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Activation of murine thymocyte subpopulations by the oxidizing mitogens: Evidence for a sialylated, mitogen-reactive site on both PNA-positive and PNA-negative cells
CELLULAR
IMMUNOLOGY
94, 28 l-284 (1985)
Activation of Murine Thymocyte Subpopulations by the Oxidizing Mitogens: Evidence for a Sialylated, Mitogen-Reactive Site on both PNA-Positive and PNA-Negative Cells *J BLANKA SHANITZKI,* ABRAHAMNOVOGRODSKY, AND KURT H. STENZEL~
AMIRAN RAVID,*
*Rogoff- Wellcome Medical Research Institute, Beilinson Medical Center, Tel Aviv University Sackler Medical School, Petah Tikva 49100, Israel; and fRogosin Institute, Cornell University Medical College, New York, New York 10021 Received March 1, 1985; accepted April 14, 1985
INTRODUCTION Immature, cortical thymocytes can be separated from the more mature medullary population by their property of binding peanut agglutinin (PNA) (1). This lectin binds with high affinity to o-galactose-p-(1 - 3)-WV-acetylgalactosamine. This site can be exposed on the mature population (PNA-negative cells) by removal of cell surface sialyl residues with neuraminidase. PNA-negative cell populations treated with the mitogenic lectins, Con A and PHA, can both respond to interleukin 2 (IL-2) and can produce interleukin 2 following treatment with interleukin 1 (IL-l) or TPA. PNA-positive populations, on the other hand, treated with these lectins, can respond to interleukin 2 but are incapable of IL-2 production (2, 3). Draber and Kisielow (4) reported that the major population of PNA-positive cells bears the typical phenotype of immature thymocytes (low content of H-2 and high content of Thy- 1, TL, Lyt-1, and Lyt-2) and is unable to respond to Con A even with the provision of IL-2. Approximately 15% of the PNA-positive cells were found to have surface antigenic phenotypes similar to those of mature thymocytes (high content of H-2, low content of Thy-l, and differentiated expression of Lyt antigens) and were the targets for IL-2 in this otherwise unresponsive population. Recently, Herron et al. (5) reported the separation of murine thymocytes using a Nacetylneuraminic acid-specific lectin, lobster agglutinin (LAg 1). This lectin agglutinates the sialic acid-rich medullary population, whereas most of the immature cortical thymocytes are not agglutinated. Cortical thymocytes purified on the basis of this lack of agglutination with LAg 1 were found to be unresponsive to IL-2 following Con A treatment. These studies suggest that the PNA-positive cells contain a minor population of sialylated thymocytes that are responsive to IL-2.
’ To whom requests for reprints should be addressed. 281 0008-8749185 $3.00 Copyright Q 1985 by Academic F’ms, Inc. All rights of mpmduction in any form reserved.
282
SHORT
COMMUNICATIONS
The target for the oxidizing mitogen, sodium periodate (104), is a surface membrane sialyl residue (6). The target for the other oxidizing mitogen, galactose oxidase, is a galactosyl residue that, in mouse splenocytes, must be exposed by removing cell surface sialic acid with neuraminidase (7). We examined the mitogenic properties of the oxidizing mitogens for murine thymocytes, since these mitogens discriminate between sialylated and nonsialylated mitogenic sites. Our studies indicate that the PNA-positive population contains sialylated cells that can respond to IL-2, but, in contrast to mature PNA-negative thymocytes, cannot produce IL-2. MATERIALS
AND
METHODS
Materials. PHA (purified HA 16) was obtained from Wellcome Research Laboratories, Beckenham, England. TPA was from Sigma Chemical Company, St. Louis, Missouri. TPA was dissolved in dimethylsulfoxide (special grade, Pierce chemical Co., Rockford, Ill.) at 1 mg/ml and diluted with PBS immediately prior to use. PNA was obtained from Sigma Chemical Company. Vibrio cholera neuraminidase, 1 U/ml, was obtained from Behringwerke AG, Marburg, West Germany (one unit will split off 1 pmol of N-acetylneuraminic acid from human cy1-acid glycoprotein in 1 min at 37°C and pH 5.5). Galactose oxidase, type V (175 U/mg protein) was obtained from Sigma Chemical Company (one unit will produce a 4A 125of 1.O per minute at pH 6.0 and 25°C in a prescribed peroxidase and O-tolidine system). Isolation of cells. Thymocytes were removed from 3- to 4-week-old Balb/c mice, minced, and washed with PBS. PNA-positive and -negative thymocytes were suspended in PBS at 8 X lo* cells/ml and an equal volume of PBS containing 0.5 mg/ml PNA was added. The cell suspension was incubated at room temperature for 10 min. Aliquots (0.5 ml) of the preparation were then layered on 20% fetal calf serum in PBS and allowed to stand for 10-l 5 min at room temperature. The top layer, containing predominantly PNA-negative cells, was removed. The agglutinated cells that settled to the bottom of the tube, containing predominantly PNA-positive cells, were removed, layered on 50% FCS, and recovered as before. Cell-bound PNA was removed by incubation with 0.2 A4 D-galactose for 10 min followed by washing and repetition of the galactose incubation. The cells were then washed X2 and suspended (2-5 X 106/ml) in RPM1 1640 medium containing 5% heat-inactivated human AB serum. The number of PNA-positive and -negative cells in these preparations was determined by incubating an aliquot of the cells with fluoresceinlabeled PNA and determining percentage labeled cells in a Zeiss uv microscope. Unfractioned thymocyte preparations contained 80-90% PNA-positive cells, preparations of PNA-positive thymocytes contained 90-98% PNA-positive cells, and preparations of PNA-negative thymocytes contained 75-85% PNA-negative cells. Cell culture and growth factors. Cells (2 X 106/ml) were suspended in RPM1 1640 medium containing 5% heat-inactivated human AB serum and cultured in 0.2-ml aliquots in flat-bottom Cooke microtiter plates at 37°C in a 95% sir/5% COz humidified atmosphere. The various additions included PHA (2 &ml), TPA 50 (50 rig/ml), and interleukin I- and interleukin 2-containing preparations. IGlcontaining preparations were prepared from human peripheral blood mononuclear cells incubated with lipopolysaccharide for 24 hr as previously described (3) [they were added at a final concentration of 20 &ml]. IG2-containing preparation were supernatants obtained from neuraminidase- and galactose oxidase-treated human
283
SHORT COMMUNICATIONS
peripheral blood mononuclear cells [(8) they were added at a final concentration of 50 &ml] or conditioned medium of the gibbon T-cell line MCA-144 [(9) they were added at a concentration of 50 &ml]. Treatment of cells with the oxidizing mitogens. Periodate: Cells (20-50 X 106/ml) suspended in PBA were incubated with NaI04 (1 mM) at 0°C for 30 min, followed by washings with PBS to remove excess reagent. Neuraminidase and galactose oxidase: Cells (20-50 X 106) suspended in PBS were treated with neuraminidase (0.1 U/ml) and galactose oxidase (4 U/ml) at 37°C for 30 min followed by washings with PBS to remove excess reagent. In some experiments cells were treated with galactose oxidase. RESULTS We determined the mitogenic effect of GO, NAGO, IO,, and PHA for PNAnegative and PNA-positive thymocyte subpopulations. PNA-negative thymocytes respond to GO only after treatment with neuraminidase (Fig. 1). This was seen without any comitogens, and upon addition of TPA, IL-l, or IL-2 (Fig. 1). This observation is consistent with the notion that the mitogenic site for GO is sialylated in PNA-negative cells. IO;, although inducing mitogenesis via oxidation of sialylated residues, was a weak mitogen for PNA-negative cells (Fig. 1). Nevertheless, its mitogenic effect was significant in the presence of the cytokines, IL-l, or IL-2. Of interest is the finding that PNA-positive cells, provided with IL-2, responded to GO only after neuraminidase treatment. IO; was also mitogenic in this subpopulation. PNA-positive cells did not respond to GO, NAGO, IO;, or PHA in the absence of IL-2, even when TPA or IL-1 was provided (data not shown). These findings indicate that the mitogenic sites for PNA-positive cells, similar to those of PNAnegative cells, are sialylated. DISCUSSION PNA-negative murine thymocyte can be induced to undergo mitogenesis by treatment with PHA and either TPA or IL-I or by PHA + IL-2. On the other hand, PNA + and IL-.?
PNA 6. TPA 150ng/mN
c. IL-l
D. IL -2
FIG. 1. Effect of different accessory stimuli proliferative responses of PNA+ and PNA- mouse thymocytes to: galactose oxidase (GO), neuraminidase/galactose oxidase (NAGO), periodate (IO;), and PHA (2 &ml). PNA+ cells did not respond beyond control values to TPA or IL-l (data not shown).
284
SHORT COMMUNICATIONS
PNA-positive cells respond only to stimulation of PHA + IL-2 (2). Shortman and his associates ( 10) claimed that the mitogenic response of the PNA-positive thymocytes could be attributed to PNA-negative contaminants. This conclusion could not be supported by our findings that PNA-positive cell preparations that respond effectively to PHA + IL-2 fail to respond to PHA + TPA. As noted above, PHA + TPA markedly stimulate PNA-negative cells. The data presented in this paper indicate that the mitogenic sites on either PNA-negative or PNA-positive cells are sialylated. This conclusion is based on the findings that IO, and NAG0 treatment renders PNA-negative and -positive cells responsive to IL-2. Treatment of cells with GO alone does not elicit this effect. As previously found cell surface sialic acid is the target for the mitogenic action of IO: (6), and galactosyl sites penultimate to sialic acid are the target for the mitogenic action of GO (7). NAG0 in the presence of TPA or IL-l is also mitogenic for PNA-negative cells and IO; is less effective. PHA is much more effective than the oxidizing mitogens in stimulating PNA-negative cells in the presence of TPA or IL- 1. Although PNA-positive cells contain galactosyl sites that are masked by sialic acid in the PNA-positive cells, they do contain a galactosyl-mitogenic site on their surface that is sialylated. When lobster agglutinin, a lectin which specifically binds sialic acid, was used to separate murine thymocyte subpopulations, the immature, lobster agglutinin-negative cell fraction did not respond to Con A + IL-2. Recently, by fractionation on bovine serum albumin gradients, a minor fraction of PNA-positive cells responsive to IL-2 was identified (4). Our data indicate the heterogeneity of PNA-positive cells and that mitogenic sites on the responsive cells are sialylated. REFERENCES 1. Reisner, Y., Linker-Israeli, M., and Sharon, N., Cell. Irnmunol. 25, 129, 1976. 2. Conlon, P. J., Henney, C. S., and Gillis, S., J. Immunol. 128, 797, 1982. 3. Ravid, A., Patya, M., Rubin, A. L., Stenzel, K. H., and Novogrodsky, A., Cancer Res. 43, 5 178, 1983. 4. Draber, P., and Kisielow, P., Eur. J. Immunol. 11, 1, 198 1. 5. Herron, L. R., Abel, C. A., VanderWall, J., and Campbell, P. A., Eur. J. Zmmunol. 13, 73, 1983. 6. Novogrodsky, A., and Katchalski, E., Proc. Natl. Acad. Sci. USA, 69, 3207, 1972. 7. Novogrodsky, A., and Katchalski, E., Proc. Natl. Acad. Sci. USA, 70, 1824, 1973. 8. Novogrodsky, A., Suthanthiran, M., Saltz, B., Newman, D., Rubin, A. L., and Stenzel, K. H., J. Exp. Med. 151, 755, 1980. 9. Henderson, L. E., Hewetson, J. F., Hopkins, R. F., Sowder, R. C., Neubauer, R. H., and Rabin, H., J. Immunol. 131, 810, 1983. 10. Wei-Feng, C., Scollay, R., and Shortman, K., J. Immunol. 129, 18, 1982.
IMMUNOLOGY
94, 28 l-284 (1985)
Activation of Murine Thymocyte Subpopulations by the Oxidizing Mitogens: Evidence for a Sialylated, Mitogen-Reactive Site on both PNA-Positive and PNA-Negative Cells *J BLANKA SHANITZKI,* ABRAHAMNOVOGRODSKY, AND KURT H. STENZEL~
AMIRAN RAVID,*
*Rogoff- Wellcome Medical Research Institute, Beilinson Medical Center, Tel Aviv University Sackler Medical School, Petah Tikva 49100, Israel; and fRogosin Institute, Cornell University Medical College, New York, New York 10021 Received March 1, 1985; accepted April 14, 1985
INTRODUCTION Immature, cortical thymocytes can be separated from the more mature medullary population by their property of binding peanut agglutinin (PNA) (1). This lectin binds with high affinity to o-galactose-p-(1 - 3)-WV-acetylgalactosamine. This site can be exposed on the mature population (PNA-negative cells) by removal of cell surface sialyl residues with neuraminidase. PNA-negative cell populations treated with the mitogenic lectins, Con A and PHA, can both respond to interleukin 2 (IL-2) and can produce interleukin 2 following treatment with interleukin 1 (IL-l) or TPA. PNA-positive populations, on the other hand, treated with these lectins, can respond to interleukin 2 but are incapable of IL-2 production (2, 3). Draber and Kisielow (4) reported that the major population of PNA-positive cells bears the typical phenotype of immature thymocytes (low content of H-2 and high content of Thy- 1, TL, Lyt-1, and Lyt-2) and is unable to respond to Con A even with the provision of IL-2. Approximately 15% of the PNA-positive cells were found to have surface antigenic phenotypes similar to those of mature thymocytes (high content of H-2, low content of Thy-l, and differentiated expression of Lyt antigens) and were the targets for IL-2 in this otherwise unresponsive population. Recently, Herron et al. (5) reported the separation of murine thymocytes using a Nacetylneuraminic acid-specific lectin, lobster agglutinin (LAg 1). This lectin agglutinates the sialic acid-rich medullary population, whereas most of the immature cortical thymocytes are not agglutinated. Cortical thymocytes purified on the basis of this lack of agglutination with LAg 1 were found to be unresponsive to IL-2 following Con A treatment. These studies suggest that the PNA-positive cells contain a minor population of sialylated thymocytes that are responsive to IL-2.
’ To whom requests for reprints should be addressed. 281 0008-8749185 $3.00 Copyright Q 1985 by Academic F’ms, Inc. All rights of mpmduction in any form reserved.
282
SHORT
COMMUNICATIONS
The target for the oxidizing mitogen, sodium periodate (104), is a surface membrane sialyl residue (6). The target for the other oxidizing mitogen, galactose oxidase, is a galactosyl residue that, in mouse splenocytes, must be exposed by removing cell surface sialic acid with neuraminidase (7). We examined the mitogenic properties of the oxidizing mitogens for murine thymocytes, since these mitogens discriminate between sialylated and nonsialylated mitogenic sites. Our studies indicate that the PNA-positive population contains sialylated cells that can respond to IL-2, but, in contrast to mature PNA-negative thymocytes, cannot produce IL-2. MATERIALS
AND
METHODS
Materials. PHA (purified HA 16) was obtained from Wellcome Research Laboratories, Beckenham, England. TPA was from Sigma Chemical Company, St. Louis, Missouri. TPA was dissolved in dimethylsulfoxide (special grade, Pierce chemical Co., Rockford, Ill.) at 1 mg/ml and diluted with PBS immediately prior to use. PNA was obtained from Sigma Chemical Company. Vibrio cholera neuraminidase, 1 U/ml, was obtained from Behringwerke AG, Marburg, West Germany (one unit will split off 1 pmol of N-acetylneuraminic acid from human cy1-acid glycoprotein in 1 min at 37°C and pH 5.5). Galactose oxidase, type V (175 U/mg protein) was obtained from Sigma Chemical Company (one unit will produce a 4A 125of 1.O per minute at pH 6.0 and 25°C in a prescribed peroxidase and O-tolidine system). Isolation of cells. Thymocytes were removed from 3- to 4-week-old Balb/c mice, minced, and washed with PBS. PNA-positive and -negative thymocytes were suspended in PBS at 8 X lo* cells/ml and an equal volume of PBS containing 0.5 mg/ml PNA was added. The cell suspension was incubated at room temperature for 10 min. Aliquots (0.5 ml) of the preparation were then layered on 20% fetal calf serum in PBS and allowed to stand for 10-l 5 min at room temperature. The top layer, containing predominantly PNA-negative cells, was removed. The agglutinated cells that settled to the bottom of the tube, containing predominantly PNA-positive cells, were removed, layered on 50% FCS, and recovered as before. Cell-bound PNA was removed by incubation with 0.2 A4 D-galactose for 10 min followed by washing and repetition of the galactose incubation. The cells were then washed X2 and suspended (2-5 X 106/ml) in RPM1 1640 medium containing 5% heat-inactivated human AB serum. The number of PNA-positive and -negative cells in these preparations was determined by incubating an aliquot of the cells with fluoresceinlabeled PNA and determining percentage labeled cells in a Zeiss uv microscope. Unfractioned thymocyte preparations contained 80-90% PNA-positive cells, preparations of PNA-positive thymocytes contained 90-98% PNA-positive cells, and preparations of PNA-negative thymocytes contained 75-85% PNA-negative cells. Cell culture and growth factors. Cells (2 X 106/ml) were suspended in RPM1 1640 medium containing 5% heat-inactivated human AB serum and cultured in 0.2-ml aliquots in flat-bottom Cooke microtiter plates at 37°C in a 95% sir/5% COz humidified atmosphere. The various additions included PHA (2 &ml), TPA 50 (50 rig/ml), and interleukin I- and interleukin 2-containing preparations. IGlcontaining preparations were prepared from human peripheral blood mononuclear cells incubated with lipopolysaccharide for 24 hr as previously described (3) [they were added at a final concentration of 20 &ml]. IG2-containing preparation were supernatants obtained from neuraminidase- and galactose oxidase-treated human
283
SHORT COMMUNICATIONS
peripheral blood mononuclear cells [(8) they were added at a final concentration of 50 &ml] or conditioned medium of the gibbon T-cell line MCA-144 [(9) they were added at a concentration of 50 &ml]. Treatment of cells with the oxidizing mitogens. Periodate: Cells (20-50 X 106/ml) suspended in PBA were incubated with NaI04 (1 mM) at 0°C for 30 min, followed by washings with PBS to remove excess reagent. Neuraminidase and galactose oxidase: Cells (20-50 X 106) suspended in PBS were treated with neuraminidase (0.1 U/ml) and galactose oxidase (4 U/ml) at 37°C for 30 min followed by washings with PBS to remove excess reagent. In some experiments cells were treated with galactose oxidase. RESULTS We determined the mitogenic effect of GO, NAGO, IO,, and PHA for PNAnegative and PNA-positive thymocyte subpopulations. PNA-negative thymocytes respond to GO only after treatment with neuraminidase (Fig. 1). This was seen without any comitogens, and upon addition of TPA, IL-l, or IL-2 (Fig. 1). This observation is consistent with the notion that the mitogenic site for GO is sialylated in PNA-negative cells. IO;, although inducing mitogenesis via oxidation of sialylated residues, was a weak mitogen for PNA-negative cells (Fig. 1). Nevertheless, its mitogenic effect was significant in the presence of the cytokines, IL-l, or IL-2. Of interest is the finding that PNA-positive cells, provided with IL-2, responded to GO only after neuraminidase treatment. IO; was also mitogenic in this subpopulation. PNA-positive cells did not respond to GO, NAGO, IO;, or PHA in the absence of IL-2, even when TPA or IL-1 was provided (data not shown). These findings indicate that the mitogenic sites for PNA-positive cells, similar to those of PNAnegative cells, are sialylated. DISCUSSION PNA-negative murine thymocyte can be induced to undergo mitogenesis by treatment with PHA and either TPA or IL-I or by PHA + IL-2. On the other hand, PNA + and IL-.?
PNA 6. TPA 150ng/mN
c. IL-l
D. IL -2
FIG. 1. Effect of different accessory stimuli proliferative responses of PNA+ and PNA- mouse thymocytes to: galactose oxidase (GO), neuraminidase/galactose oxidase (NAGO), periodate (IO;), and PHA (2 &ml). PNA+ cells did not respond beyond control values to TPA or IL-l (data not shown).
284
SHORT COMMUNICATIONS
PNA-positive cells respond only to stimulation of PHA + IL-2 (2). Shortman and his associates ( 10) claimed that the mitogenic response of the PNA-positive thymocytes could be attributed to PNA-negative contaminants. This conclusion could not be supported by our findings that PNA-positive cell preparations that respond effectively to PHA + IL-2 fail to respond to PHA + TPA. As noted above, PHA + TPA markedly stimulate PNA-negative cells. The data presented in this paper indicate that the mitogenic sites on either PNA-negative or PNA-positive cells are sialylated. This conclusion is based on the findings that IO, and NAG0 treatment renders PNA-negative and -positive cells responsive to IL-2. Treatment of cells with GO alone does not elicit this effect. As previously found cell surface sialic acid is the target for the mitogenic action of IO: (6), and galactosyl sites penultimate to sialic acid are the target for the mitogenic action of GO (7). NAG0 in the presence of TPA or IL-l is also mitogenic for PNA-negative cells and IO; is less effective. PHA is much more effective than the oxidizing mitogens in stimulating PNA-negative cells in the presence of TPA or IL- 1. Although PNA-positive cells contain galactosyl sites that are masked by sialic acid in the PNA-positive cells, they do contain a galactosyl-mitogenic site on their surface that is sialylated. When lobster agglutinin, a lectin which specifically binds sialic acid, was used to separate murine thymocyte subpopulations, the immature, lobster agglutinin-negative cell fraction did not respond to Con A + IL-2. Recently, by fractionation on bovine serum albumin gradients, a minor fraction of PNA-positive cells responsive to IL-2 was identified (4). Our data indicate the heterogeneity of PNA-positive cells and that mitogenic sites on the responsive cells are sialylated. REFERENCES 1. Reisner, Y., Linker-Israeli, M., and Sharon, N., Cell. Irnmunol. 25, 129, 1976. 2. Conlon, P. J., Henney, C. S., and Gillis, S., J. Immunol. 128, 797, 1982. 3. Ravid, A., Patya, M., Rubin, A. L., Stenzel, K. H., and Novogrodsky, A., Cancer Res. 43, 5 178, 1983. 4. Draber, P., and Kisielow, P., Eur. J. Immunol. 11, 1, 198 1. 5. Herron, L. R., Abel, C. A., VanderWall, J., and Campbell, P. A., Eur. J. Zmmunol. 13, 73, 1983. 6. Novogrodsky, A., and Katchalski, E., Proc. Natl. Acad. Sci. USA, 69, 3207, 1972. 7. Novogrodsky, A., and Katchalski, E., Proc. Natl. Acad. Sci. USA, 70, 1824, 1973. 8. Novogrodsky, A., Suthanthiran, M., Saltz, B., Newman, D., Rubin, A. L., and Stenzel, K. H., J. Exp. Med. 151, 755, 1980. 9. Henderson, L. E., Hewetson, J. F., Hopkins, R. F., Sowder, R. C., Neubauer, R. H., and Rabin, H., J. Immunol. 131, 810, 1983. 10. Wei-Feng, C., Scollay, R., and Shortman, K., J. Immunol. 129, 18, 1982.