Identification of basophils by a mAb directed against pro-major basic protein 1.

BACKGROUND: Basophils possess characteristics of both mast cells and eosinophils, and all 3 cell types often are found together, particularly during allergic reactions. A mAb (J175-7D4) generated against the recombinant pro-form of human eosinophil granule major basic protein 1 (rproMBP1) appeared to stain only basophils in tissue specimens. OBJECTIVE: We investigated J175-7D4 to characterize its specificity for basophils. METHODS: Fluid-phase immunoprecipitation, Western blotting, and immunocytochemistry and immunohistochemistry were used to establish the specificity of J175-7D4. RESULTS: First, J175-7D4 binds to various glycosylated and proteolytically processed forms of rproMBP1, but not to major basic protein. Second, cells transfected with the rproMBP1 gene and human placental tissue (known to express the pro-form of major basic protein 1 [proMBP1]) stain specifically with J175-7D4. In contrast, although mature eosinophils contain substantial major basic protein, they lack proMBP1 and do not stain. Neutrophils, lymphocytes, monocytes, and skin mast cells also are not stained. However, blood basophils are stained by J175-7D4, anti-IgE, Wright-Giemsa (metachromatically), and a previously characterized basophil-specific mAb, 2D7. Finally, formaldehyde-fixed, paraffin-embedded basophils are identically detected by J175-7D4 and 2D7, and J175-7D4 also recognizes putative basophils in formaldehyde-fixed, paraffin-embedded tissue specimens from inflammatory dermatoses, such as atopic dermatitis and delayed pressure urticaria. CONCLUSION: The J175-7D4 mAb recognizes proMBP1 as a novel marker for human basophils. J175-7D4 should prove useful for characterizing basophil involvement in human health and disease.

Interactions of eosinophil granule proteins with skin: limits of detection, persistence, and vasopermeabilization.

BACKGROUND: Eosinophil granule proteins, including eosinophil cationic protein (ECP), eosinophil-derived neurotoxin (EDN), eosinophil peroxidase (EPO), and major basic protein (MBP), are prominently deposited in skin in several cutaneous disorders and likely contribute to disease pathology. OBJECTIVE: We sought to determine the limit of detection, persistence, and vasopermeabilization activity of the eosinophil granule proteins in skin. METHODS: The eosinophil granule proteins were injected intradermally. Their minimum detectable concentrations in human surgical waste skin and their persistence in guinea pig skin were determined by indirect immunofluorescence. Vasopermeabilization activity in the guinea pig without and with H1 antihistamine (pyrilamine maleate) pretreatment was assessed by extrusion of Evans blue dye-treated plasma. RESULTS: The lowest detectable cutaneous concentrations were 0.05 micromol/L EPO, 0.1 micromol/L MBP, 0.25 micromol/L ECP, and 1 micromol/L EDN. Granule proteins persisted in guinea pig skin in vivo for 1 week (EPO), 2 weeks (ECP), 2.5 weeks (EDN), and 6 weeks (MBP). Each of the eosinophil granule proteins increased cutaneous vasopermeability in a concentration-dependent manner. The potency of vasopermeabilization induced by each granule protein was comparable with that of histamine. Pyrilamine maleate pretreatment of guinea pigs did not alter increased vasopermeability induced by ECP and EDN but significantly inhibited that induced by EPO and MBP. CONCLUSIONS: Micromolar concentrations of eosinophil granule proteins are often deposited in skin in eosinophil-associated cutaneous disorders such as atopic dermatitis. These pathophysiologically relevant concentrations of eosinophil granule proteins cause increased cutaneous vasopermeability (both by means of histamine-independent and histamine-dependent mechanisms) and might alter cutaneous function for days to weeks.

Evidence for pathogenic involvementof eosinophils and neutrophilsin Churg-Strauss syndrome.

BACKGROUND: Churg-Strauss syndrome (CSS) is a multi-organ disease with tissue and blood eosinophilia. OBJECTIVE: Our aim was to study eosinophil and neutrophil involvement in CSS. METHODS: Eight lesional skin biopsy specimens from 6 patients with CSS and serum and blister fluid from one patient were tested for eosinophil and neutrophil activity. Indirect immunofluorescence on skin specimens used antibodies to eosinophil granule major basic protein (MBP), eosinophil-derived neurotoxin (EDN), and neutrophil elastase (NE). Serum and blister fluid specimens were analyzed for granule protein levels and for eosinophil-activating cytokines. RESULTS: Indirect immunofluorescence showed prominent cellular and extracellular staining for EDN in skin biopsy specimens; MBP staining was less extensive. Five biopsy specimens showed marked cellular NE staining; 4 showed prominent extracellular NE. Serum and blister fluid specimens contained elevated MBP, EDN, and interleukin 5 levels and enhanced eosinophil survival in culture. Granulocyte-macrophage colony-stimulating factor and interleukin 5 were detected in blister fluid. Blister fluid contained more NE than normal serum. CONCLUSIONS: Both eosinophils and neutrophils likely participate in skin lesion development in CSS.