A novel human homolog of eosinophil major basic protein.

Eosinophil major basic protein (MBP) contributes to host defense and disease pathophysiology. Chromosome 11 contains the genes for human MBP1 (hMBP1) and a second novel MBP, hMBP2, in the centromere to 11q12 region. Interestingly, greater similarity exists between human and murine MBP1 and MBP2 orthologs, respectively, than between hMBP1 and hMBP2, suggesting a gene duplication event prior to the divergence of humans and mice. There is abundant mRNA for hMBP1 in both bone marrow (eosinophils and basophils) and placenta, but hMBP2 mRNA is present only in bone marrow (eosinophils). Comparison of proximal promoters for hMBP2, hMBP1, and murine MBP1 (mMBP1) shows a conserved GATA transcription factor binding site (functionally active in hMBP1). However, whether a C/EBP binding site common to hMBP1 and mMBP1 is functionally conserved in hMBP2 remains unresolved. Similarly, the role of conserved putative IK2 and STAT binding sites in MBP transcriptional control remains unknown. Like hMBP1, hMBP2 is in the eosinophil secondary granule. However, hMBP2 is two-fold less positively charged than hMBP (+8 versus +16 at neutral pH), and this difference may explain hMBP2's similar, but often less potent, in vitro biological activities. Overall, while conservation of hMBP2's amino acid sequence (63% identity with hMBP1) suggests a common function(s) with hMBP1, hMBP2's substantially reduced charge and the existence of the similar mMBP2 argue for additional, unique functions for hMBP2.

Discordant and anomalous results among cytotoxicity assays: the confounding properties of eosinophil granule major basic protein on cell viability assays.

When five cytotoxicity methods compared the toxicity of eosinophil granule major basic protein (MBP) and melittin to K562 and HL-60 cells, strikingly discrepant results were noted. Trypan blue staining, propidium iodide/CellTrackerGreen staining and incorporation of 14C-leucine assays indicated MBP damages > 75% of cells by 1 h. In contrast, 51Cr and lactic dehydrogenase (LDH) release assays indicated MBP damages most cells only at 20 h. All methods indicated melittin damages nearly all cells by 1 h. Further studies showed that without cell transfer, dye staining methods indicated MBP produces < 10% cytotoxicity after 4 h. A modified 14C-leucine assay, employing sodium dodecyl sulfate solubilization and trichloroacetic acid precipitation, showed lower cytotoxicity, 48%, at 4 h. Modified 51Cr and LDH assays showed increased cytotoxicities at 4 h, 34% and 58%, respectively. Overall, MBP's ability to cause molecular and cellular adhesion systematically confounds standard cytotoxicity measurements. However, the modified 14C-leucine assay provides a valid measure of MBP's cytotoxicity and may be useful for analyses of 'sticky' cytotoxins.

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.

The biology of the eosinophilic leukocyte.

The eosinophil is characterized by specific cytoplasmic granules that contain a series of cationic toxins able to kill many targets, including helminths, protozoa, bacteria, and other cells. In bronchial asthma, considerable evidence exists that the eosinophil releases granule proteins, especially the major basic protein (MBP), which in turn mediate tissue abnormalities. Among eosinophil-activating factors, IL-5 has been associated with helminth infection and hypersensitivity diseases and would appear to be an attractive target for pharmacological intervention.

Detection of antibodies to proteases used in laundry detergents by the radioallergosorbent test.

Two proteases, Esperase and Alcalase, derived from Bacillus licheniformis and B. subtilis, respectively, are used in laundry products. In testing for the prevalence of IgE antibodies to these enzymes in sera among 300 laundry product workers, we experienced two problems in the establishment of a reliable RAST for these antigens. The first problem was the propensity of the allergen, Esperase, to undergo autolysis, suggesting that solid-phase Esperase might also lose reactivity through degradation. Treatment of Esperase with phenylmethylsulfonyl fluoride stabilized the enzyme and permitted the synthesis of a stable solid-phase antigen. The second problem was the finding that sera reactive with Esperase in the RAST were also reactive with Savinase, an enzyme from B. licheniformis to which the workers were not exposed. Immunochemical analyses of the three enzymes with specific rabbit antisera by gel diffusion and by two-site immunoradiometric assay demonstrated that they were not cross contaminated to any appreciable extent. RAST inhibition demonstrated that solid-phase Esperase possessed unique allergenic determinants in that the reactivity of IgE antibodies was inhibited by low concentrations of Esperase and only by very high concentrations of Alcalase and Savinase. In contrast, the reactivity of solid-phase Alcalase was occasionally inhibited equally well by Esperase and Alcalase. Most strikingly, the reaction of IgE antibodies with solid-phase Savinase was always inhibited by comparable quantities of Esperase, Alcalase, and Savinase. Thus, the establishment of the RAST for these proteases appears to require the use of phenylmethylsulfonyl fluoride to retard autolysis, and the results must be interpreted with caution because IgE antibodies in certain sera demonstrate cross-reactivity with Alcalase and Savinase.

The eosinophilic leukocyte: structure and function.

The evidence reviewed here indicates that the eosinophil has the ability to kill many species of helminths and likely does so during worm infection. This toxic ability appears to be regulated by several other cells including mast cells, monocytes, and T lymphocytes. Eosinophils kill helminths through their ability to generate potent oxidants and through their content of cationic proteins, which likely achieve high concentrations at points of granule deposition. Eosinophils also participate in inflammation in human disease especially asthma, skin diseases, and heart disease. Though present concepts hold that the mast cell is the cornerstone of the allergic inflammatory response (450), the findings that eosinophils bind IgE and are activated by antigen-IgE complexes and that the eosinophil can elaborate many inflammatory mediators raise the possibility that the eosinophil might also be involved in the initiation of inflammatory responses. Finally, an eosinophil-related protein appears to play an undefined role in human reproduction.

Seasonal variation of in vitro lymphocyte proliferative response to ragweed antigen E.

Seasonal variation of symptoms and IgE response to short ragweed (SRW) allergens is well documented. Clinical symptoms generally parallel the rise and fall of the SRW-pollen count, whereas total and specific-IgE levels peak after the SRW-pollen season with a more gradual return to preseason levels. Because IgE synthesis is under T-lymphocyte control, we tested for seasonal variation in T cell-proliferative response to SRW antigen E (AgE) in vitro. Nine untreated SRW-sensitive and five nonallergic individuals were studied on 15 occasions from June 1981 through May 1982. In vitro proliferative index (SI) to AgE, serum total and specific IgE and SRW-pollen counts were measured; all persons studied kept daily symptom diaries. The mean SI was higher for the atopic group on all 15 sampling dates. The cumulative SI and the daily SI were statistically different between groups before, during, and after pollination. The peak SI for the atopic patients occurred almost 1 2/5 wk after the pollination peak, and the peak IgE antibody levels to SRW occurred at 5 2/5 wk after the pollination peak. We conclude that in vitro responsiveness to AgE is a specific response of allergic individuals and that this response demonstrates a significant seasonal component.

The mini-RAST: Comparison with other varieties of the radioallergosorbent test for the measurement of immunoglobulin E antibodies.

In the performance of the RAST a variety of different solid-phase supports for allergens are utilized. In this study we coupled allergens to microcrystalline cellulose and reduced the quantities of solid-phase allergen tenfold and of anti-IgE fourfold. These reductions actually increased the sensitivity of the RAST for IgE antibodies to several allergens, including those derived from Alternaria tenuis and rye grass pollen. We have termed this procedure the mini-RAST to distinguish it from the procedure employing larger quantities of solid-phase allergen and anti-IgE, the maxi-RAST. We then compared three RAST procedures for the detection of IgE antibodies to ragweed allergens: the maxi-RAST, the mini-RAST, and the paper disk RAST, using both commercial paper disks and disks prepared in our laboratory. The maxi-RAST and the mini-RAST yielded similar results and both appeared more sensitive than the paper disk RAST for the detection of IgE antibodies to ragweed. The results indicate that the microcrystalline cellulose RAST can be significantly scaled down without a loss of its ability to detect IgE antibodies and with a considerable saving of valuable solid-phase allergens and anti-IgE.