Characterization of macrophage proteins bearing the functional leukotriene D4-binding site of an anti-cysteinylleukotriene monoclonal antibody.
Protein conformations of the putative cysteinylleukotriene (LT) receptor of macrophages were characterized using anti-idiotypic IgG (AIAb) against an anti-LT monoclonal Ab (LTmAb). The AIAb nature of two rabbit antisera were demonstrated with titers of up to 1:1000 against F(ab')2 from the LTmAb (in an enzyme-linked immunoassay) which also inhibit LTD4 binding to the LTmAb (in a radioimmunoassay), whereas non-immunized rabbit serum was not reactive. The specific reactivity of Fc-purified AIAb towards LTmAb was measured by two fractions obtained after passage over columns of Sepharose either coupled with LTmAb (fraction A, representing immunoglobulins not absorbed to LTmAb) or coupled with homologous immunoglobins (fraction B, representing immunoglobulins not absorbed to homologous IgG). The difference in immunoreactivity between both fractions showed that fraction B contains AIAb against a LT-recognizing domain of the LTmAb (in enzyme-linked immunoassays coated with LTmAb and homologous IgG) and AIAb against the functional LT-binding site of LTmAb (in radioimmunoassay). Using the antisera, Western-blot analysis with peritoneal cell proteins detected signals at 236, 198, 118, 99, 75, 25 and 18 kDa. Dithiothreitol-reduced proteins were detected at 25 kDa and 18 kDa. In general, this suggested recognition of a 236-kDa oligomeric protein composed of subunits with molecular masses of 25 kDa and 18 kDa, including intramolecular disulfide bridges all bearing an epitope similar to the LTmAb. From these conformations, an overlay assay with [3H]LTD4 favoured a 75-kDa protein. Immunohistochemical analysis demonstrated that the recognized proteins may be located at cell membranes, because (a) in an ELISA, enriched plasma membrane preparations from peritoneal cells showed a threefold increase in reactivity to the AIAb, compared to the original cell homogenate; (b) after Western-blot analysis, the membrane-enriched protein fraction exhibited stronger protein signals than the microsomal fraction and the original cell homogenate; (c) regions of AIAb binding on the surface of cultured mouse peritoneal macrophages were detected by indirect immunofluorescence. Taken together, this study demonstrated AIAb binding to macrophage membrane-associated proteins bearing the LTD4-binding site of LTmAb, which may include identification of the putative LT receptor.
van Hilten
J A
JA
Department of Experimental and Clinical Pharmacology, University of Erlangen-Nürnberg, Germany.
Brune
K
K
Bickel
D
D
Mollenhauer
J
J
D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000276:immunology; D001665:Binding Sites; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D005260:Female; D017999:Leukotriene E4; D008264:Macrophages / Q000276:immunology*; D051379:Mice; D010529:Peritoneal Cavity / Q000166:cytology; D011487:Protein Conformation; D011971:Receptors, Immunologic / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D018077:Receptors, Leukotriene; D013189:SRS-A / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism*
D000888:Antibodies, Anti-Idiotypic; D000911:Antibodies, Monoclonal; D000939:Epitopes; D011971:Receptors, Immunologic; D018077:Receptors, Leukotriene; D013189:SRS-A; D017999:Leukotriene E4