Regulation ofin vitro human lymphocyte blast transformation associated with γ-globulin fraction containing a carbohydrate component

Abstract

Carbohydrate-enriched components were isolated from human serum y-globulin by chelation of associated transition metals. Similar to the initial y-globulin preparation, these components stimulate in vitro blast transformation of human lymphocytes from healthy donors and inhibit subsequent phytomitogen-induced lymphocyte blast transformation. Key words: y-globulin; carbohydrate component, lymphocyte blast transformationThe ability of y-globulin to maintain a definite level of human lymphocyte blast transformation (LBT) in vitro [3] is known to depend on the expression of Fc Yreceptors in cell membrane [8,12]. The effect of LBT stimulation was shown to correlate with the presence of protein aggregates in the y-globulin preparation [3].The effector functions of y-globulin also depend on the presence of carbohydrate-containing fraction bound to polypeptide chains via non-covalent bonds [1]. Carbohydrate component (CC) is associated with y-globulin molecule via metal ions of alternating valency [1]. These cations interact with specific sites of y-globulin molecule [ 13,14] and stimulate aggregation by forming bridges between protein molecules at a certain metal/IgG ratio. A positive correlation between IgG affinity to transition metal and the effect of protein on spontaneous LBT was found [2].y-Globulin aggregation is accompanied by significant changes in its conformation stability and enhances the release or exposure of components not bound covalently to polypeptide chains. MATERIALS AND METHODSMononuclear cells (MNC) were isolated from peripheral venous blood of 4 healthy men aged 20-46. The cells were separated in Ficoll-Verografin (d = 1.077 g/cm 3) one-step density gradient and MNC were harvested from the interface layer. After repeated washout with medium 199 MNC were resuspended in complete nutrient medium (CNM) consisting of RPMI-1640 (Flow) supplemented with 12% fetal calf serum (Hamaleya Institute of Epidemiology and Microbiology), 2 mM glutamine and 40 ~tg/ml gentamicin (Pharmachim) to a concentration of 2• cells/ml. To initiate spontaneous LBT, 0.1 ml MNC suspension was applied to round-bottom 96-well plates, 0.1 ml CNM and 10 ~tCi/ ml 3H-thymidine were added to each well and the plates were incubated for 18 h at 37~ in humidified atmosphere containing 5% CO 2. LBT was induced by 2 ~tg/ml phytohemagglutinin M (PHA, Serva) or 1 ~tg/ml con-0007 -4888/99/0006-0610522.00 9

Role of γ-globulin-associated fraction containing carbohydrate component inin vitro regulation of human natural killer activity

“…Our findings and previously reported data [4] indicate that LBT and NCT observed in vitro in the presence of human y-globulin [5,6] are due to the release of carbohydrate-rich components inhibiting NK cells and stimulating lymphocyte proliferation, This process results from intra-and intermolecular cross-linking in y-globulin molecule, which improves protein stability in diluted water solutions. Comparative analysis using 4x10 -2 4x10 -3 4x10 -~ a special algorithm showed that the effective dose of carbohydrate-rich components (0.1 lag/ml) inhibiting in vitro cytotoxic functions of human mononuclear cells ( Fig.…”

“…Comparative analysis using 4x10 -2 4x10 -3 4x10 -~ a special algorithm showed that the effective dose of carbohydrate-rich components (0.1 lag/ml) inhibiting in vitro cytotoxic functions of human mononuclear cells ( Fig. 1) corresponded to concentrations of 60 ng/ ml carbohydrate component and 70 ng/ml y-globulin stimulating lymphocyte proliferation [4]. Therefore, the revealed effects are mediated by y-globulin-associated fractions released during the action of chelating agents not only under rigorous [1,4], but also under neutral conditions.…”

“…Carbohydrate component-containing fractions obtained from aggregated and aggregate-free y-globulins after chelation of transition metals [1,4] were used in experiments on human cells (pH was adjusted to 7.4). These fractions were added to the suspension of mononuclear cells immediately before the test for cytotoxicity and remained in the reaction mixture to the end of incubation of mononuclears and target cells [6].…”

“…Stimulation of lymphocyte proliferation in the presence ofT-globulin inversely correlates with natural killer (NK) cytotoxicity in vitro [6]. The fraction released from y-globulin after chelation of transition metals and stimulating blast-transformation of human lymphocytes (BTL) [4], probably suppresses function of natural cytotoxicity (NCT) effectors.…”

“…y-Globulin molecule contains carbohydrate-rich components exhibiting heparin-like activity [1], inducing proliferation of human lymphocytes in vitro [4], and responsible for polyclonal activation of immunocompetent cells by aggregated proteins [4,6].…”