Demonstration that monocytes rather than lymphocytes are the insulin-binding cells in preparations of humah peripheral blood mononuclear leukocytes: implications for studies of insulin-resistant states in man.

Abstract

Insulin receptors have been demonstrated on mononuclear leukocytes prepared by centrifugation of buffy coats from normal blood donors on Ficoll-Hypaque gradients. The cell type that specifically binds insulin in this mixture of lymphocytes and monocytes has never been clearly identified, although it was assumed to be the lymphocyte since this cell constitutes about 80%/c of the population. In the present studies, insulin-binding assays were performed on the mononuclear leukocyte preparation before and 'after selective depletion or enrichment for monocytes using glass wool or Sephadex G-10 adherence columns. The amount of 'l25-labeled insulin specifically bound correlated significantly with the number of monocytes but not with the number of B or T lymphocytes.Approximately 90% of the specific insulin binding of this preparation could be accounted for by its content of monocytes. The amount of binding was unaffected by phagocytosis of latex particles or by metabolic inhibitors added to prevent endocytosis. Autoradiograms made on smears of whole peripheral blood and mononuclear leukocytes demonstrated that all of the cells that bound '251-labeled insulin were large mononuclear cells, 85-90%7 of which could be identified as monocytes by morphological criteria or by the functional criterion of latex particle ingestion. Since insulin receptor concentration may be altered in disease states in man, it is essential, when using this cell population for detecting such changes, to quantitate the number of monocytes in the preparation so that the insulin-binding data can be appropriately interpreted.Previous studies by Gavin, Archer, and their coworkers (1)(2)(3)(4) demonstrated that bone marrow-derived (B) lymphoblastoid cell lines maintained in continuous culture and peripheral blood mononuclear leukocytes from normal blood donors purified by Ficoll-Hypaque gradient centrifugation contain receptors for insulin. In the latter case, it was assumed that the binding was to receptors on lymphocytes, which comprise the majority of this population. However, a subsequent study by Krug et al. (5) failed to detect any significant insulin binding to peripheral blood lymphocytes purified by passage over tightly packed nylon wool. Since this separation procedure has recently been shown to remove B lymphocytes (6-9), it was Abbreviations: B and T lymphocytes, bone marrow-derived and thymus-derived lymphocytes, respectively.

Impaired Intracellular Calcium Mobilization and NFATc1 Availability in Tolerant Anti-Insulin B Cells

“…However, we do not detect differences in 125Tg B cell responses when either hormonally active or inactive insulin is used (not shown). In addition, previous studies have shown that mAb125 does not recognize InsR-bound insulin (52) and that resting T and B cells do not express InsR (53). However, both BCR and InsR traffic in clathrin vesicles; thus, it remains possible that some interactions in the endosomal pathway generate signals during intracellular processing.…”

Immune responses of diabetic animals

“…Insulin receptors have been demonstrated on alloimmune T cells [23] and circulating monocytes [24]. In addition, evidence has been presented that insulin can augment lymphocyte-mediated cytotoxicity [25] and modulate Fc-mediated phagocytosis by monocytes [26] and macrophages [27].…”

Insulin antibodies prevent insulin-receptor interactions

“…They were then suspended in 100 mmol/ 1 Hepes buffer containing i g/100 ml bovine serum albumin (BSA) (pH 7.8). The mononuclear cell preparation contained 85% lymphocytes and 15% monocytes [18]; since it has been demonstrated that insulin binding depends primarily upon monocytes [21,22] 0012-186X/80/0019/0118/$01.00 Relationship between percent labelled insulin combined to antibody and subsequent binding of labelled insulin (0.2 ng/ml) to placenta membranes (150 ~xg/ml). Maximal receptor binding (i. e. 100%) was evaluated using labelled insulin pre-incubated for 48 h at 4 ~ in sodium phosphate buffer and was 31.2% of total radioactivity.…”