646 A. Novogrodsky Eur. J. Immunol. 1974.4: 646-648

A. Novogrodsky Selective activation of mouse T and B lymphocytes by periodate, galactose oxidase and soybean agglutinin

Department of Biophysics, The Weizmann Institute of Science, Rehovot Modifications of surface saccharides of mouse spleen cells wirh periodate

(NaIO4) or with neuraminidase and galactose oxidase induced extensive blastogenesis. The mitogenic effects of these agents on purified thymus- derived lymphocytes (T cells) and bone marrow-derived lymphocytes (B cells) were investigated. Thymus cells were stimulated after treatment with NaIO, or with neuraminidase and galactose oxidase. The stimulation of these cells was markedly enhanced upon addition of a lymphocyte-activating factor ob- tained from human leukocyte culture. Spleen cells from congenitally athymic mice did not respond to treatment with Na1O4 or with neuraminidase and ga- lactose oxidase. Spleen cells from mice deprived of T cells by thymectomy, irradiation and bone marrow reconstitution, responded poorly t o the above mentioned agents. It was concluded that periodate or treatment with neura- minidase and galactose oxidase stimulates exclusively T cells. Soybean agglu- tinin (SBA) had selective effects on T and B mouse lymphocytes. Neuramini- dase treatment rendered T cells highly responsive to stimulation by SBA. On the other hand the small but significant stimulation of B cells by SBA is re- duced after treatment of the cells with neuraminidase. SBA stimulates B cells and neuraminidase-treated T cells by interaction with galactose-like sites.

1. Introduction

Various mitogens induce selectively the proliferation of thymus- derived (T) and bone marrow-derived (B) mouse lymphocytes. Phytohemagglutinin (PHA) and concanavalin A (Con A) in their soluble form stimulate T lymphocytes [ 1-31 whereas lipopolysaccharide (LPS) stimulates exclusively B lympho- cytes [ 2 , 41. Pokeweed mitogen stimulates both B and T cells [3]. Recently we have shown that certain modifications of membrane saccharides induced lymphocyte transformation. Lymphocytes treated with periodate (NaIO4) [5] or after sequential treatment with neuraminidase and galactose oxi- dase underwent extensive blastogenesis [6]. In addition we have found that treatment of lymphocytes with neuramini- dase exposed galactose-like sites on the cell surface and ren- dered them responsive t o stimulation by the lectin soybean agglutinin (SBA) [7]. We have postulated that the chemical oxidizing agent, periodate, the enzymic oxidizing agent galac- tose oxidase and the chemically inert lectin SBA trigger lym- phocytes t o undergo transformation by affecting the same membrane site. In this study we investigated the mitogenic effects of the above-mentioned agents on T and B murine lymphocytes. Purified T and B cell suspensions have been used in this study. As a source of purified T lymphocytes we have used mouse thymus cells. The latter were incubated in the presence of a lymphocyte-activating factor obtained from human leukocyte cultures. The factor potentiated the T lymphocyte response t o mitogens [8-lo]. As a source of purified B lymphocytes we have used: (a) spleen cells from mice which were thymectomized, lethally irradiated and re- constituted with syngeneic bone marrow (TxBM mice); (b) spleen cells from congenitally athymic mice (nude mice).

[I 7941

Correspondence: Abraham Novogrodsky, Department of Biophysics, The Weizmann Institute of Science, Rehovot, Israel

Abbreviations: T cells: Thymus-derived lymphocytes B cells: Bone marrow-derived lymphocytes Con A: Concanavalin A PHA: Phyto- hemagglutinin SBA: Soybean agglutinin LPS: Lipopolysaccharide TxBM: Thymectomized, lethally irradiated and bone marrow restored

2. Materials and methods

2.1. Materials

Concanavalin A (Con A), twice crystallized, was obtained from Miles-Yeda Ltd., Rehovot, Israel. Phytohemagglutinin-P (PHA) was obtained from Difco Laboratories, Detroit, Mich. Soybean agglutinin (SBA), purified by affinity chromatogra- phy [ 111 was kindly supplied by N. Sharon, H. Lis and R. Lotan of our department. Sodium periodate, analytical reagent was obtained from BDH Chemical Ltd., Poole, Eng- land. Lipopolysaccharide from E. coli 055:B5 (LPS) was obtained from Difco Laboratories. Saccharides were obtained from Pfanstiehl Inc., Waukegan, Ill., USA. Neuraminidase from Vibrio comma was obtained from Behringwerke AG., Marburg/Lahn, Germany. Galactose oxidase, 25 units/mg, was obtained from Worthington Biochemical Corp., Freehold, N.J., USA. (Meth~I-[~H]thymidine) (5 Ci/mmol) was obtained from the Nuclear Research Center, Negev, Israel.

2.2. Mice

Normal (control) mice: CBA/LAC and C3H/HeJ mice, aged 6-12 weeks were used. T cell-depleted mice (TxBM): C3H/ HeJ mice were thymectomized (T) a t 4 weeks of age. Two weeks later they were irradiated with 850 R and reconstituted with 5 x 1 O6 syngeneic bone marrow cells (BM). Mice were used 6-8 weeks after reconstitution. Congenitally athymic mice (nude) were obtained from the Weizmann Institute breeding center and used a t 6 weeks of age.

2.3. Preparation of lymphocytes

The mice were killed b y ether. Spleens o r thymus glands were removed and minced in phosphate-buffered saline (PBS). The large pieces of connective tissue were allowed t o settle and the supernantant suspension was harvested.

2.4. Lymphocyte cultures

Cells were suspended at a final concentration of 5 x 106/ml in Dulbecco’s modified Eagle’s medium containing fetal calf serum (5 %, heat inactivated at 56 OC for 30 min), and sup- plemented with penicillin ( 1 00 units/ml) and streptomycin

Eur. J. Immunol. 1974.4: 646-648 Selective activation of T and B lymphocytes 647

(100 pg/mlj. Cultures of 1 ml were prepared in triplicate in polystyrene tubes (17 x 100 mm), loosely capped, and in- cubated at 37 "C in a n atmosphere of 95 % air - 5 % CO, for 72 h.

2.5. Preparation of lymphocyte-activating factor

Lymphocyte activating factor was prepared according to Gery e t al. [8] as follows. Human peripheral leukocytes (2 x 1 06/ml Dulbecco's modified medium supplemented with 5 7% human serum, heat inactivated at 56 OC for 3 0 min) were incubated in the presence of LPS (50 pg/ml) for 24 h. The cells were then centrifuged and the supernatant was used as the lymphocyte activating factor.

2.6. Treatment of cells with different agents

(a) NaI04. Cells were suspended (1 3 x 1 06/ml) in PBS con- taining NaIO, ( 1 x 1 0 - 3 ~ ) and incubated at 0 OC for 30 min. The cells were then centrifuged and suspended in culture medium. (b j Neuraminidase. Cells (8 x 107/ml) in PBS were treated with neuraminidase (50 units/ml) for 30 min at 37 O C

with shaking. The cells were then washed with PBS and sus- pended in culture medium. (c) Neuraminidase and galactose oxidase. Cells were incubated with neuraminidase under con- ditions specified above in the presence of galactose oxidase (0.5 unit/ml).

2.7. [3H]thymidine incorporation into DNA

[3H]thymidine (2.5 pCij was added t o cell cultures (1 ml) that had been incubated for 72 h. After additional incubation of the cell cultures for 2 h with shaking, incorporation of [3H]thy- midine into DNA was determined [ 121.

3. Results

3.1. Response of mouse thymus cells to various mitogens

The effect of various mitogens on mouse thymus cells was in- vestigated. Mouse thymus cells, incubated in the presence of lymphocyte-activating factor, are markedly stimulated by Con A, PHA and NaIO4. The stimulation of the thymus cells by these mitogens in the absence of the factor is much less pronounced (Table 1, I). Similar results have been reported previously [9, 10, 131. Thymus cells were stimulated after treatment with neuraminidase and galactose oxidase. Either one of these enzymes alone had practically no stimulatory effect on the cells. The stimulation of the enzymically treated cells was markedly enhanced upon incubation in the presence of lymphocyte-activating factor (Table 1 , I I ) .

Neuraminidase-treated thymus cells are stimulated by SBA upon incubation in the presence of lymphocyte-activating factor. SBA hardly stimulates thymus cells which were not treated with neuraminidase or cells which were treated with the latter but incubated in the absence of the lymphocyte activating factor (Table 1,111). The response of thymus cells to PHA, in contrast to SBA, is only slightly enhanced after treatment with neuraminidase.

3.2. Response of normal TxBM and congenitally athymic (nude) mouse spleen cells to various mitogens

The effect of various mitogens on normal, TxBM and nude mouse spleen cells was investigated. The response of TxBM mouse spleen cells t o Con A is markedly reduced and that

Table 1. Response of mouse thymus cells to various mitogens

i3H] thymidine incorporation

Lymphocyte-activating factor +a) -

Treatment (cprn+~E) < pb) ( c p m f ~ ~ ) < P

I . None Con A (2 pg/ml) PHA (20 pg/ml) ~ a 1 0 ~ 11.

Neuraminidase Gahctose oxidase Neuraminidase, galactose oxidase 111. SBA (50 pglml) Neuraminidase, SBA (50 pg/ml)e) Neuraminidase, PHA (20 pg/ml)e)

225 f 43') 4833 5 161 1638 f 325 3770 f 104

290f. 17 306' 56

4548 f 209

473f 59

982f. 41

2010 f 212

-

0.001 0.02 0.001

n.s.d) ns.

0.001

0.05

0.001

0.002

2811* 511 80532f 651 32565 * 1468 33605 f 501

3450+ 389 33.502 220

37794 f 1526

30902 89

34582f-2178

36920 f 1860

- 0.001 0.001 0.001

n.s. n.s.

0.001

n.s.

0.001

0.001

a) 50 pl of lymphocyte activating factor were added. b) P, level of significance of observed differences from controls, un-

treated cells. c) Mean f standard error of triplicate cultures. d) Not significant, P > 0.05. e) Where indicated, SBA or PHA were added to the cell cultures after

treatment of the cells with neuraminidase.

of nude mouse spleen cells is completely abolished (Table 2). This indicates a marked depletion of T cells in the TxBM spleen and their absence in the nude spleen. The response of TxBM and nude spleen cells to LPS, a known B mitogen, is retained. TxBM spleen cells are markedly reduced in their blastogenic response t o NaIO4 or t o treatment with neura- minidase and galactose oxidase. The stimulatory effect of SBA on neuraminidase-treated TxBM spleen cells is also marked- ly reduced. A small but significant stimulation of nude mouse spleen cells by SBA was noted. The response of nude mouse spleen cells t o SBA was reduced after treatment of the cells with neuraminidase. In this respect B cells differ from T cells. As was shown above, the latter are rendered highly responsive to SBA after treatment with neuraminidase. The SBA-induced stimulation of nude mouse spleen cells was markedly inhibited by N-acetyl-D-galactosamine and D-galaCtOSe. The other sac- charides tested had essentially no effect (Table 3). The specific inhibition by N-acetyl-D-galactosamine and D-galactose of SBA- induced stimulation of neuraminidase-treated normal spleen cells was previously reported 173.

4. Discussion

The results presented above show that N a I 0 4 0 r treatment of the cells with neuraminidase and galactose oxidase selectively stimulates mouse T cells. The small stimulatory effect of these agents on TxBM mouse spleen cells is due probably t o the pre- sence of some T cells in these spleens. The unrCsponsiveness of B cells t o NaI040r t o treatment with neuraminidase and galactose oxidase might be due either to a failure of these agents to modify the surface saccharides on B cells or to an inability of the chemically and enzymically modified B cells to undergo blastogenesis. The former possibility seems, how- ever, unlikely. Neuraminidase probably does expose galactose- like sites on B cells as shown by its ability t o reduce the trans- formation of these cells which is induced by SBA. In this

648 A. Novogrodsky

Table 2. Response of noimal, TxBM and nude mouse spleen cells to various mitogens

[3H]thymidine incorporation Nor ma I

Treatment (cpm f SEP) < pb)

None 1632f 179 Can A (2 pg/rnl) 107827 2 9224 0.001 LPS (50 pg/ml) 19420 f 2144 0.002 ~ a 1 0 ~ 40679 f 1660 0.001 Neuraminidase 16322 105 Neuraminidase, galactose oxidase 15 1957 f 245 3 0.001 SBA (50 pg/ml) 3078f 355 0.05 SBA (100 pglrnl) 5138 f 644 0.01 Neuraminidase, SBA (50 pg/mlY) 35812 f 815 0.001 Neuraminidase, SBA (100 pg/ml)c) 39155 f 2259 0.001

Tx BM (cpm f SE)

1408+ 46 3342 f 289

18617 k 757 3354 2 191 865 + 42

3589 * 105 2791 k 235 3129 f 224

2304 f 266

2635 2 338

< P

0.005 0.001 0.001 0.001

0.001

0.005 0.005

0.05

0.05

Nude (cpm f SE)

10682 110 7 7 7 2 57

22441f1206 .546f 53

758k 18

1047f 83 51792 272 7659 f 1017

1990f 118

3490 f 150

a,) Mean 2 standard error of triplicate cultures. b) P, level of significance of observed differences from control, untreated cells. c) SBA was added to the cell cultures after treatment of the cells with neuraminidase.

Table 3. Effect of saccharides on SBA-induced stimulation of nude mouse spleen cells

Additions [3H]thymidine incorporation S B A ~ ) Saccharidesb) (cpm 2 SE)

1472 f 322C) 6515 2 230 +

+ D-Galactose 2189 f 232 + N-Acetyl-D-galactosamine 1764 5 95 + N-Acetyl-D-glucosamine 5778 2 740 + (Y-Met hyl-D-mannose 5086 2 574 + C-FUCOS~ 665 1 f 560

- - -

a) SBA was added to the cell cultures at a final concentration of 100 pg/ml.

b) Saccharides were added to the cell cultures at a final concentration of 5 mg/ml.

c) Mean 2 standard error of triplicate cultures.

connection it is pertinent to no te that Con A binds equally to T and B cells [ 141. However, this lectin, in i ts soluble form, fails t o stimulate B cells. Our results show selective effects of SBA on T and B mouse lymphocytes. Neuraminidase treatment renders T cells highly responsive t o st imulation b y SBA. On the other hand, the small bu t significant st imulation of B cells by SBA is reduced after treatment of the cells with neuramini- dase. SBA effects T and B cells by interaction with galactose- like sites. Since sialic acid in glycoproteins is always found t o occupy a nonreducing terminal position and t o b e glycosidical- ly linked t o a galactosyl residue [ 151, it is possible that the SBA sites which are involved in triggering of lymphocytes are “masked” b y sialic acid in T cells and are exposed in B cells and thus can interact directly with the lectin. Neuraminidase reduces the blastogenic response of B cells to SBA. I t is thus possible that SBA has an inhibitory effect on B cells upon interaction with the galactose-like sites which are exposed b y

Em. J. Immunol. 1974.4: 646-648

< P

n.s. 0.001 0.02 0.05

n.s. 0.001 0.005

0.005

0.001

neuraminidase. Neuraminidase might also affect t he blastogenic response of the cells t o SBA b y reducing their net surface charge.

The skillful technical assistance of Ms. Segula Halmann is gratefully acknowledged.

Received March 24, 1974.

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