Immunology 1979 37 555

Cell electrophoretic studies on mouse lymphocytes treated with concanavalin A: possible existence of two types of receptors on cell surface

K. B. SAINIS, A. N. BHISEY*, K. SUNDARAM & G. P. PHONDKE Bio-Medical Group, Bhabha Atomic Research Centre, Modular Laboratories, Trombay, Bombay, India and *Biology Division, Cancer Research Institute, Tata Memorial Centre, Parel, Bombay, India

Received 21 September 1978; acceptedfor publication 17 January 1979

Summary. Concanavalin A (Con A) induced biphasic changes in the electrophoretic mobility (EPM) of splenic lymphocytes of AKR mice. At low concentrations of Con A (2-12 5 pg/ml) there was a significant increase in the EPM which could be attributed to the redistribution and endocytosis of Con A receptors. At higher concentrations (> 15-0 pg/ml) a reduction in EPM below that of untreated cells was possibly due to the binding of excess Con A after redistribution. The biphasic profile of EPM was observed only in respect of Con A-treated T lymphocytes. The EPM of B lymphocytes, despite binding of Con A, remained unaltered at all concentrations of Con A. On the basis of these data, the existence of two types of Con A receptors on the cell surface is proposed and their status vis-a-vis the redistribution is discussed. INTRODUCTION Concanavalin A (Con A) induces redistribution of its receptors on mammalian cell surfaces (De Petris, Raff & Malluci, 1973; Nicholson, 1974; Yahara & Edelman, 1975; De Petris, 1975). This process involves the formation of clusters, patches and caps and a part of Correspondence: Dr. G. P. Phondke, Bio-Medical Group, Bhabha Atomic Research Centre, Trombay, Bombay 400 085, India.

0019-2805/79/0700-0555$02.00

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1979 Blackwell Scientific Publications

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the membrane may be subsequently endocytosed or internalized. Con A has been shown to induce modifications of cell-surface charge density (Yamada & Yamada, 1973; Wioland, 1974; Wioland, Donner & Neauport-Sautes, 1976). Yamada & Yamada (1973) observed that the electrophoretic mobility (EPM) of rat ascites hepatocarcinoma cells increased at concentrations of Con A between 5 and 40 yg/ml, but decreased below that of untreated cells at higher concentrations (100-300 yg/ml). The authors attributed the change in surface charge density to the binding of Con A and suggested that surface distribution of glycoproteins might have been altered. The conditions of treatment, however, favoured redistribution of Con A receptors. It was, therefore, not clear whether binding of Con A alone or the induced redistribution was responsible for the altered EPM. Wioland (1974) reported that the binding of Con A under non-capping conditions (50) did not alter the EPM of rat thymocytes. Under capping conditions, however, the mean EPM of mouse thymocytes increased with increasing concentration of Con A, reached a maximum at 10 Mg/ml and decreased to a value close to that of untreated cells at higher concentrations (Wioland et al., 1976). While the initial increase in the surface charge density was attributed to capping and endocytosis, the subsequent decrease has been hypothetically attributed to innate inhibition of the same process (Wioland et al., 1976).

K. B. Sainis et al.

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The striking difference between rat tumour cells and murine thymocytes vis-a-vis their interaction with Con A has been that while the mean EPM of the former cell type decreased below the control value at higher concentrations of Con A, the latter cell type did not exhibit this phenomenon. Whereas these differences may emanate from different cell sources, the role of Con A at high concentrations in the process of receptor redistribution, and hence the change in EPM, required a thorough investigation. The interaction of Con A with splenic lymphocytes of AKR mice was, therefore, studied to elucidate the mechanism of action of Con A at high concentrations.

of Mount Sinai Medical Centre, Milwaukee, Wisconsin, U.S.A. A stock solution of 0 2 M in saline was used.

Cell electrophoresis Electrophoretic measurements were made in a cylindrical microelectrophoresis apparatus described by Bangham, Heard, Flemens & Seaman (1958). Details of the method are described elsewhere (Sundaram, Phondke & Ambrose, 1967; Phondke & Sundaram, 1971). The EPM of human RBC was always measured to check the performance of the instrument. Measurements of EPM were made either at 5 or 250. The values at 5° were corrected for changes in viscosity and reported as at 25°. In addition the absolute values of EPM at 5° are also reported.

MATERIALS AND METHODS Cells Spleen cells from healthy AKR mice of either sex were suspended in standard saline pH 7-2 (0-146 M NaCl + 0-01 M KCl). RBC were removed by treatment with ammonium chloride (Yata, Desgranges, Tachibana & de The, 1973). Viability was measured by trypan blue dye-exclusion and cell density adjusted to

107 cells/ml. Lymphocyte sub-populations The constituent sub-populations of lymphocytes were separated by differential adhesion to nylon wool according to the method of Julius, Simpson & Herzenberg (1973) as modified by Chollet, Chasagne, Sauvezie, Codegnat & Plagne (1976). The nylon nonadherent cells were T-cells as tested by cytotoxicity with anti-Thy 1 1 (C3H anti-O-AKR) antiserum. About 90% of the adherent sub-population were characterized as B cells as they were found to be Thy 1 1 negative and Ig positive. Con A Pure, thrice crystallized Con A was a gift from Dr J. A. Forrester of Chester Beatty Research Institute, London, U.K. A stock solution of Con A (100 pg/ml) was stored in small aliquots at -20°. Sodium azide (NaN3) NaN3 (Sigma), a metabolic inhibitor (Loor, 1973) was used at a final concentration of 2 mM.

a-Methyl glucopyranoside (aMG) a MG (Schwarz-Mann, U.S.A.), a competitive inhibitor of Con A binding was a gift from Prof. R. F. Barth

Fluorescence microscopy Fluorescein labelled Con A (Fl-Con A) was prepared as described by Sainis, Bhisey, Sundaram & Phondke (1979). 107 cells and 100 pg of Fl-Con A in 1 0 ml PBS (phosphate-buffered saline, pH 7-2, without Ca2+ or Mg2+) were incubated at 50 for 2 min. After the incubation cells were washed thrice in cold PBS, reincubated in 1-0 ml PBS at room temperature (220) for 30 min, washed and examined under a fluorescence microscope using an HBO 200 source. RESULTS

Electrophoretic mobilities of Con A-treated lymphocytes The mean EPM of untreated splenic lymphocytes was found to be -1 19 pm sl V- cm. When incubated with Con A at 22 or 37° for 1 h (hereinafter referred to as 'capping conditions'), the mean EPM of lymphocytes showed a biphasic profile (Fig. 1). There was no significant difference in the EPM profiles of cells treated at 220 and 370. The mean EPM of splenic lymphocytes was significantly increased from the control value (P < 0 05) when incubated with 2 pg/ml Con A, remained at a plateau level up to 12 5 pg/ml and decreased significantly (P

Cell electrophoretic studies on mouse lymphocytes treated with concanavalin A: possible existence of two types of receptors on cell surface.

Immunology 1979 37 555 Cell electrophoretic studies on mouse lymphocytes treated with concanavalin A: possible existence of two types of receptors on...
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