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 selective eosinophil infiltration preferentially in the dermis, we speculated that dermal fibroblasts might represent a potential cellular source of eosinophil-selective attractants. Cultivated dermal fibroblasts treated with tumor necrosis factor alpha secreted, not before day 3 of stimulation, eosinophil-specific chemotactic activity. Purification of this activity revealed a heparin-binding protein with an apparent molecular mass of 13 kDa in SDS/polyacrylamide gel electrophoresis. Peptide mapping with subsequent amino acid sequence analyses revealed it to be human eotaxin. Natural eotaxin preparations contain 50% N-terminally truncated 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 natural 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. Neither neutrophils, monocytes or lymphocytes responded towards natural eotaxin preparations indicating that N-terminal truncation and O-glycosylation did not affect the cell-specificity of chemotactic activity. Treatment of eosinophils with natural eotaxin desensitizes chemotactic responses towards eotaxin, regulated an normal T-lymphocyte expressed and secreted (RANTES) and monocyte chemotactic protein 3 (MCP-3), whereas RANTES and MCP-3 were unable to desensitize natural eotaxin-dependent responses.
Noso
N
N
Department of Dermatology, University of Kiel, Germany.
Bartels
J
J
Mallet
A I
AI
Mochizuki
M
M
Christophers
E
E
Schröder
J M
JM
D000595:Amino Acid Sequence; D002478:Cells, Cultured; D054413:Chemokine CCL11; D018946:Chemokine CCL5 / Q000494:pharmacology; D054410:Chemokine CCL7; D019742:Chemokines, CC; D002631:Chemotactic Factors, Eosinophil / Q000096:biosynthesis* / Q000737:chemistry / Q000494:pharmacology; D002634:Chemotaxis, Leukocyte / Q000187:drug effects; D016207:Cytokines / Q000096:biosynthesis* / Q000737:chemistry / Q000494:pharmacology; D004804:Eosinophils / Q000187:drug effects / Q000502:physiology; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D018945:Monocyte Chemoattractant Proteins / Q000494:pharmacology; D011994:Recombinant Proteins / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D012867:Skin / Q000187:drug effects* / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
C515456:CCL11 protein, human; C515450:CCL7 protein, human; D054413:Chemokine CCL11; D018946:Chemokine CCL5; D054410:Chemokine CCL7; D019742:Chemokines, CC; D002631:Chemotactic Factors, Eosinophil; D016207:Cytokines; D018945:Monocyte Chemoattractant Proteins; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha