Role of protein phosphatases in the regulation of nonspecific cytotoxic cell activity

Pergamon

Developmental and Comparative Immunology, Vol. 18, No. 2, pp. 13%146, 1994 Copyright © 1994 Elsevier Science Ltd Printed in the USA. All fights reserved 0145-305X/94 $6.00 + .00

0145-305X(94)E0011-3

ROLE OF PROTEIN PHOSPHATASES IN THE REGULATION OF NONSPECIFIC CYTOTOXlC CELL ACTIVITY Donald L. Evans and Liliana Jaso-Friedmann Department of Medical Microbiology, College of Veterinary Medicine, University of Georgia, Athens, GA 30602 (Submitted December 1993; Accepted February 1994)

rqAbstract--In vitro and in vivo experiments were conducted to determine the effects of the protein phosphatase (PPase) inhibitors sodium fluoride (fluoride), sodium orthovanadate (vanadate), and lithium chloride on nonspecific cytotoxic cell (NCC) lysis of target cells. Both vanadate and fluoride stimulated NCC activity. Lithium chloride had no effects. Optimum enhancement for "normal" NCC was at low effector:target cell ratios and at least 30 rain treatment was required to achieve maximum activation effects. Fluoride, but not vanadate activation effects were largely reversible. Vanadate, 2.5-10 raM, produced a 5-10-fold increase in cytotoxicity at 25:1 E:T, whereas less than twofold increases were produced by these concentrations at 100:1. NCC activity from "stressed" fish that had essentially no cytotoxic activity were also activated by vanadate. In vitro preincuhation of NCC with 10-20 mM fluoride or 2.5-10 mM vanadate produced up to a 20-fold increase in stressed cytotoxicity. Combined treatments with 2.5 mM vanadate and 20 mM fluoride produced even greater responses. In vivo responses to vanadate were also determined. Treatment of catKsh by immersion in 50 pM vanadate produced significant increases in cytotoxicity by 24-48 h posttreatment. Activation of cytotoxicity was not accompanied by increases in percentage of NCC (small lymphocyte content) or in total cell numbers in anterior kidney tissue. These studies indicated that levels of NCC activity are partially regulated by control of dephosphorylation of membrane proteins. Inability of NCC from stressed fish to lyse IM-9 target cells was reversed (probably) by disruption of an equilibrium between kinase and phos-

phatase activities. Normal NCC were "superactivated" only under conditions were they were in limiting numbers. These data show that phosphatases must be considered as active participants in regulation of signal transduction processes. DKeywords--Protein phosphatases; Cytotoxic cell activity; Signal transduction; Ictalurus punctatus.

Introduction Important posttranslational modifications of certain proteins occur subsequent to ligand/receptor binding. T h e s e events m a y consist of phosphorylation of serine, threonine, or tyrosine amino acids. Although n u m e r o u s kinases h a v e b e e n described that participate in the production of these phosphoproteins, attention has not b e e n given to the role(s) of p h o s p h a t a s e s in establishing regulatory control of cytotoxicity. Natural killer (NK) cell studies have not previously considered p h o s p h a t a s e s as activators o f cytotoxicity. We p r o p o s e that this class of e n z y m e s not only exists in equilibrium with kinases to control levels o f amino acid phosphorylation, but p e r h a p s as important, they m a y participate as activators o f cytotoxicity. D e p h o s p h o r y l a -

Address c o r r e s p o n d e n c e to Donald L. Evans.

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tion of certain amino acids on kinases may initiate enzyme activity and significantly alter cytotoxicity. In the present study we propose that phosphatases initiate positive regulatory actions in nonspecific cytotoxic cells (NCC) resulting in increased cytotoxicity. We have previously described (1,2) a putative receptor molecule on catfish nonspecific cytotoxic cells that, following monoclonal antibody (mAb) or target cell binding triggers a c t i v a t i o n responses. The evidence that this putative receptor is a signalling molecule comes from other studies showing activation of cytotoxicity by phorbol ester and calcium ionophore treatment (3), modulation of cytotoxicity by mAb 5C6 (specific for the putative receptor) including release of significant levels of cytosolic free calcium (4) and redirected lysis of mAb 5C6 hybridoma cells by NCC (5). We have also shown that mAb 5C6 binding to NCC produces increased phosphorylation of several different membrane proteins (2). These experiments demonstrated that both tyrosine (2) and serine (unpublished data) were phosphorylated following binding. Tyrosine appeared to be the major amino acid involved in these reactions. Further evidence for the participation of this amino acid in NCC triggering responses was obtained using the protein tyrosine kinase (PTK) inhibitor genistein (2). Previous studies (6) have demonstrated that tyrosine phosphorylation is an obligatory intermediate step in NK signalling. Genistein almost completely inhibited NCC activity (2) indicating that similar to NK cells, activated NCC utilized tyrosine phosphorylation of membrane proteins as a step proximal to the initiation of a competent lyric cycle. Phosphatase inhibitors (zinc chloride, okadaic acid, etc.) have been previously utilized to demonstrate the role of phosphoproteins, including kinases, in signalling responses. Sodium fluoride and sodium orthovanadate appear to have pref-

D.L. Evans and L. Jaso-Friedmann

erential binding specificities for serine and tyrosine, respectively (7,8), although by no means are these inhibitors restricted to these phosphorylated amino acids. Phosphatases have previously not been considered by anyone to directly participate in NK/NCC cytotoxic reactivity. In the present study, we demonstrate that fluoride and/or vanadate significantly increase NCC lysis of IM-9 target cells. This occurs following either in vitro or in vivo treatment. The level of increase in cytotoxicity is inversely proportional to the native state of NCC activity, for example, maximum augmentation occurs under conditions of low NCC numbers.

Materials and Methods

Fish Outbred channel catfish (Ictalurus punctatus) of both sexes weighing 5-60 g, were obtained from local commercial farms. Fish were maintained as previously described (9).

Media and Chemicals All cell culture was done using RPMI 1640 (Cellgro, Mediatech, Washington, DC) as previously described (10). All media contained 10% fetal bovine serum (Sigma Chemical Co., St. Louis, MO). Phosphatase inhibitors were sodium fluoride (Sigma; S 6521; lot #41H04052) and sodium o r t h o v a n a d a t e (Sigma; S-6508; lot #52H0326) and lithium chloride (Sigma).

Preparations of Cell Suspensions Fish were lightly anesthetized with ethyl m-aminobenzoate (Sigma) in water and sacrificed. Anterior kidney cells

Phosphatase regulation of cytotoxicity were removed and single cell suspensions were prepared (10).

Target Cells Target cells used in cytotoxicity assays were IM-9 human EBV transformed B lymphocytes. Cells were grown in stationary cultures.

Cytotoxicity Assay A 51chromium release assay was used to determine cytotoxicity. Target cells were labeled with 100 i~Ci sodium chromate (Amersham, Arlington Heights, ILL NCC effector cells were prepared as previously described (10). Assay incubation time was 4 h. The results are expressed as percent specific release.

Inhibitor Treatment NCC were harvested and preincubated for 3 h in RPMI-1640 (10% FBS). Cells were counted, washed, and different concentrations of inhibitor were added to 8 x 106 or 4 X 10 6 cells/mL and incubated for 90-120 min (37°C). Following incubation, the inhibitors were removed from the NCC or the labeled IM-9 target cells were added without removing the inhibitor.

In Vivo Treatment With Sodium Orthovanadate Fish were treated by immersion in 10 gal (static flow) tanks containing the appropriate inhibitor concentrations in 15 L declorinated water (19-20°C). All fish were acclimated to tank conditions 24 h prior to treatment. Twenty fish (approxi m a t e l y 4 - 5 in. l e n g t h ) / t a n k w e r e treated. At 24-h intervals 50% of the water was changed.

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Results

Characterization of Effects of Phosphatase Inhibitors on NCC Activity The phosphatase inhibitors NaF and Na3VO 4 were previously shown (2) to significantly increase NCC activity in a dose-dependent fashion. In the present study additional characteristics of these activities were evaluated. We first determined the incubation times required to produce optimum effects on normal NCC lytic activity. NCC were treated with fluoride and vanadate for 15, 30, or 60 min and effects on the killing of IM-9 target cells was determined in a 4-h microcytotoxicity assay (Table 1). For healthy fish, less than a twofold increase was produced. More than 15-min treatment time was required to achieve maximum increases in cytotoxicity. To determine if the activating effects of the protein phosphatase (PPase) inhibitors were reversible, NCC were treated for 90 min with NaF and vanadate, and then the inhibitors were removed prior to addition of the labelled target cells. Normal NCC produced significantly increased cytotoxicity following treatment with vanadate (Fig. I) or NaF (Fig. 2) in the presence of the inhibitors. Removal of vanadate produced slightly reduced Table 1. Treatment Kinetics for Optimum Effects of Protein Phoaphataea Inhlbltora on NCC Lysla of IM-9 Target Cells. Percent Specific Releaser (min)$ Treatment*

15

30

60

Control NaF (20 mM) Na3V04 (5 mM)

49 81 71

52 90 89

55 92 90

* Inhibitors were not removed prior to addition of the target cells. t 20:1 effector:target cell ratio, -1:Time NCC were treated prior to the addition to the target cells.

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D.L. Evans and L. Jaso-Friedmann

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Concentretion (mU) Figure 1. Effects of removal of NaaVO4 on NCC activation. NCC were harvested, incubated for 3 h, washed, counted, and added to duplicate tubes containing either media alone or media containing 2.5, 5, and 10 mM vanadate. Following a 90-rain incubation time, the cells were either (0) washed and resuspended in media or (0) added directly to wells containing Slchromium labelled IM-9 target cells (E:T ratio 40:1).

levels of cytotoxicity, whereas N a F removal almost completely obliterated activation (Fig. 2). Toxicity control experiments (data not shown) revealed that 20 m M N a F was not toxic to labelled IM-9 targets and 10 m M vanadate produced less than 10% n o n s p e c i f i c t o x i c i t y (e.g., r e l e a s e of 5~chromium from IM-9 targets).

Experiments were next conducted to determine if the PPase inhibitors had effects on NCC from fish that had virtually no cytotoxicity (i.e., stressed fish). A c o m m o n observation (data not shown) in fish exposed to toxicants, drastic temperature changes, handling, etc. is the temporary loss of NCC activity. Normal activity does not return to this type of

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media, washed, counted, and added to duplicate tubes containing media alone or in the presence of 5, 10, and 20 mM NaF. Following a 90-min treatment time, NCC were either (0) washed or (©) added directly to wells containing labelled IM-9 target cells (E:T ratio 40:1).

Phosphatase regulation of cytotoxicity

stressed fish for several days to weeks. Control (stressed) fish had essentially no cytotoxicity (Table 2). Both fluoride (group I) and vanadate (group II) (individually) augmented cytotoxic activity from essentially zero to relatively high levels. The combined treatment effects (groups I l l - V ) were additive. Lithium chloride (5 and 10 mM) either individually or in combination had no effects on cytotoxicity (data not shown). The relationship between inherent levels of cytotoxicity and NCC responsiveness to inhibitor treatment was further examined by comparing the effects of the PPase inhibitors on fish with low, medium, and high cytotoxicity levels. In Figure 3, NCC from catfish with 2.0% control Specific Release were highly responsive to vanadate treatment. A similar relationship was observed following treatment with fluoride (Figure 4). Much higher concentrations of fluoride than vanadate were required to achieve activation responses. High responders were almost refractory to inhibitor treatment. T a b l e 2. Effects o f P h o s p h a t a s e Inhibltors on N C C Activity F r o m Stressed Fish.

141

In Vivo Responses of NCC to Inhibitor Treatment To determine if phosphatase inhibitors produced similar in vivo actions compared to in vitro treatments, fish were treated by immersion in vanadate, and cytotoxicity was determined at 10, 24, and 48 h posttreatment. Figure 5 shows that 50 I~M vanadate produced large increases in cytotoxicity by 48 h. Fluoride was not used in these experiments because of the high concentrations required to produce in vitro effects; and also because in vivo toxicity reactions in general by the phosphatase inhibitors were produced by much lower concentrations than in vitro. In vivo, levels of 125 ixM and higher were toxic to fish (data not shown); 2.5 and 5.0 mM were immediately lethal. To determine if the increased cytotoxicity was associated with changes in percent composition of small lymphocytes (i.e., NCC) and in total numbers of cells, total cell counts and percent NCC in anterior kidney tissue were determined. Table 3 shows that although some changes occurred in both parameters, notable large fluctuations were not seen.

Treatment

Group Control I

II

III

IV

V

NaF (mM)

NasVO 4 (mM)

Percent Specific Release*

-5 10 20 ---5 10 20 5 10 20 5 10 20

----2.5 5.0 10.0 2.5 2.5 2.5 5.0 5.0 5.0 10.0 10.0 10,0

0.9 1.4 4.4 22.0 7.3 9.9 13.1 14.0 22.5 36.1 16.4 22.6 27.4 20.1 17.1 20.1

* 100:1 effector:target cell ratio. Four-hour cytotoxicity assay.

Discussion The notion that phosphatases might be involved in the process of regulation of NCC cytotoxicity was derived from several different observations. Phosphatase inhibitors (LiCI) are required in experiments to detect increased levels of phosphatidylinositol metabolites (i.e., PIPs) that appear during cell triggering responses. Lithium chloride specifically inhibits phosphatidylinositol phosphate phosphatase activity. Other phosphatase inhibitors (zinc chloride, sodium fluoride, okadaic acid, etc.) have been used in numerous studies to demonstrate the

142

D.L. Evans and L. Jaso-Friedmann

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build-up of phosphoproteins following agonist binding. We have previously shown that mAb 5C6 binding to NCC activates cytotoxicity (3), initiates the re-

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lease of free cytoplasmic calcium (4,5), and causes increased levels of IP3 production (unpublished results). Increasing and prolonging levels of protein phos-

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Concentration (mM) Figure 4. Effects of NaF on NCC with different levels of cytotoxicity. NCC were harvested from anterior kidneys of fish from three different sources. The cells were incubated in media for 3 h, washed, counted and added to media or media with 5, 10, and 20 mM NaF. Following a 2 h incubation, NCC were added to wells containing Slchromium labelled IM-9 target cells for a final E:T ratio of 40:1.

Phosphatase regulation of cytotoxicity

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144

Table 3. Effects of In Vlvo Vanedate Treatment on NCC Content of Anterior Kidney Tissue.

D.L. Evans and L. Jaso-Friedmann

augments phospholipase C-gamma-1 activity in the membrane of activated cells, further supported our hypothesis that Treatment (% NCC) phosphatases may participate directly in Hours Control NCC lytic responses. Posttreatment % NCC 50 ixM 125 t~M We also have preliminary data sug3 60 (3.2)* 51 (3.1)* 53 (6.5)* gesting that p56 lCk is constituitively ex24 55 (2.6) 60 (2.8) 52 (3.2) pressed in NCC and that levels of this 48 52 (2.1) 66 (3.5) 73 (2.7) cytoplasmic kinase increase significantly 72 70 (2.3) 72 (3.1) 61 (1.9) 96 61 (3.6) 78 (1.6) -following mAb 5C6 binding (unpublished data). Previous studies of mammalian or * Total cells/mL × 10e. cold-blooded vertebrate NK/NCC have not considered the possibility that phosphorylation could thus be associated phatases are directly associated with cytotoxicity. In the present study we have with increased cytotoxicity. Other studies that suggested that al- shown (indirectly) that these enzymes terations in dephosphorylation by phos- are responsible for downregulation of cyphatase inhibitors might affect cytotox- totoxicity. Inhibition of this effect by icity comes from data demonstrating that vanadate produces a significant increase vanadate-stimulated cellular carbohy- in cytotoxicity. This action is produced drate biosynthetic pathways could possi- under conditions where inherent levels bly progress to cell activation responses. of in vivo cytotoxicity are low because of Vanadate has insulin-minetic activity stressful conditions, or the activity of the (11-14) as d e m o n s t r a t e d by studies phosphatase inhibitors can be experishowing: augmentation of insulin binding mentally demonstrated by producing in(14); prolongation of insulin activity (14); creased cytotoxicity (in vitro) at low efstimulation of glucose oxidation (11); fector:target cell ratios (Tables 1, 2). Unand stimulation of glycogen synthetase der experimental conditions, such as activity (12). The study that suggested higher E:T ratios, numbers of cytotoxic that alterations of phosphatase activity cells are not limiting in the presence of might affect cytotoxicity was, in trans- 104 targets, thus the maximum possible formed human B-cells (15) and rat adipo- lyric activity occurs. At low E:T ratios cytes (16), vanadate directly activated the numbers of effectors are reduced in the presence of the same concentration kinases. Additional models to suggest that of targets (i.e., 104 cells). An activated phosphatases might participate in signal- NCC could, under these conditions, ling responses in NCC are studies of the have an increased recycling capacity to glycoprotein CD45 (although this mole- enable a single NCC to kill significantly cule does not exist on NCC). CD45 par- more target cells. The in vitro effect(s) of vanadate on ticipates in T-cell activation processes (16-23). Specifically, the substrate rec- NCC from stressed catfish could indicate ognized by CD45 appears to be p56 Ick that the phosphatase substrate(s) con(21,23). This kinase is probably activated taining the retained phosphotyrosine(s) by dephosphorylation of Tyr 5°5 that pre- might occupy a common position in sigcedes autophosphorylation, activation of nalling pathways required for elicitation the zeta chain (22), etc. These data, com- of cytotoxicity. More likely, because the bined with studies showing that vanadate effects of vanadate are heterogenous, produces relatively large increases in several protein tyrosine (and serine) phosphoinositol hydrolysis (17) and also phosphatases are probably involved at

Phosphatase regulation of cytotoxicity

145

different stages of the lytic activation pathways. Perhaps the most surprising results were that vanadate treated catfish had increased cytotoxicity 24-48 h posttreatment (Fig. I). This observation might hold great promise for future studies of the ability of PPase inhibitors to activate nonspecific immunity in fishes as both

preventative and therapeutic measures. Studies are in progress to answer these important questions. Acknowledgements--Research supported by U S D A grant 1-7924-01; by U n i v e r s i t y of Georgia, College of Veterinary Medicine, Veterinary Medical Experiment Station Grant 93-221-95004.

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21. Mustelin, T.; Coggeshall, K. M.; Altman, A. Rapid activation of the T-ceU tyrosine protein kinase pp56~¢k by the CD45 phosphotyrosine phosphatase. Proc. Natl. Acad. Sci. USA 86: 6302-6306; 1989. 22. Salcedo, T. W.; Kurosaki, T.; Kanakaraj, P.; Ravetch, J. V.; Perussia, B. Physical and functional association of p56~ck with Fc-gamma-

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RIIIA (CDI6) in natural killer cells. J. Exp. Med. 177:1475-1480; 1993. 23. Ostergaard, H. L.; Shackelford, D. A.; Hurley, T. R.; et al. Expression of CD45 alters phosphorylation of the lck-encoded tyrosine protein kinase in murine lymphoma T-cell lines. Proc. Natl. Acad. Sci. USA 86:8959; 1989.