HAPTOGLOBIN PHENOTYPING BY NEWLY DEVELOPED MONOCLONAL-ANTIBODIES - DEMONSTRATION OF HAPTOGLOBIN UPTAKE INTO PERIPHERAL-BLOOD NEUTROPHILS AND MONOCYTES

We have generated two IgG murine mAbs that recognize native human hapt oglobin (Hp), These mAbs, 3A8 and 482, efficiently bind to the Hp comp lex regardless of the serum donor's phenotype, The specificity of mAb 3A8 was confirmed by immunoaffinity purification of 3A8-binding materi al from human serum and subsequent N-terminal amino acid sequencing of the invariant 40-kDa chain, mAb 3A8 and 482 were also reactive with c ell-associated Hp when studied by immunocytochemistry, When human peri pheral blood leukocytes were tested, 90% of granulocytes and a lesser (and variable) fraction of monocytes displayed an intense intracytopla smatic granular staining, This was confirmed by flow cytometric analys is of permeabilized leukocytes and by demonstrating the presence of Hp (of the expected Hp serum phenotype) in extracts of washed granulocyt es by immunoblotting, Leukocytes obtained from a Hp phenotype 2-2 dono r, incubated in culture medium supplemented with 10% serum from a dono r possessing the Hp 1-1 phenotype, contained both Hp phenotypes when a nalyzed by immunoblotting after a 6-h incubation period, In addition, Hp was actively exocytosed by granulocytes following their exposure to Candida albicans, These observations suggest that exogenous Hp is con centrated within granulocytes and not synthesized de novo and is, in t urn, exocytosed following neutrophil activation, Northern blotting ana lysis is consistent with the lack of haptoglobin gene transcription in granulocytes, These findings together with the earlier observations t hat Hp modulates granulocyte activity suggest that Hp levels may be en hanced locally at sites of inflammation to modulate granulocyte activi ty.