Binding of histamine and histamine analogs to lymphocyte subsets analyzed by flow cytometry.
The binding of histamine, 4-methylhistamine (a histamine type 2 receptor agonist), cimetidine (a histamine type 2 receptor antagonist), and telemethylhistamine (an inactive analog) to human peripheral blood mononuclear cell subsets was investigated by flow cytometry by using conjugates of these ligands coupled to fluorescein-labeled human serum albumin. Our results indicate that binding of fluorescent protein conjugates of histamine and its analogs does not selectively identify a lymphocyte subset(s) that mediates the immunomodulatory effects of histaminergic ligands. Conjugates with both low (2.5 to 2.8:1) and high (28 to 57:1) ligand to protein coupling ratios were used. No binding above background could be detected for the low mole ratio reagents. The high mole ratio reagents were bound by 95 to 99% of all lymphocytes when used at ligand concentrations of 50 microM or greater. At lower ligand concentrations, the number of lymphocytes exceeding a set fluorescence threshold was decreased, but fluorescence distributions remained unimodal at all concentrations used (1 to 500 microM). Monocytes also bound the high mole ratio reagents and gave rise to a second high-intensity peak in the fluorescence distribution unless they were excluded by other means. Levels of conjugate binding detected by flow cytometry did not parallel ligand potencies at classical histamine type 2 receptors; at equivalent ligand concentrations, approximately equal amounts of histamine or 4-methylhistamine conjugate were bound per lymphocyte, and only 30% less telemethylhistamine conjugate was bound. Competition with free ligands (10(2)- to 10(4)-fold excess histamine, 4-methylhistamine, cimetidine, or telemethylhistamine) did not significantly decrease the level of binding observed for the high mole ratio reagents at bound ligand concentrations of 1 to 25 microM. Dual staining with fluorescein-labeled conjugate and phycoerythrin-labeled monoclonal antibodies Leu-3ab (anti-helper T), Leu-2a (anti-suppressor T), Leu-M3 (anti-monocyte), or anti-HLA-DR (B cells and monocytes) was also carried out. The extent of conjugate binding to helper and suppressor cells was identical for each of the ligands used, but higher levels of conjugate binding were seen for monocytes and B cells than for T cells in every case. Our data do not exclude the possibility of enhanced conjugate binding to small numbers of activated (HLA-DR positive) T cells that might be involved in mediation of histamine effects.(ABSTRACT TRUNCATED AT 400 WORDS)
K
K
Muirhead
P
P
Bender
N
N
Hanna
G
G
Poste
D001665:Binding Sites / Q000187:drug effects; D001667:Binding, Competitive; D003080:Cold Temperature; D005434:Flow Cytometry / Q000379:methods; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives*; D005452:Fluoresceins / Q000378:metabolism; D006632:Histamine / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008214:Lymphocytes / Q000145:classification / Q000378:metabolism*; D008761:Methylhistamines / Q000378:metabolism; D012710:Serum Albumin, Bovine / Q000378:metabolism; D013194:Staining and Labeling
D005452:Fluoresceins; D008761:Methylhistamines; C039217:fluorescein isothiocyanate bovine serum albumin; D012710:Serum Albumin, Bovine; C025566:4-methylhistamine; D006632:Histamine; D016650:Fluorescein-5-isothiocyanate; C020404:tele-methylhistamine