DELAYED PRODUCTION OF BIOLOGICALLY-ACTIVE O-GLYCOSYLATED FORMS OF HUMAN EOTAXIN BY TUMOR-NECROSIS-FACTOR-ALPHA-STIMULATED DERMAL FIBROBLASTS

Since a number of inflammatory skin diseases are characterized by sele ctive eosinophil infiltration preferentially in the dermis, we specula ted that dermal fibroblasts might represent a potential cellular sourc e of eosinophil-selective attractants. Cultivated dermal fibroblasts t reated with tumor necrosis factor a secreted, not before day 3 of stim ulation, eosinophil-specific chemotactic activity. Purification of thi s activity revealed a heparin-binding protein with an apparent molecul ar mass of 13 kDa in SDS/polyacrylamide gel electrophoresis. Peptide m apping with subsequent amino acid sequence analyses revealed it to be human eotaxin. Natural eotaxin preparations contain 50% N-terminally t runcated forms missing two or three amino acids. It is O-glycosylated at Thr71, resulting in at least two sialylated O-glycosylated variants . Electrospray ionization mass spectrometric analyses revealed the nat ural eotaxin preparation to be heterogeneous with principal masses of 9033 Da and 9317 Da. Natural eotaxin stimulated eosinophil chemotaxis with identical potency and efficacy as recombinant human eotaxin. Neit her neutrophils, monocytes or lymphocytes responded towards natural eo taxin preparations indicating that N-terminal truncation and O-glycosy lation did not affect the cell-specificity of chemotactic activity. Tr eatment of eosinophils with natural eotaxin desensitizes chemotactic r esponses towards eotaxin, regulated an normal T-lymphocyte expressed a nd secreted (RANTES) and monocyte chemotactic protein 3 (MCP3). wherea s RANTES and MCP-3 were unable to desensitize natural eotaxin-dependen t responses.