or EPX (P<0.05 and P<0.0004, respectively) was detected. By subdivision of the patients into allergic and nonallergic subjects, significant differences were found for serum MBP values only(mean 1382.2 microg/l vs. 770.5 microg/l; P<0.0001), but not for ECP or EPX serum levels or for eosinophil proteins in sputum. Although no differences between atopic and nonatopic CF patients in ECP and EPX were found, serum MBP levels were higher in patients sensitized to inhalant allergens than in nonsensitized subjects. These results indicate differential release of eosinophil granule proteins in peripheral blood from eosinophils, and they also indicate that MBP in serum likely is to be a better discriminator of atopy in CF.     

The role of eosinophil major basic protein in angiogenesis

Background:  Eosinophil-derived major basic protein (MBP) plays an active role in allergic inflammation and tissue remodelling. However, its role in angiogenesis has not been established as yet. Therefore our objective was to investigate whether MBP exhibits any direct pro-angiogenic effects.
Methods:  Rat aortic endothelial cells and human umbilical vascular endothelial cells were cultured with different concentrations of MBP and their viability (Trypan blue exclusion test), proliferation (thymidine incorporation) and capillary-like structure formation (matrigel assay) were investigated in vitro . The angiogenic activity of MBP was then tested in vivo using the chick chorio allantoic membrane (CAM) assay.
Results:  Subcytotoxic concentrations of MBP induce endothelial cell proliferation and enhance the pro-mitogenic effect of vascular endothelial growth factor (VEGF), but do not affect their VEGF release. MBP promotes capillarogenesis by endothelial cells seeded on matrigel and sprouting formation in the CAM assay. Furthermore, we have shown that the pro-angiogenic effect of MBP is not due to its cationic charge since stimulation of the CAMs with the synthetic polycation, poly- l -arginine does not induce any angiogenic effects.
Conclusions:  These data demonstrate that MBP has pro-angiogenic effects in vitro and in vivo , providing a novel mechanism whereby MBP can participate in tissue inflammation and remodelling in atopic diseases.     

Preparation and characterization of monoclonal antibodies to human neutrophil cathepsin G, lactoferrin, eosinophil peroxidase, and eosinophil major basic protein

This report describes the production and characterization of five murine monoclonal antibodies that react with granule proteins of human granulocytes. Monoclonal antibody AHN-11 (IgG2a) reacted specifically with neutrophil cathepsin G; no reactivity with the homologous neutrophil neutral proteases, elastase, proteinase 3, or esterase N was detected. Antibodies AHN-9 (IgG1) and AHN-9.1 (IgG2b) each reacted with different epitopes on human lactoferrin, but not with the homologous protein transferrin. Two IgG1 antibodies, AHE-1 and AHE-2, reacted specifically with eosinophils; AHE-1 reacted strongly with eosinophil peroxidase but not eosinophil major basic protein while AHE-2 recognized eosinophil major basic protein but not eosinophil peroxidase. All five antibodies could detect their respective antigens in alcohol-fixed cytospin preparations. These antibodies should be useful for immunolocalization and quantification of their respective antigens as well as for other studies of the roles of these proteins in granulocyte function and differentiation.     

Deposition of eosinophil granule major basic protein onto microfilariae of Onchocerca volvulus in the skin of patients treated with diethylcarbamazine

We investigated the association between eosinophil degranulation, as evidenced by the deposition of granule major basic protein (MBP), and the killing of microfilariae of Onchocerca volvulus in vivo following treatment with diethylcarbamazine (DEC). Utilizing an immunofluorescence procedure for the cellular and extracellular localization of eosinophil MBP in formalin-fixed, paraffin-embedded tissues, we studied skin biopsies from onchocerciasis patients before and during treatment with topically or orally administered DEC. Before DEC, there was little or no inflammatory response in either dermis or epidermis and microfilariae were essentially intact. Immunofluorescent staining for MBP revealed some filamentous fluorescence associated with dermal collagen fibers, very few eosinophils, and no fluorescence in association with intact microfilariae. In contrast, during treatment with DEC, immunofluorescent staining for MBP revealed extensive eosinophil infiltrates in both dermis and epidermis with numerous intraepidermal eosinophil abscesses containing degenerating microfilariae. An intense extracellular immunofluorescence for MBP surrounded degenerating microfilariae in the dermis and epidermis in both the presence and absence of eosinophil infiltrates as early as 4.5 hours after starting therapy. Many intact nondegenerating microfilariae were also present, but they did not show immunofluorescent staining for MBP nor a surrounding inflammatory infiltrate. The results show that immediately following administration of DEC, eosinophils localize and degranulate around microfilariae in the skin and release granule MBP onto or in close proximity to the parasite's surface. Because of the striking association between eosinophil localization, degranulation, and deposition of MBP onto microfilarial surfaces, and the degeneration of microfilariae in the skin, these observations support the hypothesis that the eosinophil, through helminthotoxic granule proteins such as MBP, damages the microfilariae of O. volvulus.