Human B-cell TNF-beta microheterogeneity.
The production of TNF-alpha and TNF-beta by human B-cell lines was studied at both the molecular and biological levels. The 24 B-cell lines studied included EBV+ cell lines (n = 13), EBV- cell lines (n = 8), and AIDS-associated B-cell lines (AABCL) (n = 3) which are EBV+/HIV-. Whereas radioimmunoprecipitation using TNF-alpha antisera detected 17-kDa TNF-alpha as expected, similar studies with anti-TNF-beta antisera revealed TNF-beta microheterogeneity. In the AABCL three bands with approximate MW of 26, 24, and 22 kDa were detected under reducing conditions, and in the non-AABCL, two bands only with 26 and 22 kDa were observed. To determine whether the size heterogeneity of TNF-beta is due to glycosylation, TNF-beta deglycosylation studies were done in two AABCL (PA682BM-2, PA682PE-1) and one non-AABCL (IM-1178). As control, the normal lymphoblastoid B-cell line RPMI-1788, which is known to secrete TNF-beta with MW 25 and 20 kDa, has been used. Deglycosylation studies using N-glycanase + neuraminidase + O-glycanase reduced the various bands in all cell lines to one band with 18.6 kDa, which is compatible with the TNF-beta backbone. In attempt to determine whether the differential glycosylation of TNF has any functional significance, all 24 cell lines were studied for TNF secretion and for TNF neutralization by monoclonal antibodies and polyclonal antibodies to TNF-alpha and TNF-beta. Constitutive secretion of TNF-alpha and TNF-beta has been detected only in the three AABCL. Following activation with the tumor promoter teleocidin, the secretion of both TNFs has been triggered in 2/8 EBV- cell lines and in 8/13 EBV+ non-AABCL. Using rabbit polyclonal antibodies to human TNF-alpha and to human TNF-beta, only little if any neutralization of these TNFs has been shown. Our data suggest that the differences in glycosylation of B-cell-derived TNFs may account for the incomplete neutralization, and may influence the cytotoxic biological activity of this lymphokine.
Benjamin
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D
Lymphokine Research Laboratory, Arthur G. James Cancer Hospital, Ohio State University, Columbus 43210.
Kofler
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G
Tschachler
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E
D000581:Amidohydrolases / Q000378:metabolism; D000906:Antibodies / Q000276:immunology; D001402:B-Lymphocytes / Q000276:immunology*; D002460:Cell Line; D002461:Cell Line, Transformed; D006031:Glycosylation; D006801:Humans; D008233:Lymphotoxin-alpha / Q000096:biosynthesis* / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D008970:Molecular Weight; D009439:Neuraminidase / Q000378:metabolism; D009500:Neutralization Tests; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011233:Precipitin Tests; D011863:Radioimmunoassay; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis* / Q000737:chemistry / Q000276:immunology / Q000378:metabolism
D000906:Antibodies; D008233:Lymphotoxin-alpha; D014409:Tumor Necrosis Factor-alpha; D009439:Neuraminidase; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D013755:Tetradecanoylphorbol Acetate