CELLULARIMMUNOLOGY144, 155-168(1992)

Agonistic Effects of Tyrphostins on Human Peripheral Mononuclear Cells HARRY M. LANDER,*'?,' DANIEL M. LEVINE,*%? ANDABRAHAMNOVOGRODSKY*'$$~~ *The Rogosin Institute and the Departments of tBiochemistry and *Medicine, Cornell Universit) Medical College, 1300 York Avenue, New York, New York 10021 Received April 14, 1992; acceptedJune 29, 1992 Tyrosine kinases of the src family, ~56”~ and p59@“,were implicated in the transduction of signals via the T-cell receptor complex. These kinasesare negatively regulated by phosphorylation of a carboxyl-terminal tyrosine residue.Tyrphostins are synthetic low molecular weight compounds that selectively inhibit different protein tyrosine kinases. We report here on the agonistic and antagonistic effects of tyrphostins on human peripheral blood mononuclear cells (PBM). At low concentration, the tyrphostins enhanced glucose uptake and maximal stimulation was attained at a concentration characteristic for each of the tyrphostins used. Higher concentrations were less effective.The tyrphostins AG126 and AG183 were also found to enhanceIL-2-induced cytotoxicity in human PBM in a biphasic manner. In contrast, the tyrphostin AGl7 markedly inhibited IL2-induced cytotoxicity at low AG17 concentration and no stimulation was observed. The tyrphostins tested had selective effects on [3H]thymidine incorporation induced by the mixed lymphocyte culture and different agents.The most potent inhibitor was AG 17. Tyrphostins also affect cytokine secretion by human PBM. AG 126and AG I83 enhanced TNF-a secretion and this effect was more prominent in the presenceof IL-2. AG126 enhanced IFN-y. IL- I, and IL-6 production in PBM that were costimulated with the stressstimuli heat shock and phenylarsine oxide. The stimulatory effects of the tyrphostins on cytokine secretion and induction of cytotoxicity might be interrelated. The agonistic and antagonistic effects of tyrphostins on lymphocyte functions may have therapeutic potential. 0 1992 Academic PESS, IIIC.

INTRODUCTION Protein tyrosine phosphorylation was implicated to mediate signals inducing lymphocyte activation (1). Tyrosine phosphorylation of several intracellular substrates is seen as early as 5 set after stimulation while inositol phosphates are detected only after a lag of approximately 30 set (2). Recent studies indicate that tyrosine phosphorylation activates phospholipase C-r 1 which is involved in the hydrolysis of phosphatidylinositides (3) leading to an increase in intracellular Ca” and to activation of protein kinase C. Two tyrosine kinases of the SK family are implicated to play a role in transduction of signals via the T-cell receptor complex (TCR). p.561ck, which is a Tcell-specific tyrosine kinase, and ~59~~“,which is found in a variety of tissues, are ’ To whom correspondence should be addressedat Cornell University Medical College, 1300 York Ave.. Box 135, New York, NY 10021. ’ Current address:The Rogoff Institute, Beilinson Medical Center. Petah-Tikva. 49 100, Israel. 155 0008-8749/92 $5.00 Copyright 0 1992 by Academic Press, Inc. All n&s of reproduction in any form reserved.

156

LANDER,

LEVINE,

AND

NOVOCRODSKY

members of the SK gene family. ~56”~ interacts with CD4 and CD8 and ~59~~”with the TCR (4-7). Tyrosine kinases of the SK family are negatively regulated by phosphorylation of a carboxyl-terminal tyrosine residue. Thus, dephosphorylation of a tyrosine residue in ~56’~~is associatedwith enhanced tyrosine kinase activity (8). The leukocyte-common antigen, CD45, is a tyrosine phosphataseuniquely and abundantly expressedby cells of hematopoietic origin, including T-cells (9). CD45 was implicated in mediating TCR signaling possibly via dephosphorylation of SK tyrosine kinases leading to their activation (10). Tyrphostins are specific inhibitors of protein tyrosine kinases whose structures were designedto interfere with the substrate-binding site of tyrosine kinases and thus exhibit selectivity in their ability to inhibit different protein tyrosine kinases. Tyrphostins were previously reported to exclusively inhibit proliferation of cells induced by a variety of ligands and also to inhibit polyclonal activation of B and T lymphocytes (11, 12). The various tyrphostins exhibit differential inhibitory activity toward different tyrosine kinases ( 13- 16). In view of the fact that tyrosine kinases which are involved in lymphocyte activation are negatively controlled by tyrosine phosphorylation, we evaluated the possibility that tyrphostins may have inductive effects on lymphocyte functions. MATERIALS AND METHODS Cell Isolation and Culture Human PBM were obtained from healthy normal subjects by Ficoll-Hypaque density gradient centrifugation. For mitogenicity assays,PBM were resuspended( 1 X 106/ ml) in RPM1 1640 plus 5% FCS supplemented with 10 pg/ml gentamicin. Cells were distributed (0.2 ml/well) in flat-bottomed microwells and various additions were made. The cells were then incubated at 37°C in a 95% air, 5% CO2 atmosphere for 68 hr, and [3H]thymidine incorporation (2 pCi/well) into DNA during the final 20 hr of incubation was determined. Triplicate cultures were used and the means determined. For the mixed lymphocyte culture (MLC), responder PBM were resuspended to 1 X lo6 cells/ml in RPM1 1640 containing 10% FCS, 25 mA4 Hepes, and 10 pg/ml gentamicin and 50 ~1 was added to 50 ~1 of irradiated (3000 rad) PBM from a different donor in flat-bottomed microwells. One hundred microliters of medium was then added and the plates were incubated for 120 hr before pulsing as described above. Tyrphostins, generously provided by Dr. A. Levitzki, Hebrew University, Jerusalem, Israel, were dissolved to stock concentrations of 50 mM in DMSO. All other reagents were purchased from Sigma Chemical Co. (St Louis, MO). Cytotoxic Assay Target cells were labeled with 400 PCi of sodium chromate-5 1 for 1 hr in complete medium, as defined above. They were then washed twice with PBS and resuspended in complete medium at lo5 cells/ml. Effector cells, prepared as above, were washed and mixed in a volume of 0.15 ml with target cells (0.05 ml) at various effector to target ratios and were incubated in round-bottomed microtiter plates for 3 hr at 37°C in a 5% CO* incubator. The culture supernatants were harvested and counted in a liquid scintillation counter. Maximum isotope release (MR) was produced by incubation of the targets with Triton X-100. Spontaneous release (SR) was measured by

TYRPHOSTINS

STIMULATE

HUMAN

PBM

157

incubation of the targetswith medium alone. The percentageof cell lysis was calculated by ER - SR/MR - SR X 100, where ER is the experimental effector-induced release. MLC-induced cytotoxic T-lymphocyte (CTL) generation was assessedas follows. The MLC was established as described above for mitogenicity except that 5 ml of responder cells were mixed with 5 ml stimulator cells before assayin the cytotoxic assay described above. 2-Deoxy-D-[l-3H]Glucose Uptake Assay Fresh PBM were resuspended to 10 X lo6 cells/ml in PBS containing 1 mg/ml BSA and 5 mM lactate. After treatment, cells (0.5 ml per data point) were put on a shaker and 10 ~1 of 5 mM 2-deoxy-D-glucose containing 5 &i 2-deoxy-D-[ l-3H]glucose (Amersham, Arlington Heights, IL) was added. After 5 mitt, 200 ~1was removed and layered on top of dibutyl phthalate:mineral oil (4:l) and spun in a microfuge for 30 sec.The tip (with the cell pellet) was cut and placed in 1 ml of 1% Triton X-100 and 0.1 NNaOH, left overnight, and counted with 15 ml Ultrafluor (National Diagnostics, Manville, NJ) in a liquid scintillation counter. The assaywas linear at 5 min. Cytokine Measurements PBM were suspended at 5 X 106/ml in complete medium (1 ml) with the various experimental reagentsand incubated at 37°C for 48 hr. The supernatants were assayed directly for cytokine content. TNF-(r, IL-l, and IL-6 levels were determined using ELISA kits (R & D Systems, Minneapolis, MN) and IFN--r levels determined using an RIA kit (Centocor, Malvern, PA). Analysis of hsp70 mRNA Production The procedure was carried out as follows: PBM (20 X 106) were lysed in 4 it4 guanidinium thiocyanate containing 0.5% sarkosyl, 0.1 M 2-mercaptoethanol, and was layered atop 5.7 A4 CsCl and spun at 35,000 RPM for 16 hr at 15°C in a SW50.1 rotor. Total RNA pellets were collected and dissolved in H20. Five micrograms of RNA was denatured in 1X Mops, 3% formaldehyde, and 50% formamide for 15 min at 55“C. Samples were run on a 1.2% agarose/formaldehyde gel, capillary blotted to nitrocellulose in 20X SSC, and then baked for 2 hr at 80°C. Nitrocellulose was then prehybridized for 4 hr in 1 M NaCl, 50 mM Tris, pH 7.5, 10% dextran sulfate, 1% SDS, and 100 pg/ml denatured salmon sperm DNA at 65°C. Afterward, 0.6 pmol (approximately 3 X lo6 cpm) of 32P5’-end-labeled DNA probe (Oncogene Science, Manhasset, NY) to the human hsp70 gene (17) was added. After an overnight incubation at 65”C, membranes were washed with 2X SSCand were exposedto x-ray film. DNA-Protein Binding Assay PBM (40 X 106/ml) in 1 ml were treated with or without tyrphostin for 4 hr at 37°C in complete medium. Cells were pelleted, washed once with cold PBS, and were resuspended in 250 ~1 of 10 mM Hepes, pH 7.9, 10 mM KCl, 1.5 mA4 MgC12, 1 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF, and 10%glycerol. Cells were left on ice for 10 min and NP-40 was added to a final concentration of 0.04%. After 5 min on ice, nuclear extracts were pelleted at 1OOOgfor 5 min. Supernatants were removed and 100 ~1 of 20 mA4 Hepes, pH 7.9, 50 mM KCI, 20% glycerol, 0.5 mM PMSF, and 1

158

LANDER, LEVINE, AND NOVOGRODSKY TABLE 1 Effect of Tyrphostins on the Rate of Glucose Uptake Glucose uptake (pmol/106 cells/min)

(PM)

AG17

AC126

AGl83

0

16.3 51.9 30.5 n.d.

13.8 17.5 33.8 20.4

12.7 10.9 35.3 34.0

1 IO 50

Note. Human PBM were incubated for 1 hr with the indicated concentrations of tyrphostin followed by determination of the rate of glucoseuptake asdescribed under Materials and Methods. n.d., not determined.

mM EDTA was added to the pellets. After another hour on ice, pellets were spun at 100,OOOgfor 20 min. Supernatants were stored at -70°C until ready for use. Protein concentrations were determined by the method of Lowry as modified by Markwell ( 18). The NF-KB DNA probe used was the -206 to - 195 region of the IL-2 promoter. Two-tenths nanogram (approximately lo4 cpm) of S-end-labeled DNA was added to

B. Con A

100

100

50

50

0

0 0

0.1

1

Tyrphostin

10 (JIM)

AC126

100

0

0.1

1

Tyrphostin

AC183

10 ()rM)

100

0

1 Tyrphostin

10

100 (JIM)

AG17

FIG. I. Effect of tyrphostins on lymphocyte mitogenesis. Human PBM were treated with the indicated concentrations of tyrphostin AC I26 (0), AG 183 (O), or AC 17 (m) in the presenceof 2 pg/ml phytohemagglutinin (PHA, A), 2 &ml concanavalin A (Con A, B), or 500 U/ml interleukin-2 (IL-2, C). Data represent means of triplicate cultures from one representative experiment. The cpm of cells unexposed to tyrphostins but treated with PHA or Con A was between 53,000 and 6 1,000 and for IL-2 between 17,000 and 20,000 and set equal to 100%.The structures of the various tyrphostins are shown.

TYRPHOSTINS

0

STIMULATE

20

10 Time

after

PHA

HUMAN

40

30 addition

159

PBM

50

(hr)

FIG. 2. Effect of tyrphostin AC 17 added at different time intervals on PHA-induced [3H]thymidine incorporation. Human PBM were treated with PHA (2 &ml) at time zero. Tyrphostin AG I7 (1 PM) was added at time of culture (0 hr) or after the indicated time elapsed from PHA addition. [3H]Thymidine was added at 48 hr and cells assayedfor mitogenicity as described under Materials and Methods. The mean f SD cpm for untreated cells was 805 f 148 and with PHA alone was 53,778 f 5623.

5 pg nuclear protein and 2 pg Poly(dI-dC) in 20 ~1 of 10 mM Tris, pH 7.5, 50 mM NaCl, 1 mM EDTA, 1 mM DTT, and 5% glycerol at room temperature for 20 min. Protein-DNA complexes were resolved on 4% polyacrylamide gels in 0.25X TBE at 150 V for 1.5 hr at room temperature. Gels were dried and exposed to x-ray film. RESULTS Effect of Tyrphostins on Glucose Uptake An early event of lymphocyte activation is the increase in the rate of glucose uptake. We have found the tyrphostins AG 126, AG 183, and AG 17 to be potent enhancers of glucose uptake in human PBM (Table 1). The most potent of these was AG 17, which maximally stimulated at 1 PA+?AG126 and AG183, on a molar basis, were approximately 1O-fold lesspotent but maximally stimulated glucoseuptake to the samedegree. Efect of Tyrphostins on Stimulation of [3H]Thymidine Incorporation Induced by D@erent Agents The effect of the tyrphostins AG126, AG183, and AG17 on [3H]thymidine incorporation in human PBM induced by different agents was investigated. AG126 had little effect on PHA stimulation, had a modest enhancing effect on Con A stimulation, and inhibited IL-2-induced stimulation. AG183 had a slightly inhibitory effect on PHA and Con A stimulation and like AG 126, inhibited in a dose-dependent manner

160

LANDER, LEVINE, AND NOVOGRODSKY TABLE 2 Effect of Tyrphostins on MLC and MLC-Induced CTL Generation Treatment A AC17

[‘H]Thymidine incorporation (c.p.m.)

0 l/,rM

2806 + 600 f 780 f 419 f 388 f 1752 f 2632 f 993 f 392 f

2d4 5lrM 10j1M AC126 1 rul/l

21J4 5pM 10pM

345 240 64 65 233 642 232 385 243

Specific 5’Cr release(%) Treatment B

5O:l

25:l

0 1pM IOjtM AC126 I pM 10 pM

7.6 1.8 0.4 5.0 5.8

3.4 2.4 1.0 3.8 2.8

AC17

Note. Treatment A: [‘HlThymidine incorporation induced in the MLC using human PBM as responder cells and irradiated PBM from another donor as stimulator cells was assessedas described under Materials and Methods. Treatment B: MLC-induced CTL generation in human PBM using irradiated PBM from another donor was assessedas described under Materials and Methods.

IL-2 stimulation. AG17 was most potent, on a molar basis, in inhibiting stimulation by PHA, Con A, and IL-2 (Fig. 1). Efect of Tyrphostin AG17 Added at Dlxerent Time Intervals on PHA-Induced [3H]Thymidine Incorporation The effect of adding tyrphostin AG 17 at different time intervals after stimulation with PHA was investigated (Fig. 2). AG17 was added at time of culture (0 hr) or at 2, 4, 8, 18, 24, or 48 hr after addition of PHA. The 48-hr cultures were treated with tyrphostin just before the addition of [3H]thymidine. The results suggestthat addition of tyrphostin AG 17 interferes with late events involved in PHA-induced mitogenicity. Efect of AGI 7 on the MLC and on A4LC Induction of CTL We examined whether AG17, a potent inhibitor of mitogenicity and cytotoxicity induced by various mitogens, could inhibit the MLC and MLC-induced CTL generation (Table 2). The data show that tyrphostin AG 17, at low dose, effectively inhibited the MLC. In addition, CTL generation was also effectively blocked. Efect of Tyrphostins on IL-2-Induced Cytotoxicity The effectsof tyrphostins AG 126, AG 183, and AG 17 on IGZ-induced cell-mediated cytotoxicity was investigated. We have found that both AG 126 and AG 183 augmented

TYRPHOSTINS

STIMULATE

HUMAN 0

0

I

161

PBM

, Cl AG126

I Tyrphostin

(PM)

FIG. 3. Effect of AC126 and AC183 on IL-2-mediated cytotoxicity. Human PBM effector cells were incubated with IL-2 (500 U/ml) and the two tyrphostins at the indicated concentrations. After 48 hr, effector cells were mixed with RC29 target cells at the indicated ratios and the cytotoxicity assaywas performed as described under Materials and Methods.

cytotoxicity with a maximal effect seen at concentrations of 2 puMfor AG126 and 10 pLM for AG183. Higher concentrations of the tyrphostin were less stimulatory (Fig. 3). AG 17 markedly inhibited IL-2-induced cytotoxicity and 50%inhibition wasattained at concentrations of 2 pA4 (Fig. 4). 40 0 0

0

Control IL-2 500

U/ml

-\ 0

y\

0

\

.-------.

o--o\ O\n/O

0 0

2

4

8

6 AG17

10

12

(PM)

FIG. 4. Effect of AC17 on IL-2-mediated cytotoxicity. Human PBM effector cells were incubated with or without IL-2 and AC I7 at the indicated concentrations. After 48 hr. effector cells were mixed with RC29 target cells and the cytotoxicity assay was performed as described under Materials and Methods. The data shown are for a 10:1 effector:target ratio.

162

LANDER, LEVINE, AND NOVOGRODSKY

EJect of Tyrphostins on Cytokine Production We investigated the effects of tyrphostins on their ability to induce cytokine formation by human PBM in vitro. We found that AG126 stimulated TNF-a releaseat concentrations of lo-50 PM (Fig. 5). AG183 also induced TNF-a release but to a lesser extent. Coincubation of PBM with the tyrphostins and IL-2 yielded increased levels of TNF-a in the supernatant than with the tyrphostins alone (Fig. 5). In contrast, AG126 alone did not stimulate IL-l secretion by human PBM but did so in the presence of phenylarsine oxide (PAO), an oxidant known to induce protein tyrosine phosphorylation and stressproteins (19) (Fig. 6). PA0 by itself stimulated IL- 1 proliferation by these cells. AG 126 had only a marginal stimulatory effect on PBM treated with heat shock. AG 126 by itself had no effect on IL-6 secretion. Heat shock synergized with AG126 in inducing IL-6 secretion (Fig. 7). PA0 induced very high levels of IL6 secretion by itself and there was no further induction by AG 126.All of the tyrphostins tested negative for endotoxin in the limulus amoebocyte lysate assay(data not shown). IFN-y secretion is also affectedby tyrphostins. PAO, and to some extent heat shock, synergized with AG126 at low dose (2 p*M) in eliciting IFNq secretion. Higher dose ( 10 PM) inhibited secretion induced by PA0 (Fig. 8). There is a disparity in the ability of the tyrphostins AG 126 and AG 183 to inhibit IL-2-induced EN-7 secretion. AG 183 had no effect up to 50 FM, whereas AG126 inhibited above 20 PM (Fig. 9). Effect of Tyrphostin AG126 Added 48 hr after IL-2 Stimulation on IFN-7 Production We examined whether AG126 could block the inductive effects of IL-2-induced IFN-7 secretion. The data indicate (Table 3) that AG126 inhibits early inductive effects culminating in IFN--r production. Addition of tyrphostin 48 hr following IL2 stimulation did not inhibit secretion of IIN- between 48 and 72 hr. This indicates that cells already committed to produce IFN-y were tyrphostin resistant. 2500

2000 0 No Addition W IL-2 10 u/ml

1500

1000

500

0

10’

100 Tyrphostin

102

(JJM)

FIG. 5. Effect of tyrphostins on TNF-ol production. Human PBM were inqbated in the presence or absenceof IL-2 and tyrphostins at the indicated concentrations. After 48 hr, supernatants were assayedfor cytokine content as described under Materials and Methods.

TYRPHOSTINS

STIMULATE

HUMAN

163

PBM

3500 3000 2500 -i \ Et i 2

2000 1500 1000

0

AG126

(FM)

FIG. 6. Effect of AC I26 on IL- I production. Human PBM were treated with various stressstimuli in the presence of the indicated concentrations of AG126. Heat shock denotes heat treating cells for 30 min at 40°C and then placing at 37°C. After 48 hr, supernatants were assayedfor cytokine content as described under Materials and Methods.

Efect of Tyrphostins on Nuclear Events The ability of tyrphostins to induce cytokine secretion, especially upon costimulation with certain stressstimuli led us to examine whether a transcription factor known to

3000 /

2000 -

0

loo0 ./; 0 0

I 100

10' AG126

n 102

(uM)

FIG. 7. Effect of AG 126 on IL-6 production. Human PBM were treated with various stressstimuli in the presence of the indicated concentrations of AG126. Heat shock denotes heat treating cells for 30 min at 40°C and then placing at 37°C. After 48 hr, supernatants were assayedfor cytokine content as described under Materials and Methods.

164

LANDER, LEVINE, AND NOVOGRODSKY

T \ 3 iI t-

FIG. 8. Effect of AG I26 on IFN-+yproduction. Human PBM were treated with various stressstimuli in the presence of the indicated concentrations of AG126. Heat shock denotes heat treating cells for 30 min at 40°C and then placing at 37°C. After 48 hr, supematants were assayedfor cytokine content as described under Materials and Methods.

play a role in regulating cytokine gene transcription (20) was activated in PBM treated with the tyrphostins. We found that AG126 at 2 FLMinduced NF-KB activation in human PBM and higher dose (50 p.M) was less stimulatory (Fig. 10). The tyrphostin AG 17 and to a lesserextent AG 126 were found to induce the expression of hsp70 mRNA in human PBM (Fig. 11).

150

0 n

0

No Addition IL-2 10 u/ml

10’

100 Tyrphostin

102

(jrM)

FIG. 9. Effect of AG 183 and AG 126 on IL-2-induced IFN-7 production. Human PBM were treated with or without IL-2 (10 U/ml) and tyrphostins at the indicated concentrations. After 48 hr, supematants were assayedfor cytokine content as described under Materials and Methods.

TYRPHOSTINS STIMULATE

HUMAN

165

PBM

TABLE 3 Effect of AG 126 Added 48 hr after IL-2 Stimulation on IFN-7 Production

- AG126 (50 /.&f)

+ AC126 (50 PM)

0 IL-2

0 92

0 0

0 IL-2

0 38

0 42

Treatment A.

B.

Note. Treatment A: Human PBM were untreated or treated with IL-2 (10 U/ml) for 72 hr in the absence (-) or presence(+) of AG126 (50 pAJ). Treatment B: Human PBM were untreated or treated with IL-2 (10 U/ml) for 48 hr, washed twice, and then exposed to AG126 (50 PM) for 24 hr. Supernatants were collected and assayedfor IFN--y as described under Materials and Methods.

DISCUSSION Triggering of the T-cell receptorcomplex by either antigens or mitogens has been shown to increase tyrosine phosphorylation of several substrates in both murine and human T cells (1). Tyrosine phosphorylation was implicated to be crucial for the transduction of these signals. AG 126 (PM):’ 0

2 50’

NF-KB FIG. 10. Effect of AG 126on NF-KB activation. Human PBM were treated for 4 hr with or without AG 126 at the indicated concentrations.The nuclear proteins were isolated and assayedfor NF-KB binding as described under Materials and Methods.

166

LANDER, LEVINE, AND NOVOGRODSKY

0

B

H.S.: ‘-

AG17 T

AG126 T

2.6 kb -

FIG. 11. Effect of tyrphostins on hsp70 mRNA expression. Human PBM were incubated at 37°C for 3 hr or heat shocked at 42°C for 30 min subsequent to a 3-hr incubation at 37°C with or without 50 PM of the indicated tyrphostin. Cells were then analyzed for hsp70 mRNA content as described under Materials and Methods. (A) The ethidium bromide-stained gel prior to transfer and (B) the autoradiograph of the probed blot.

Recently, a seriesof protein tyrosine kinase inhibitors were synthesized and shown to be able to inhibit a variety of tyrosine kinase activities including those of the insulin, epidermal-derived growth factor and platelet-derived growth factor receptors ( 13- 16, 2 1). Tryphostins were also shown to inhibit tyrosine phosphorylation and proliferation of human B cells induced by the SAC mitogen (12). In this report, we examine the effect of severaltyrphostins on human PBM functions and induced responses. We have found that tyrphostins have inductive effects on resting and mitogen-stimulated human lymphocytes. In addition, inhibitory properties toward mitogen-stimulated lymphocytes were revealed. At low concentrations, the tyrphostins enhanced glucose uptake and maximal stimulation was attained at a concentration characteristic for each of the tyrphostins used. Higher concentrations were less effective. The tyrphostins AG 126 and AG 183 were also found to enhance IL-2induced cytotoxicity in human PBM in a biphasic manner. In contrast, the tyrphostin AG17 markedly inhibited IL-2-induced cytotoxicity at low concentration as well as MLC-induced CTL generation and no stimulatory effect was observed. The tyrphostins tested had selective effects on mitogenicity of PBM to different agents as measured by [3H]thymidine incorporation. The most potent inhibitor was AG17. IL-2-induced mitogenesis was more susceptible to inhibition as compared to Con A- or PHA-induced mitogenesis. IL-2 is considered the ultimate mitogen involved in stimulation of T lymphocytes induced by a variety of agents. Therefore, it is most plausible that the selective effects of tyrphostins on mitogenicity induced by IL-2 versus the lectins is related to their effect on the generation of the ~55 subunit of the IL-2 receptor necessary for the formation of the high-affinity form of the receptor.

TYRPHOSTINS STIMULATE

(inactive)

HUMAN

r-tPTKl

PBM

167

Tyr-P

I!

$

Biological Response FIG. 12. Possible mechanism of agonistic properties of tyrphostins. Tyrphostins which block the protein tyrosine kinase (PTK?) responsible for inactivation of the src family PTK (PTK,) may induce agonistic properties similar to those induced by classical activation signals.

Tyrphostin AG 17 also inhibited late events associatedwith PHA-induced mitogenicity. Cell viability was greater than 95% as evidenced by trypan blue exclusion. Tyrphostins also affect cytokine secretion by human PBM. AG126 and AG 183 enhance TNF-LUsecretion and this effect was more prominent in the presence of IL2. AG126 enhanced IFN-7, IL-l, and IL-6 production in PBM that was costimulated with the stressstimuli heat shock and PAO. All tyrphostins testednegative for endotoxin in the limulus amoebocyte lysate test. The stimulatory effects of the tyrphostins on cytokine secretion and induction of cytotoxicity might be interrelated. Our data demonstrated that the tyrphostins by themselves could elicit nuclear events such as NFKB activation establishing the agonistic nature of these reagents. It should be noted that NF-KB was previously shown to be activated by a variety of agents inducing cell proliferation and cytokine production. The mechanism of the stimulatory effect of tyrphostins on PBM functions and induced responses is unknown. The tyrosine kinases of the src family, p561Ckand p59fy”,were implicated to play a role in transduction of triggering signalsin lymphocytes (4-7). These tyrosine kinases are negatively regulated by phosphorylation of a carboxylterminal tyrosine residue (8). Thus, it is possible that inhibitors of tyrosine kinases may lead to activation of the src kinases (Fig. 12). CD45, a phosphotyrosine phosphatase, was also implicated to be involved in activation of ~56’~~and ~59~Y”tyrosine kinases (10). Another possibility is that the tyrphostin effect is mediated by inhibiting tyrosine phosphorylation of a regulatory protein leading to modulation of its function. The agonistic and antagonistic effects of tyrphostins on lymphocyte functions may have therapeutic potential. AG 17,which at micromolar levels inhibits multiple immune functions in vitro as well as the MLC and MLC-induced CTL generation, could be evaluated as an immunosuppressive agent in vivo. The immune stimulatory properties of the tyrphostins such as AG 126 and AG 183 could also be further evaluated for their potential antitumor effects. ACKNOWLEDGMENTS We thank Dr. Alexander Levitzki, Hebrew University, Jerusalem, Israel, for generously providing the tyrphostins and helpful discussions, Dr. Prabodh Sehajpal for help with the DNA-protein binding assay,

168

LANDER, LEVINE, AND NOVOGRODSKY

and Rachel Schwartz for her excellent technical assistance.This work was supported in part by a grant from the HRC Foundation.

REFERENCES 1. Klausner, R. D., and Samelson, L. E., Cell 64, 875, 1991. 2. June, C. H., Fletcher, M. C., Ledbetter, J. A., and Samelson, L. E., J. Immunol. 144, 1591, 1990. 3. Nishibe, S., Wahl, M. I., Hernandez-Sotomayor, S. M. T., Tonks, N. K., Rhee, S. G., and Carpenter, G., Science 250, 1253, 1990. 4. Rudd, C. E., Immunol. Today 11,400, 1990. 5. Luo, K., and Sefton, B. M., Mol. Cell. Biol. 10, 5305, 1990. 6. Chalupny, N. J., Ledbetter, J. A., and Kavathas, P., EMBO .I. 10, 1201, 199I. 7. Cooke, M. P., Abraham, K. M., Forbush, K. A., and Perlmutter, R. M., Ceil 65, 28 1, 1991. 8. Amrein, K. E., and Sefton, B. M., Proc. Natl. .4cad. Sci. USA 85, 4247, 1988. 9. Trowbridge, I. S., Ostergaard, H. L., and Johnson, P., Biochim. Biophys. Acta 1095, 46, 199 I. 10. Ostergaard, H. L., and Trowbridge, I. S., J. Exp. Med. 172, 347, 1990. Il. Roifman, C. M., Chin, K., Gazit, A., Mills, G. B., Gilon, C., and Levitzki, A., J. Immunol. 146, 2965, 1991.

12. Stanley, J. B., Gorczynski, R., Huang, C-K., Love, J., and Mills, G. B., .I. Immunol. 145, 2189, 1990. 13. Lyall, R. M., Zilberstein, A., Gazit, A., Gilon, C., Levitzki, A., and Schlessinger,J., J. Biol. Chem. 264, 14503, 1989. 14. Shechter, Y., Yaish, P., Chorev, M., Gilon, C., Braun, S., and Levitzki, A., EMBO .I. 8, 1676, 1989. 15. Shiraishi, T., Owada, M. K., Tateuka, M., Yamashita, T., Watanabe, K., and Kakunaga, T., Cancer Rex 49, 2374, 1989. 16. Yaish, P., Gazit, A., Gilon, C., and Levitzki, A., Science 242, 933, 1988. 17. Wu, B., Hunt, C., and Morimoto, R., Mol. Cell. Biol. 5, 330, 1985. 18. Markwell, M. A. K., Haas, S. M., Bieber, L. L., and Tolbert, N. E., Anal. Biochem. 87, 206, 1978. 19. Garcia-Morales, P., Minami, Y., Luong, E., Klausner, R. D., and Samelson, L. E., Proc. Natl. Acad. Sci. USA 87, 9255, 1990. 20. Lenardo, M. J., and Baltimore, D., Cell 58, 227, 1989. 21. Bilder, G. E., Krawiec, J. A., McVety, K., Gazit, A., Gilon, C., Lyall, R., Zilberstein, A., Levitzki, A., Perrone, M. H., and Schreiber, A. B., Am. .I. Physiol. 260, C721, 1991.

Agonistic effects of tyrphostins on human peripheral mononuclear cells.

Tyrosine kinases of the src family, p56lck and p59fyn, were implicated in the transduction of signals via the T-cell receptor complex. These kinases a...
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