Eotaxin is a potent and selective CC chemokine for eosinophils and basophils. We established several monoclonal antibodies (Mabs) allowing the neutralization and measurement of human eotaxin. Using the Mabs as probes, we demonstrated that normal eosinophils contained intracellular granule-associated eotaxin. Quantification of cell-associated eotaxin in different leukocyte subsets revealed that it was principally expressed in eosinophils. Finally, we showed that normal eosinophils released eotaxin upon stimulation with either of two secretagogues, C5a or ionomycin. These findings raise the possibility that eosinophil-derived eotaxin contributes to the local accumulation of eosinophils at the site of inflammation.z 1998 Federation of European Biochemical Societies.

The cytoplasmic plaque of the spot desmosome or macula adhaerens mediates the attachment of bundles of intermediate filaments to the plasma membrane. We have isolated from a bovine epidermal desmosome preparation a fraction that is highly enriched in the non-glycosylated desmosomal proteins. Plastic-embedded and thin-sectioned high-speed pellets of this fraction reveal closely packed filaments that resemble plaque regions of the low pH whole desmosome preparation from which they are derived. NaDodSO4/polyacrylamide gel electrophoresis reveals four major, non-glycosylated proteins of 240, 210, 81, and 77 kDa. In agreement with a previous study, we find the 240-and 210-kDa proteins (desmoplakins I and II) to be closely related, whereas the 81-and 77-kDa proteins are unique. This is shown both immunologically and by one-dimensional proteolytic peptide mapping. Monospecific, polyclonal rabbit antibodies were prepared against the 81-kDa protein and used, in conjunction with protein A-complexed colloidal gold particles (PAG), to immunolocalize this antigen on ultrathin sections of bovine muzzle epidermis. On antibody-labeled sections, PAG particles were associated principally with the desmosomal cytoplasmic plaque. Sections exposed to preimmune serum showed little or no labeling. We conclude that the 81-kDa protein, like the 240/210-kDa protein family, is one of the major components of the desmosomal plaque. We designate it as "desmoplakin III." The location of the 77-kDa protein remains to be definitively established.

In a previous post-embedding hnmunogold elecaon microscopic (EM) studies, localization of d o u s desmosomal antigens was possible at high but not at low magnification. We developed a method for simultaneous demonstration of epidermal d e s " a l antigens at both low-and high-powet EM magnificltions by a method based on a y d i t i o n and acetone ayosubstitution and the use of a 1-nm gold probe with silver enhancement. Ultra-thin sections of Lowiayl K l l M were incubated with primary antibodies against desmoplakin, desmmllin, or desmoglein, followed by 1-nm goldanjugated seoondary antibody. Silver enhancement for 12 min provided the ideal labeling size for low-power visualization, whereas silver enhancement for 4-6 min was ideal

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ABSTRACT. Here we report a new method of isolating epidermal desmosomes from Xenopus laevis, and a major constituent of desmosomes designated as Xenopus desmogleins (XDsg). Isolation of desmosomes from Xenopus laevis epidermis was carried out by a two step-incubation with different concentrations of NP-40. After discontinuous sucrose gradient centrifugation at 30,000 g for 60 min, a pure desmosomal fraction was obtained at 30%/40% interface. In the SDS-PAGE of isolated desmosomes, at least 12 bands (XDB1 to XDB12) were observed over a 75 kD region. Among them, three bands (XDB3, XDB7, XDB8; estimated MW 175, 124, and 112 kD respectively) were recognized as glycoproteins based on ConA binding. Monospecific polyclonal antibody against XDB3 cross-reacted with bovine Dsgs and vis-a-vis anti-bovine Dsgs with XDB3. By contrast, monospecific antibody against bovine Dsc a/b did not cross-react with either XDB7 or XDB8. Heterogeneous molecular constituents of desmosomal adhesion molecule, which have been observed among different bovine tissues, were confirmed in a phylogenetically different animal, Xenopus laevis. Combined results with other evidence could suggest an alternative system for desmosome-mediated cell adhesion.