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Eosinophil-derived neurotoxin (EDN) and eosinophil cationic protein (ECP) are generally regarded as eosinophil-specific proteins. We tested whether EDN and ECP are present in mature neutrophils. By indirect immunofluorescence, both eosinophils and neutrophils stained with antibodies to EDN and ECP. Lysates of purified (<0.1% eosinophil contamination) neutrophils contained EDN, 112+/-4 ng/10(6) cells, and ECP, 163+/-2 ng/10(6) cells, whereas eosinophil major basic protein (MBP) was not detectable. Electron microscopic examination of immunogold-labeled buffy coat cells stained with EDN antibody showed that EDN is localized to neutrophil granules. Finally, EDN mRNA was detected in lysates of highly purified neutrophils (0.001% eosinophil contamination) by the reverse transcription-polymerase chain reaction. We conclude that proteins that are either identical to or immunologically cross-reactive with EDN and ECP are present in neutrophils and that EDN is synthesized and localized to neutrophil granules. Thus, caution must be exercised in interpreting the presence of EDN and ECP as specific markers of eosinophil-associated inflammation in human disease.

Eosinophils, neutrophils, and mast cells have all been implicated in the pathogenesis of bullous pemphigoid (BP). To comparatively characterize the involvement of these cells in BP, 10 lesional skin biopsy specimens were identified retrospectively and studied for tissue localization of eosinophil, neutrophil, and mast cell granule proteins. Subsequently, multiple skin biopsies of lesions in various developmental stages were obtained from 3 patients with untreated BP. Involved and uninvolved skin specimens were also obtained from 2 patients. Using indirect immunofluorescence, retrospectively identified lesions showed eosinophils and extracellular granule protein deposition prominently in areas of blistering. Evolving lesions showed eosinophil granule protein deposition in all stages but was most marked in early erythematous and prebullous (urticarial) lesions and was minimal in uninvolved skin. Vascular cell adhesion molecule-1, E-selectin, and P-selectin were detected on vessels and very late activation antigen-4 was detected on mononuclear cells and eosinophils by immunoperoxidase staining of lesions. Eosinophil granule proteins were increased in the peripheral blood, urine, and blister fluid. Blister fluids caused increased eosinophil survival that was inhibited by antibodies to interleukin-5 and interleukin-3. Although neutrophil and mast cell infiltration was observed, extracellular granule protein deposition from these cells was minimal except in two specimens. These results demonstrate that eosinophils infiltrate and deposit granule proteins early in the development of BP lesions, that eosinophil-activating cytokines are present in blister fluid, and that eosinophil-selective adhesion molecules are present. These studies strongly support a role for eosinophils in blister formation in BP.

Urticarial dermographism and delayed pressure urticaria are two forms of physical urticaria which are well defined clinically and histologically. Previous studies have shown eosinophil granule protein deposition in urticarial reactions, including chronic urticaria, solar urticaria and delayed pressure urticaria. To evaluate and compare the involvement of granulated inflammatory cells in urticarial dermographism and delayed pressure urticaria, we studied sequential biopsies of induced lesions of urticarial dermographism and delayed pressure urticaria by indirect immunofluorescence, to detect eosinophil granule major basic protein (MBP) and neutrophil granule elastase. Biopsies from dermographic lesions at time 0, 5 min, 15 min, 2 h and 24 h, showed few infiltrating eosinophils, with minimal extracellular MBP deposition, and a few infiltrating neutrophils, with minimal neutrophil elastase deposition, throughout the evolution of the lesions. Sequential biopsies of delayed pressure urticaria at time 0, 20 min, 6, 12 and 24 h, showed eosinophil infiltration with extensive MBP deposition beginning at 20 min, and neutrophil infiltration with variable elastase deposition beginning at 20 min. Control tissue specimens from normal volunteers showed neutrophil infiltration and slight degranulation, but no eosinophil infiltration or degranulation. Comparison of urticarial dermographism with delayed pressure urticaria showed marked differences in the patterns of infiltration. Delayed pressure urticaria, with eosinophil and neutrophil degranulation, was strikingly similar to the IgE-mediated late phase reaction. In contrast, eosinophil and neutrophil involvement in urticarial dermographism was minimal. Considering the extent of eosinophil granule protein deposition and the biological activities of the eosinophil granule proteins, the findings in delayed pressure urticaria point to an important pathophysiological role of eosinophils in the disease.

Previous studies showed that lung and skin mast cells do not contain eosinophil granule major basic protein (MBP). However, MBP has been localized by immunofluorescence to mast cells from a recently established human mast cell line. Analysis of MBP in human mast cell-1 cell lysates by radioimmunoassay showed immunochemical similarity to eosinophil MBP as judged by comparison of dose-response regression lines. Based on these findings and other new information about mast cell heterogeneity, we tested whether mast cells contain MBP. Mast cells were preserved in Carnoy's fixative and were identified by staining with rhodamine-conjugated avidin or for chloroacetate esterase or aminocaproate esterase activity. MBP was localized by immunofluorescence to mast cells in 6 of 7 nasal polyps, 4 of 4 ileal tissue specimens, and 12 of 14 cutaneous mastocytosis specimens. Furthermore, by immunoelectron microscopy MBP was localized to mast cell granules in cutaneous mastocytosis lesions. In contrast, normal skin mast cells preserved in Carnoy's fixative did not contain MBP. After injection of MBP into normal skin and fixation in Carnoy's fluid, mast cells became MBP-positive within 3 minutes, suggesting that endocytosis of MBP by mast cells had occurred. These results suggest that human mast cells in several tissues may sequester toxic eosinophil proteins by endocytosis.

The tendency of lactoferrin to associate with other proteins at low salt concentrations defeated attempts to fractionate the acetate-extracted granule proteins of guinea pig granulocytes by ion-exchange chromatography. This problem was overcome by using copper-chelate chromatography at high salt concentrations to separate lactoferrin from the other granule proteins. Lysozyme and a 'cationic protein' were purified to apparent homogeneity in the same operation. The chromatographic profile of proteins extracted from purified secondary granules differed from that of proteins extracted from total granules chiefly in the absence of a substantial 'cationic protein' peak.

We show how two-dimensional gel electrophoresis can be used to monitor protein components in effluent fractions from a displacement column. A minor protein in human serum, of interest in studies on psoriasis and highly enriched by using carboxymethyldextrans as displacers on DEAE-Sephacel, was easily detected in the effluent fractions with the two-dimensional gel method because its concentration was sufficiently high and there was no interference by the carboxymethyldextrans or salt.