Reversal of feline retroviral suppression by indomethacin

Leukemia Research Vol. 9, No. 12. PP. 1451~1456. 1985, Printed in Great Britain.

0145-2126/8553.00 ÷ ,00 (c~ 1985 P e r g a m o n Press Ltd.

REVERSAL OF FELINE RETROVIRAL SUPPRESSION BY INDOMETHACIN* MARK G. LEWlS,I" RICHARD H. FERTEL~ and RICHARD G. OLSENI"§ tDepartment of Veterinary Pathobiology, :l:Department of Pharmacology and §Department of Microbiology and Comprehensive Cancer Center, The Ohio State University Columbus, OH 43210, U.S.A. (Received 20March 1985. Accepted 30May 1985)

Abstract--The immunosuppressive effect of feline leukemia virus (FeLV) and its 15,000 dalton envelope protein (pl5E) were studied to determine if the mechanism of action was due to an increase in prostaglandin production. We examined the effects of exogenous PGE, and PGE2 on the normal Con A response of feline peripheral blood lymphocytes (PBL) and found them to be inhibitory. The addition of the cyclooxygenase inhibitor indomethacin to cells incubated with FeLV or FeLV DI5E and Con A completely abrogated the viral suppressive effects. This reversal was titratable and time-dependent. Other non-steroidal anti-inflammatory (NSAI) drugs were found to have similar actions, lndomethacin was also able to increase the suppressed Con A response of PBL from FeLV-infected cats. Upon measurement of PGE2 levels from PBL cultured with FeLV, we found a decrease in PGE2 accumulation associated with FeLV presence during the first 24 h of culture. These findings indicate that FeLV does not cause its immunosuppressive effects by increasing PG production and suggests that indomethacin and the other tested NSAI drugs do not produce their effect by PG inhibition. Key words: Indomethacin, feline leukemia, prostaglandin E, lymphocyte, immunosuppression, retrovirus, Con A activation.

INTRODUCTION FELINE leukemia virus (FeLV), like other retroviruses, can cause a variety of disease syndromes including lyrnphoproliferative disease, anemias, hemopoietic degenerative disease and immune deficiency [12]. The association between retroviral infection and retroviral immunodeficiency has been reinforced with the recent recognition of human T-cell leukemia virus llI (HTLV-III) as the causative agent of human acquired immune deficiency syndrome (AIDS) [2]. The inherent immunosuppression caused by retroviruses has been well documented (for 1-evie~ see [3]). Mathes et al. [4] and Hebebrand et al. [5] sho~ed that ultraviolet light-inactivated FeLV, as well as

*Supported in part by NIH grants CA-31547-02 and 30338-03 and American Cancer Society grant 1N-16W. Abbreviations: FeL l. feline leukemia virus: HTL 11, human T-cell leukemia virus; AIDS, acquired immune deficiency syndrome: pl5E, 15,000 dalton envelope protein: indo, indomethacin; Con A, concanavalin A; KT-FeL V, KawakamiTheilen FeLV: LBT, lymphc, blast transformation test: u.v., ultraviolet: PBL~ purified blood lymphocytes; PG, prostaglandin: NSAI, non-steroidal anti-inflammatory: NDGA. nordehydroguararetic acid. Correspondence to: Dr M. G. Lewis, The Ohio State Uni\ersit\, Department of Veterinary Pathobiolog3, 1925 Coffex Road, Columbus, OH 43210, U.S.A.

the purified 15,000 dalton FeLV envelope protein (pl5E) can inhibit lectin and antigen-induced blastogenesis. Additional studies found that FeLV and FeLV-pl5E inhibited lectin cap formation and interleukin 2 stimulation of Con A activated lymphocytes [6-8]. Work from several other laboratories has confirmed these results and has further implicated p l 5 E as an inducer of in-vivo immune dysfunction [9-11]. The mechanism by which feline p l 5 E causes its immunosuppressive action has yet to be established. Since feline p l 5 E is a lipophilic protein [12], its association with, and integration into cellular membranes may cause an interference with essential membrane signals. Nichols et al. [13] found that the inhibitory effect of FeLV on surface receptor mobility could be reversed by adding cholchicine, suggesting a microtubular interaction at the cell membrane. There are a number of important cellular growth modulating systems associated with the cell membrane. In particular, the prostaglandin system is known to cause immune modulation (for review see [14]). In this report we have investigated the possibility that pro8taglandin production may play a role in the action of FeLV. We found that the addition of a prostaglandin synthesis inhibitor, indomethacin (indo), was able to completely abrogate the suppressive effects of FeLV on Con A stimulation o f peripheral blood lymphocytes. However, measurements of PGE2 levels suggest that the reversing ability of indomethacin may not be related to its inhibition of cyclooxygenase.

1451

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MARK G. LEwis, RICHARD H. FERTELand RICHARD G. OLSEN MATERIALS

AND

METHODS

Animals Adult cats from the specific-pathogen-free cat colony for the Department of Veterinary Pathobiology, Ohio State University were used as blood donors. For the study of FeLV viremic animals, four cats received a 20% (W/v) cell-free tumor homogenate containing 8 x 1@ focus forming units/ml of in vivo passed Richard FeLV [15]. All 4 cats demonstrated chronic viremia as tested by an indirect immunofluorescence test [16].

Blood preparation Venous blood was drawn in 0.4% Na citrate. Mononuclear cells were isolated using a Ficoll-Hypaque (Pharmacia, Piscataway, N J) density gradient. Cells were removed and washed in 1640 medium (GIBCO, Grand Island, NY) with 0.4o70 Na Citrate (Sigma, St Louis MO) and 1.0% antibiotics (penicillin G, streptomycin sulfate).

Virus isolation

.....

The purification of Kawakami-Theilen FeLV (KT-FeLV) from tissue culture media has been described (17). Sucrosebanded virus was dialyzed twice in Hank's Balanced Salt Solution (GIBCO) and once in 1640 (GIBCO) with 1.0% antibiotics. The virus was inactivated by using ultraviolet light according to the method of Yohn et al. [18]. Protein determination was performed using a BioRad Protein Kit (BioRad, Richmond, CA).

Purification of pl5E T h e ~ u r i f i c a t i o n of FeLV pl5E from KT-FeLV has been described previously [4]. A few modifications were used. Briefly, the purified virus was extracted with Triton X containing buffer (1.0°70 Triton X100, 0.05M Tris-HCl, 0.6M KCt, 0.01M EDTA, 0.01M Dithiothreitol) and ultracentrifuged to remove undissolved elements. The supernatant fraction was extracted with ether and dialyzed against distilled water. The precipitate was collected by centrifugation, resuspended in Triton X buffer and added to a Sephacryl 200 (Pharmacia) column. Fractions containing the protein were collected, ether extracted and dialyzed against distilled water. Purity was determined by polyacrylamide electrophoresis. The purified protein was then dialyzed and u.v. irradiated.

Lymphoblast transformation test The lymphoblast transformation test (LBT) is a modification of one which has been previously described [5]. Briefly, purified blood lympbocytes (PBL) were added at a concentration of 1 x 10~ cells per well to a 96-well plate. The Con A was titrated against normal PBL for maximal stimulation. Con A (4 big, Miles Biochemicals, Elkhart, IM), FeLV (40 fag), FeLV pl5E (2 ,g), a n d / o r prostaglandin inhibitors were added to bring the total volume per well to 200 I.tl. Indomethacin, Nordihydroguararetic acid (NDGA), aspirin, tranylcypromine and imidazole (Sigma) were dissolved in 95% ethanol and diluted in culture media; a control solution of ethanol alone had no effect on this assay system. Na flunixin (Shering Corp., Kenilworth, N J) was dissolved in media. Five percent fetal calf serum (Sterile Systems, Salt Lake City, UT) was added and the suspension was incubated 72 h. Eighteen hours before harvest the cultures were pulsed with [~H] thymidine (New England Nuclear. Boston,

MA). The plates were harvested on glass wool using a Mash unit (Brandel, Gathersburg, MD) and assessed for thymidine uptake using a liquid scintillation counter IPackard Instruments. Downers Grove. ILl.

Assay for PGEz Purified blood lymphocytes (5 × 10"/well) were plated in a 24-wall plate (CoStar) with RPMI 1640 media containing 1.0% glutamine, Hepes, antibiotics and 5.0°'o fetal calf serum. Con A (20 ~g), FeLV (200 ~tg) a n d / o r Indo (0. i ~M) were added to a total of 1.0 ml of media per well. At the specified times the cells were removed and centrifuged. The supernatant was removed and frozen for later prostaglandin quantitation. The concentration of PGE2 was assessed by radioimmunoassay (RIA). Antibodies were obtained from chicken egg yolks after injections of PG coupled to keyhole limpet hemocyanin [19]. The concentration of antibody used was sufficient to bind 30-40% of its specific PG ligand. Twelve concentrations of unlabeled PG standard ranging from 1 to 10,000 pg/0.1 ml were utilized. All reagents were adjusted to the appropriale concentration with 50 mM Tris buffer (0. l% bovine serum albumin, 0.05% sodium azide, pH 7.5). Each 10 × 75 mm glass tube contained 0.05 ml of antibody, 0.05 ml o f ' H - P G ( 1 7 pg or 15,000 dpm) (New England Nuclear) and either 0.01 ml of PG standard or medium sample. The final volume of 0.2 ml was mixed by vortexing and incubated at 4°C for 18 h. The unbound PG was removed by mixing with a 0.5-ml suspension of dextran-coated charcoal (250 mg Norit A, 25 mg Dextran Y-70, 100 mt Tris buffer) and centrifuged at 2 0 0 0 g for 15 min. The supernatant containirtg the antibody-bound PG was decanted into a 3-ml scintillation vial containing 3 ml Scinti Verse 11 scintillation cocktail (Fisher Scientific Co., Fair Lawn, N J) and counted on a Beckman 7000 LSC. The results were expressed as the percentage of labeled PG bound vs the concentration of unlabeled PG present. The sample PG content was extrapolated from the standard displacement curve which had a m i n i m u m sensitivity of I pg/0.1 ml. RESULTS

Effect o f exogenous prostaglandins on the feline L B r P r o s t a g l a n d i n s o f t h e E series d e c r e a s e s l e c t i n - i n d u c e d b l a s t o g e n e s i s o f h u m a n a n d m u r i n e l y m p h o c y t e s [14]. T o d e t e r m i n e if PGE~ h a d s i m i l a r e f f e c t s o n feline P B L , we a d d e d it to C o n A - s t i m u l a t e d ce!!s (Fig. 1). T h e c o n c e n t r a t i o n r a n g e d b e t w e e n 1.0 a n d 0.01 ~tM o f p r o s t a g l a n d i n per l0 g l y m p h o c y t e s . T h e n o r m a l P B L r e s p o n s e to C o n A r a n g e d b e t w e e n 4 8 , 0 0 0 to 9 0 , 0 0 0 c o u n t s / r a i n . C o n c e n t r a t i o n s o f 0.1 uNl P G E : c a u s e d significant (p<0.001) suppression of the 'H-thymidine u p t a k e . T h i s s u p p r e s s i o n was n o t a l t e r e d if F e L V was p r e s e n t . F e L V itself c a u s e d 2 7 . 6 % s u p p r e s s i o n o f t h e Con A response without added PGE. When PGE2 was t i t r a t e d in t h e c u l t u r e t h e r e s p o n s e c u r v e s h o w e d n o significant difference from the response curve without F e L V (Fig. 1). S i m i l a r r e s u l t s were o b s e r v e d w h e n P G E , w a s u s e d (results n o t s h o w n / .

Effect o f indornethacin on l.vmphocyte blastogenesis In view o f t h e ability o f e x o g e n o u s P G E , a n d P G E 2 to d e c r e a s e L B T , b o t h in t h e p r e s e n c e a n d a b s e n c e o f F e L V , we tested i n d o m e t h a c i n (indo), a n i n h i b i t o r o f

lndomethacin and feline leukemia virus suppression

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PGE~ CONCENTRATION (~M) FIG. 1. Effect of PGE: on the normal feline Con A LBT of 10~ peripheral blood tymphocytes (_~) and normal PBL with 40 l.tg FeLV present (A). PGE was added at time 0. The normal response of PBL ranged between 45,000 and 90,000 tn = 7). The cultures were harvested after 72 h of incubation following an 18-h terminal pulse with 3H-thymidine.

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FIG. 3. Time-dependent effect of indomethacin addition to the Con A LBT. Figure represents one of four.tests, lndo was added at fixed concentrations between 10 and 0.01 laM at the time of Con A addition ( o ) , 24 h after Con A (--), and 48 h after Con A (/~). 10' Con A-treated cells were used. FeLV (40 lag/well) was added at time 0. The cultures were harvested after 72 h of incubation following an 18-h 3H-thymidine pulse.

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[NDOMETHACIN CONCENTRATION {,.,.,.M) Fl(~. 2. The effect of indomethacin on FeLV (40 lag) In =20) and

FeLV pl5E (2 /ag) tn=4)-induced suppression of the Con A LBT. Results expressed as comparison of I0' normal control cells (*p<0.01 vs control) (**p'<0.001 vs control). Control range between 48.000 and 90,000 counts/min. Various concentrations of indo were added to triplicate wells containing Con ( o ). Con A and FeLV / i: I. and Con A and FeLV pl 5E ( A ). The cultures were harvested after 72 h of incubation, following an 18-h 3H-thymidine terminal phase.

e n d o g e n o u s prostaglandin p r o d u c t i o n , for its effects on the same system, l n d o alone, at c o n c e n t r a t i o n s of 0.1 ~tm and above caused a small but significant increase in the normal Con A-induced thymidine uptake (Fig. 2). Howe~er, the observed decrease in Con A-induced thymidine uptake caused by FeLV or FeLV p l 5 E was completely reversed in the presence o f indo at concentrations above 1.0 ~tM. The effect of indo was observed

only if it was added before 24 h o f culture time (Fig. 3). Other k n o w n non-steroidal a n t i - f l a m m a t o r y drugs, nordihydroguararetic acid, aspirin and flunixin, had similar effects (Table 1). The concentrations necessary for complete reversal o f FeLV-induced suppression by these drugs are shown in Table 1. Two drugs, tranylcypromine, an inhibitor o f t h r o m b o x a n e synthesis, and imidazole, and inhibitor o f prostacycline synthesis, showed no effect on the FeLV-induced suppression o f blastogenesis.

Measurement o f PGE2 levels in cells treated with FeL V The reversal of FeLV-induced suppression by indo and other cyclooxygenase inhibitors suggested that FeLV may cause its effect by increasing prostaglandin p r o d u c t i o n . However, m e a s u r e m e n t o f PGE2 in the culture fluid o f PBL incubated with FeLV did not sho~' any F e k V - i n d u c e d increase. In fact, FeLV decreased the

1454

MARK G.

LEwIs, R I C H A R D

H. FERTEL and RICHARD G. OLSEN

ranging from 3034 to 29,000). Our results show that indo enhances the activity o f Con A-induced LBT o f FeLV-infected cats (Fig. 5). C o n c e n t r a t i o n s between 0.01 and 1.0 ~.M caused a significant e n h a n c e m e n t of thymidine uptake.

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FIo. 4. Prostaglandin E2 levels in culture media of PBL from one of 4 cats tested. 5 x 10' PBL were incubated for various lengths of time with media ( o ), media + Con A (~), media + 200 lag FeLV (Zx), or media + Con A + 200 lag FeLV (V). After the cultures were incubated for the specified period, cells were removed and the supernatant was tested for PGE~ levels by RIA. Data for graph A and graph B were generated in two separate tests due to the limitation in cell numbers, lndomethacin, when present, blocked any additional PGE: ac cumulation with the levels remaining at <-N100 pg/ml. This level was not dependent on the presence of Con A or FeLV.

a m o u n t o f PGE2 found in the media tFig. 4). !n 4 o f 4. cats tested, FeLV caused a decrease in PGE~ in the culture fluid. The inhibition was most visible during the first 8 h o f culture and by 24 h the levels showed no significant difference. Limitations in total cell numbers did not allow for a continuous test running between 0 and 72 h. Indo blocked any PGE2 accumulation after 1 h o f culture, with level remaining ~< 100 p g / m l .

Effect o f indo on Con A L B T o f virol-infected cats Since indo reversed the inhibition by p l S E and FeLV o f Con A-induced LBT o f normal cells, we tested the effect o f the agent on PBL collected from FeLV-infected cats. These ceils were shown to have a decreased Con A response, as has been previously reported ( c o u n t s / m i n

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FIG. 5. The effect of indomethacin upon Con A stimulation of l& cells from FeLV-infected cats. Expressed as percent increase above control cells receiving Con A alone. Each point represents the mean __ S.E. of 8 experiments. Control Con A stimulation ranged between 3034 and 29,000 counts/min. Indo at concentrations between 1 × 10-' and 1.0 ~M were added to triplicate wells containing 10' cells and Con A (4.0 ~g/well). The cultures were harvested after 72 h of incubation following an 18-h 3H-thymidine terminal pulse.

DISCUSSION Retroviral induced i m m u n o s u p p r e s s i o n has been well d o c u m e n t e d , especially with the recent linking o f H T L V - l l l with AIDS [2]. The suppressive effect o f disrupted retroviruses has been associated with an intrinsic envelope protein o f approx. 15,000 daltons in weight [4-111. The mechanism o f the viral suppression has not been well studied, but in one case the observed suppression was suggested to be prostaglandin-related [I 11. Prostaglandins o f the E series have been found to inhibit a variety o f immune functions [14] and our studies indicate that they are capable o f suppressing feline lymphocyte functions in a similar manner. Since prostaglandins act as immune suppressors in several normal and disease states [20-22], we examined th'e possibility that they were a factor in the observed suppression with F e L V - p l 5 E , We considered the possibility that the p l 5 E might be enhancing the p r o d u c t i o n o f end o g e n o u s PGE. We chose to examine this both by treating the cells with the agent indo and other similar drugs, which inhibit the enzyme that is responsible for PG synthesis, and by measuring PGE_~ accumulation in the presence o f FeLV. The present study shows that indo can increase the thymidine uptake by Con A-stimulated feline PBL,

lndomethacin and feline leukemia virus suppression which is in agreement with studies using PBL from other species [23-25]. In the presence of suppressing levels of FeLV or FeLV pl5E, indo was able to completely abrogate the virus interference of the Con A stimulation. This effect was titratable with complete reversal of FeLV inhibition occurring at a concentration of 0.24 I~M indo per well. When other drugs, classified as NSAI drugs, were added in place of indo, similar results were observed. Extrapolation of the titration curves generated gave relative concentrations necessary for complete reversal of FeLV suppression (Table 1). Flunixin proved to be the most effective, while aspirin was the least effective of the drugs tested. Two drugs, tranylcypromine, an inhibitor of thromboxane synthetase [26], and imidazole, an inhibitor of prostacyclin synthetase [27], had no effect on FeLV suppression. This may indicate that thromboxane and prostacyclin are not involved in the mechanism of FeLV action. The time dependence of indo (Fig. 3) showed that the drug was only effective at reversing FeLV suppression when added during the first 24 h of culture. This is also the period when Con A has been shown to have its effect on cell blastogenesis [28] and is also the period of time when PGE effects T suppressor cell induction [29]. This data, when combined with the NSAI drug data, appeared to imply that the mechanism of action of FeLV and indo was prostaglandin-related and, to prove this, direct measurement of PGE2 production in the presence of both substances was needed. However, upon measurement of PGE: produced by normal feline PBL during incubation with and without FeLV we found that FeLV could decrease PGE2 accumulation in culture during the first hours, again the period of time critical for Con A stimulation [28]. These results suggest that FeLV is not causing its effect by increasing PGE production and that indo's reversal may not be due to the inhibition of PG production. The mechanism of action of FeLV on PGE2 accumulation could be by a number of events; first, FeLV may interfere with PG production by changing the availability of arachidonic acid or altering enzymes essential for PG production such as cyctooxygenase or phospholipase A; second, FeLV may act by causing an increase in PGE catabolism; third, FeLV may interfere with the detection of PGE in the culture media. Of these possibilities, the most likely is a disruption of PGE production. An increase in PG catabolism is unlikely and we have found no interference by FeLV in our RIA. An alteration in PG production is possible, since we have previously reported that FeLV plSE can interfere with lymphocyte membrane function [13]. FeLV pl5E, due to its hydrophobic nature [30], may interact with the plasma membrane of the lymphocyte and cause a disruption in PG metabolism. Further testing will be necessary to determine the exact action of FeLV on PG production. lndomethacin-mediated enhancement of T-lymphocyte responses has been observed in a number of disease states and it has also been shown to increase normal mitogenic response in a number of other mammalian cultures [23-25] leading to the speculation that indo and other non-steroidal anti-inflammatory drugs may be useful as immune res-

1455

ponse modifiers, lndomethacin has been used in vivo in humans and shown to enhance the secondary antibody response [25]. Our observation of the enhancement of FeLV-infected cat PBL Con A responses by indomethacin indicates that it may, in fact, be useful as a treatment for FeLV infections and possibly in other retroviral infections. Since the reversing ability of the NSAI drugs does not appear to be prostaglandin-related, there must be another common mechanism of action. Although NSAI drugs are normally used as and reported to be cyclooxygenase inhibitors, most have also been shown to have a number of other actions on normal cellular metabolism, including inhibition of leukotriene production [31], cAMP phosphodiesterase [28], cAMP-dependent kinase [32], certain amino acid transport across the membrane [33], and cellular growth [34]. Although many of these studies used high levels of the drug (>i 10-'M), these effects could also occur at lower doses as used in our studies. Further investigation will be necessary to determine the exact mechanisms of FeLV suppression and of NSAI drugs' effects. The reversing ability of NDGA is noteworthy in that the drug is a strong leukotriene inhibitor and a weak prostaglandin inhibitor, suggesting that the lipoxygenase pathway may be associated with FeLV suppression. Several types of experiments will be necessary to further determine the mechanism of indo action. Leukotriene and cyclic nucleotide metabolism changes in the presence of FeLV will indicate if the mechanism is through a change in endogenous leukotriene production and/or alteration in cyclic nucleotide levels. The effect of FeLV on specific areas of PG metabolism may also be informative. In addition, further work is also necessary to determine the effectiveness of in vivo indomethacin or other non-steroids as treatments for FeLV-infected cats. REFERENCES 1. Teich N., Wyke J., Mak T., Berstein A. & Hardy W. (1982) In Molecular Biology of Tumor Viruses: RNA Tumor Viruses (Weiss R., Teich N., Varmus H., Eoffin J., Eds), p. 785. Cold Spring Harbor Laboratory, N.Y. 2. Popovic M., Sarngadharan M. G., Read E. & Gallo R. C. (1984) Detection, isolation, and continuous production of cytopathic retroviruses (HTLV-IlI) from patients with AIDS and pre-AIDS. Science, N.Y. 224, 497. 3. Rojko J. L. & Olsen R. G. (1984) The immunobiology of the feline leukemia virus. Vet. lmmun. Immunopath. 6, 107. 4. Mathes L. E., Olsen R. G., Hebebrand L. C., Hoover E. A. & Schaller J. P. (1978) Abrogation of lymphocyte blastogenesis by a feline virus protein. Nature, Lond. 2"/4, 687. 5. Hebebrand L. C., Mathes L. E. & Olsen R. G. (1977) Inhibition of concanavalin A stimulation of feline lymphocytes by inactivated feline leukemia virus. Cancer Res. 37, 4532. 6. Hebebrand L. C., Olsen R. G., Mathes L. E & Nichols W. S. (1979) Inhibition of human lymphocyte mitogen and antigen response by a 15,000 dalton protein from feline leukemia virus. Cancer Res. 39, 443. 7. Dunlap J. E., Nichols W. S., Hebebrand L. C., Mathes L.

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MARK G. LEWIS, RICHARDH. FERTELand RICHARD(3. OLSEN

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