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Effects of SK&F 105685, a novel anti-arthritic agent, on immune function in the dog
0192-0561/93 $6.00 + .0o PergamonPress Ltd. ©1993 InternationalSocietyfor Immunopharmacology.
Int. J. lmmunopharmac., Vol. 15, No. 2, pp. 113-123, 1993. Printed in Great Britain.
EFFECTS OF SK&F 105685, A NOVEL ANTI-ARTHRITIC AGENT, ON IMMUNE FUNCTION IN THE DOG JOHANNE M. KAPLAN, ALISON M . BADGER, ELIZABETH V. RUGGIERI, BARBARA A. SWIFT a n d PETER J. BUGELSKI* Departments of Toxicology and Cell Sciences, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406-0939, U.S.A. (Received 21 July 1992 and in final forrn 19 October 1992)
Abstract - - SK&F 105685 (N,N-dimethyl-8,8-dipropyl-2-azaspiro[4,5]decane-2-propauamine dihydrochlo-
ride) is a novel azaspirane with beneficial activity in animal models of autoimmune disease such as adjuvantinduced arthritis and experimental encephalomyelitis in the Lewis rat and lupus-like disease in the MRL mouse. The activity of SK&F 105685 in these models is associated with the induction of non-specific suppressor cell (SC) activity as defined by the ability of cells from drug-treated animals to inhibit the proliferative response of lymphocytes from control animals to concanavalin A. To evaluate the immunotoxicologic potential of SK&F 105685, the effect on immune function of one month of dosing with 1 mg/kg/day of SK&F 105685 was examined in the dog. Differential blood cell counts and ex vivo immune function assays were performed using blood collected before dosing on days 1 (baseline), 15 and 29, of the study. Immune function assays were performed on spleen cells on day 30. Under the conditions of the study, SK&F 105685 displayed pharmacological activity as demonstrated by the induction of splenic SC activity. The drug did not affect the total number or relative percentages of the various white blood cell types present in peripheral blood and did not cause generalized immunosuppression. The ability of peripheral blood lymphocytes or spleen cells to produce IL-2 or proliferate in response to mitogenic stimulation was not affected by drug treatment. SK&F 105685 also failed to affect the candidacidal activity of polymorphonuclear leucocytes and spleen cells indicating that it is unlikely to compromise nonspecific resistance to infection. SK&F 105685 however, was able to inhibit the generation of a specific in vitro antibody response to sheep red blood cells (SRBC) by splenocytes from treated animals. Inhibition of the anti-SRBC antibody response was also observed upon addition of the drug to normal spleen cells. Addition of the drug at different time points during the culture period indicated that SK&F 105685 was interfering with an event(s) occurring during the first 72 h of culture. Taken together, these results suggest that, in a therapeutic setting, SK&F 105685 is unlikely to compromise the immune status of the host as it can down-regulate a specific immune response without causing generalized immunosuppression.
SK&F 105685 (N,N-dimethyl-8,8-dipropyl-2-azaspiro[4,5]decane-2-propanamine dihydrochloride) (Fig. 1) is a novel azaspirane with beneficial activity in a n i m a l models o f a u t o i m m u n e disease such as adjuvant-induced arthritis and experimental encephalomyelitis in the Lewis rat a n d lupus-like disease in the M R L m o u s e (Badger et al., 1989; A I b r i g h t s o n - W i n s l o w et al., 1990). T h e activity of SK&F 105685 a n d related analogs in these models was f o u n d to correlate with their ability to induce non-specific splenic suppressor cell (SC) activity as defined by the ability o f splenocytes f r o m drug-
treated animals to suppress the c o n c a n a v a l i n A (Con A)-induced p r o l i f e r a t i o n o f n o r m a l spleen cells (Badger et al., 1989; A l b r i g h t s o n - W i n s l o w et al., 1990; Badger et al., 1990b). In the rat, the SC activity appears to reside within a n o n - T , n o n - B , n o n - N K , s e m i - a d h e r e n t spleen cell p o p u l a t i o n with a density ~ 1 . 0 7 g / m l (Badger et al., 1990a). While the i n d u c t i o n o f SC activity by SK&F 105685 m a y be beneficial in the suppression of a u t o i m m u n e responses, the i m m u n o m o d u l a t o r y activity o f the c o m p o u n d also raises concerns a b o u t the potential i n d u c t i o n o f generalized i m m u n o s u p p r e s s i o n . T o
*Author to whom correspondence should be addressed: SmithKline Beecham Pharmaceuticals, P.O. Box 1539, Department of Toxicology, L-60, King of Prussia, PA 19406-0939, U.S.A. 113
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address this issue in a non-rodent species, the effects of SK&F 105685 on immune function were examined in male dogs, a species commonly used in toxicology studies. SK&F 105685 has recently been shown to induce splenic SC activity in the dog thereby supporting the biological, in addition to the toxicological, relevance of this species (Thiem, Kaplan, Bugelski, Ruggieri & Badger, 1992). Dogs were treated orally with a non-toxic dose of SK&F 105685 (1 m g / k g / d a y ) or vehicle for one month. To assess the immune status of the animals, differential blood cell counts and various parameters of immune function were evaluated ex vivo using blood collected before closing on days 1 (baseline), 15 and 29 of the study. Immune function assays were performed on spleen cells on day 30.
placed in ice-cold phosphate-buffered saline (PBS). The spleen was then minced and forced through a 60-mesh sieve to form a single cell suspension. For the suppressor cell assay, spleen cells were washed 3 times in PBS and resuspended in culture medium. For all other assays involving spleen cells, red blood cells were first lyzed with 0.16 M Tris-ammonium chloride buffer, pH 7.4. Blood cell counts
Automated differential blood cell counts were performed on samples of whole blood using a Technicon H*I instrument (Technicon Instrument Corporation, Tarrytown, NY). Suppressor cell assay
EXPERIMENTAL PROCEDURES Animals
Purebred male beagle dogs (Marshall Animal Farms, Inc., North Rose, NY), 9.5 - 10.5 months old were housed individually in stainless steel cages. A physical examination, as well as hematologic and serum biochemical evaluations, were performed to ensure that only healthy animals were included in the study. The dihydrochloride salt of SK&F 105685 (MW 381.473; supplied by Johnson Matthey, West Chester, PA) was administered orally in gelatin capsules, once daily for 29 consecutive days, to 3 dogs at a dosage of 1 m g / k g / d a y . A control group, consisting of 3 dogs, received empty capsules. Cell preparation
Heparinized blood was collected from each dog before dosing on days 1 (baseline), 15 and 29 of the study. Peripheral blood lymphocytes (PBL) were isolated by density gradient centrifugation on a F i c o l l - Hypaque gradient (1.077 g/ml density) and polymorphonuclear leucocytes (PMN) were obtained by dextran sedimentation (Boyum, 1968). Cytospin preparations of the PBL and PMN fractions from each dog were made for differential analysis. Varying amounts of contaminating P M N were found to be present in the PBL preparations and therefore, values obtained in experiments using PBL were adjusted for the actual number of lymphocytes present in the assay. PMN preparations were 91 - 99070 pure. On day 30 of the study, a portion of spleen (approximately 25 g) was collected aseptically from each dog as soon as the abdomen was opened and
Suppressor cell activity was evaluated using a coculture system which measures the ability of irradiated effector (E) cells to suppress the proliferative response of normal responder (R) cells to the mitogen concanavalin A (Con A) (Badger, Mirabelli & DiMartino, 1985). Briefly, 100/A of effector spleen cells from individual drug-treated or control animals were seeded into 6 replicate wells of a round bottom microtiter plate and serially diluted 2-fold ( 2 . 5 - 0 . 0 8 x 10~ cells/well) in RPMI-1640 medium supplemented with 2 mM glutamine, I00 U/ml penicillin, 100/~g/ml streptomycin and 10070 heat-inactivated fetal calf serum (FCS, Hyclone, Logan, UT). Plated cells were irradiated (3000 Rads) using a Gammacell 40J37Cs irradiation unit (Atomic Energy of Canada Ltd, Ottawa, Canada). Fifty microliters (2.5 x 10~) of responder spleen cells from control animals and 50 ~1 of Con A (5 ~g/ml final concentration) were added to each well. Effector spleen cells from each of the 6 study dogs were tested for suppressor cell activity against responder cells from each of the 3 control dogs thus giving rise to 18 co-culture combinations. The cultures were incubated at 37°C/5% CO2 for 72 h, pulsed with 0.5/~Ci of tritiated thymidine (specific activity 6.7 Ci/mmol, New England Nuclear, Boston, MA) for the final 18 h of incubation and harvested onto glass fiber filters with a 96 well plate harvester (Skatron Inc., Sterling, VA). Cellassociated radioactivity was measured by liquid scintillation counting (Beckman Instruments, Irvine, CA). The percent suppression at each E : R ratio was calculated according to the following equation: °70 suppression= 1 counts/min of heterologous co-culture counts/rain of autologous co-culture x 100.
Immune Function and Anti-arthritic Agent Total "units of suppression" for each co-culture combination were derived from the area under the curve (AUC) of a plot of the percent suppresssion versus the log of the number of effector cells (Badger et al., 1989). Results shown are the mean units of suppression +_ standard error of the mean (S.E.M.) of the control and drug-treated groups tested against each of the control dogs.
Mitogen responses of peripheral blood lymphocytes and spleen cells The mitogenic responsiveness of spleen cells and of PBL after isolation or as present in whole blood was determined. In the whole blood assay, heparinized blood from each dog was diluted 5-fold in serum-free Iscove's Modified Dulbecco's culture medium (Gibco, Grand Island, NY) supplemented with 100 U / m l penicillin, 100/ag/ml streptomycin and 5 × 10 -5 M 2-mercaptoethanol (2-ME). Twenty five microliters of the diluted blood was added in triplicate to the wells of a 96 well round bottom plate with 100/A of serial 5-fold dilutions of mitogen. For isolated PBL and spleen cells, 100/A of cell suspension was added in triplicate to the wells of a 96 well round bottom plate (2.5 x 105 cells/well) with 100 ~1 of mitogen in Iscove's culture medium supplemented with 10°70 heat-inactivated FCS. The mitogens used were: phytohemagglutinin (PHA, Difco Laboratories, Detroit, MI), pokeweed mitogen (PWM, Gibco Laboratories) and Con A (Pharmacia, Piscataway, N J). Cultures were incubated for 72 h at 37°C/5% CO2, pulsed with 0.5/aCi tritiated thymidine for the final 18 h of incubation and tritium incorporation measured.
IL-2 production by peripheral blood lymphocytes and spleen cells To evaluate IL-2 production, 2 × 106 isolated PBL or 4 × 106 spleen cells from each dog were added to the wells of a 24 well plate in a 1 ml volume with 10/al of Con A (10/ag/ml final) or 10/A of medium as a control. Cultures were done in triplicate and the culture medium consisted of RPMI-1640 medium supplemented with 10% heat-inactivated FCS. Culture supernatants were collected after 24 h of incubation at 37°C/5% COy The IL-2 content of the supernatants was determined using the IL-2dependent CT20 murine cytotoxic T-cell line. Triplicate assay cultures in the wells of a 96 well flat bottom plate contained 100~1 of CT20 cells (1 × 104/well) and 100/al of either diluted test supernatant, serial 2-fold dilutions of recombinant mouse IL-2 (Genzyme Corporation, Cambridge,
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MA) or culture medium as a control. The cultures were incubated overnight at 37°C/5°70 CO2, pulsed with 0.5/aCi of tritiated thymidine for 6 h and cellassociated radioactivity measured. The number o f IL-2 units/ml of supernatant was derived by linear regression analysis from a standard curve of the counts/rain versus recombinant IL-2 units/ml.
Candidacidal activity of polymorphonuclear leucocytes and spleen cells Constitutive microbicidal activity against the facultative intracellular pathogen Candida albicans was measured by a micro-colony formation assay adapted from Karbassi, Becker, Foster & Moore (1987) and Hasimoto (1983). Briefly, C. albicans strain B792 (gift of Dr G. Livi, Biopharmaceutical Sciences, SmithKline Beecham) was maintained on Sabouraud's dextrose (YEPD) agar at 28°C. Yeasts were opsonized by incubating for 2 h at 4°C in RPMI-1640 with 20°70 dog serum. PMN or spleen cells from each dog were resuspended in RPMI-1640 with 10% heat-inactivated FCS and added to quadruplicate wells of a 24 well plate in a 1 ml volume (1 × 10 6 cells/well). The cells were allowed to adhere for approximately 90 min at 37°C/5% CO2 and the supernatant removed. Each well then received 0.2 ml of culture medium and 0.2 ml of opsonized C. albicans (approximately 100 colonyforming units). Control quadruplicate wells contained yeasts without phagocytes. The plates were incubated at 37°C/5% CO2 for 2 h and candidacidal activity was stopped by the addition of 1 ml of YEPD agar to each well. The plates were then incubated overnight to permit the growth of surviving yeasts and subsequent enumeration of the number of colonies/well under a dissecting microscope. The " % kill" was derived from the following formula:
~No.
colonies in experimental well~ o70 kill= 1 - ~ h/~..c--~-~oni'~n~ w e l ~ s J × 100.
Primary antibody response against sheep red blood cells The ability of spleen cells from individual control and drug-treated dogs to mount a primary antibody response against the antigen sheep red blood cell (SRBC) was measured after 29 days of dosing. In in vitro studies, SK&F 105685 ( 0 . 5 - 2 0 0 nM) was added directly to cultures of normal dog spleen cells at different time points during the incubation period. Immunization with SRBC was performed according
116
J. M. KAPLANet at.
to a modification of the method of Mishell & Dutton (1967). Spleen cells were resuspended in Iscove's Modified Dulbecco's culture medium with 10070heatinactivated FCS. Two milliliters of the spleen cell suspensions were added to the wells of a 24-well plate (1 × 107 cells/well) and stimulated with 50/~1 of 1070 SRBC or medium as a background control. Cultures were done in quadruplicate. The plates were incubated for 5 days at 37°C/5% CO, to allow for the development of an anti-SRBC response. The cultures were fed on the second day of incubation with 1 ml of fresh medium containing the appropriate concentration of SK&F 105685 in the case of drug-containing wells. The anti-SRBC response was assessed on the fifth day of culture by the localized hemolysis in gel technique (plaque assay; Wortis, Taylor & Dresser, 1968). Cells harvested from each individual well were resuspended in 0.5 ml of RPMI-1640 medium. A 100/~1 aliquot of cell suspension was mixed with 100/A of a 20070 suspension of SRBC and 0.8 ml of 0.625070 agarose (FMC Bioproducts, Rockland, ME) at 45°C. At this point, SK&F 105685 was also added to aliquots of control cells to look at its effect on antibody secretion. The mixture was then poured into 35 × 10 mm petri dishes pre-coated with 1 ml of 0.5°70 agarose and the dishes were incubated at 37°C for 2 h. At the end of this incubation period, 0.5 ml of guinea pig complement (Cedarlane Laboratories Inc., Ontario, Canada) diluted 1 : 10 in RPMI-1640 medium was poured onto the surface of the gel and the dishes were incubated for an additional hour to allow for the development of IgM plaques.
Stat&tical analysis Results for the control and dosed groups are expressed as the mean _+ S.E.M. of the values obtained for each individual dog. Statistical significance between the 2 groups was determined using the 2-sample Student's t-test. A P value equal to or lower than 0.05 was considered significant.
RESULTS
Splenic suppressor cell activity Splenic suppressor cell (SC) activity was measured after 29 days of dosing, to confirm the pharmacological activity of SK&F 105685 administered at 1 mg/kg/day. SC activity was defined by the ability of irradiated effector spleen cells to suppress the proliferative response of control responder spleen
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Fig. 2. Suppressor cell activity of spleen cells on day 30. Irradiated effector spleen cells from dogs treated with vehicle (I--1) or 1 mg/kg/day of SK&F 105685 ([]) were incubated with responder spleen cells from each of the 3 control dogs (responder 1,2,3) in the presence of Con A. Mean suppressor cell activity for the control and drugtreated groups is expressed as AUC units __+S.E.M. cells to concanavalin A (Con A). Responder cells (R) from each control dog were incubated with irradiated effector cells (E) from the same animal (reference autologous co-culture), each of the other 2 control or 3 dosed dogs. Various E : T ratios were tested. Units of suppression were derived from the area under the curve (AUC) of a plot of the °7o suppression versus the log of the number of effector cells. As shown in Fig. 2, when tested against each of
117
Immune Function and Anti-arthritic Agent Table 1. Hematology parameters in control dogs and dogs treated with SK&F 105685 Time point
Cell type
Control
SK&F 105685 (1 mg/kg)
WBC Neut Lymph Mono Eos Baso
9.2 5.6 2.6 0.4 0.4 0.06
+0.1" ± 0.1 ± 0.1 ± 0.02 ± 0.1 +_ 0.01
9.4 5.6 2.7 0.5 0.4 0.05
± 1.1 +_ 0.7 +_ 0.3 ± 0.1 ± 0.1 ± 0.01
Day 15
WBC Neut Lymph Mono Eos Baso
9.1 5.5 2.6 0.5 0.4 0.06
+ 0.3 +_0.2 +_ 0.1 ± 0.1 ± 0.1 ± 0.01
8.6 4.8 2.7 0.6 0.5 0.05
± 1.5 ± 1.1 ± 0.2 +- 0.1 +- 0.1 ± 0.01
Day 29
WBC Neut Lymph Mono Eos Baso
10.0 6.3 2.7 0.6 0.3 0.06
_+ 0.8 ± 0.8 _+ 0.3 _+ 0.2 +0.1 ± 0.01
8.8 5.1 2.7 0.4 0.5 0.05
± 0.9 _ 0.8 ± 0.2 +_ 0.1 _+0.1 ± 0.01
Day 1 Baseline
* × 103//al
Neut = neutrophil; Lymph = lymphocyte; Mono = monocyte; Eos = eosinophil; Baso = basophil. the 3 control dogs, cells f r o m drug-treated animals showed greater SC activity than spleen cells from control animals. Statistical significance however, was achieved against only 2 of the control dogs (responders 1,2), the third one being a poor target for suppression. The SC activity observed nonetheless confirmed that SK&F 105685 was pharmacologically active under the conditions of the study.
Blood cell counts Differential blood cell counts were performed on blood collected prior to dosing on days 1 (baseline), 15 and 29 o f the study. Results shown in Table 1 indicate that SK&F 105685 did not affect the total number or relative percentages of the various white blood cell types present in peripheral blood.
Mitogen responses Mitogen-induced proliferation of blood lymphocytes collected before dosing on days 1 (baseline), 15 and 29 of the study was measured to provide a gross index of lymphocyte function. Results f r o m these experiments indicated that SK&F 105685 did not affect the response of unfractionated blood cells or isolated peripheral blood lymphocytes (PBL) to varying concentrations o f the B- and T-cell mitogen
pokeweed mitogen (PWM) and the T-cell mitogens phytohemagglutinin ( P H A ) and Con A. There was no statistically significant difference in the proliferative responses of whole blood [Figs 3(A), (B), (C)] and isolated P B L [Figs 4 (A), (B), (C)] from drug-treated and control animals to optimal concentrations of mitogens. Similar results were obtained with suboptimal and supraoptimal concentrations of mitogens (results not shown). SK&F 105685 also failed te affect the mitogen responses o f spleen cells after 29 days o f dosing (Fig. 5). No significant difference was observed between the proliferative responses of splenocytes from control and drug-treated animals after stimulation with optimal (Fig. 5), suboptimal and supraoptimal (not shown) concentrations of mitogens.
IL-2 production IL-2 production by P B L and spleen cells was evaluated as a parameter of T-cell function. SK&F 105685 did not inhibit the ability of T-cells from the spleen or peripheral blood to produce IL-2 upon stimulation with Con A. There was no significant difference in the a m o u n t of IL-2 produced by PBL from control and drug-treated animals on days 1 (baseline value), 15 and 29 of the study [Fig. 6 (A)]. Similarly, SK&F 105685 had no significant effect on
118
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Fig. 3. Mitogenic responses of whole blood. Unfractionated peripheral blood cells from control (Fq) and drug-treated ([]) dogs were stimulated with optimal concentrations of mitogens. (A) PWM (1/250 dilution), (B) PHA (1/7500 dilution), (C) Con A (1.6/ag/ml). Proliferative responses were measured after 72 h of incubation. IL-2 production by spleen cells after 29 days of dosing [Fig. 6 (B)].
Candidacidal activity
The non-specific microbicidal activity of polymorphonuclear leucocytes (PMN) and macrophages represents the first line of defense against invading
DAY 1
DAY 15
DAY 29
Fig. 4. Mitogenic responses of isolated PBL. Isolated PBL from control ([~) and drug-treated dogs (g'A)were stimulated with optimal concentrations of mitogens. (A) PWM (1/250 dilution), (B) PHA (1/7500 dilution), (C) Con A (1.6/~g/ml). Proliferative responses were measured after 72 h of incubation.
microorganisms. To examine the effect o f SK&F 105685 on this aspect of immune defense, the ability of peripheral blood P M N and spleen cells to kill the yeast form o f C. albicans was measured. As depicted in Fig. 7 (A), the same high levels o f candidacidal activity were displayed by P M N from the control and drug-treated groups on days 1 (baseline), 15 and
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Fig. 5. Mitogenic responses of spleen cells on day 30. Spleen cells from control (1-1) and drug-treated (~) dogs were stimulated with optimal concentrations of the mitogens PWM, PHA and Con A. Proliferative responses were measured after 72 h of incubation. 29 of the study. The candidacidal activity of spleen cells (mediated by PMN and/or macrophages) also remained unaffected after 29 days of dosing [Fig. 7 (B)].
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Primary antibody response of spleen cells to sheep erythrocytes The ability of spleen cells from control and drugtreated animals to mount an antibody response to the antigen sheep red blood cells (SRBC) was measured ex vivo in a hemolytic plaque-forming cell (PFC) assay after 29 days of dosing. As shown in Fig. 8, there was a significant reduction in the ability of spleen cells from SK&F 105685-treated animals to mount an anti-SRBC PFC response indicating that the drug was able to selectively suppress a specific immune response. The inhibitory effect of SK&F 105685 on the generation of an anti-SRBC response was also observed in vitro. Addition of SK&F 105685 to normal dog spleen cells at the beginning of culture resulted in a dose-dependent inhibition of the antiSRBC PFC response (Fig. 9). The suppression was not due to drug cytotoxicity since there was no significant difference in the number of viable cells recovered at the end of the culture period in control and drug-treated wells (not shown). Addition of SK&F 105685 at different time points showed that
DAY 30
Fig. 6. IL-2 production by PBL and spleen cells. PBL (A) and spleen cells (B) from control (to) and drug-treated (~) dogs were stimulated with Con A for 24 h and the amount of IL-2 released in the supernatant measured. the suppressive effect was no longer observed when addition of the drug was delayed by 72 h indicating that SK&F 105685 was affecting an event(s) occurring during the first 72 h of culture (Fig. 10). Addition of SK&F 105685 at the time of the hemolytic PFC assay indicated that the drug did not interfere with secretion of preformed antibodies (not shown). DISCUSSION SK&F 105685 administered for one month at a dosage of 1 mg/kg/day displayed pharmacological activity as defined by the induction of splenic
120
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Fig. 7. Candidacidal activity of peripheral blood PMN and spleen ceils. The candidacidal activity of peripheral blood PMN (A) and spleen cells (B) from control (gl) and drugtreated (VI) dogs was assessed before dosing on days 1, 15, 29 and on day 30 of the study, respectively.
suppressor cell activity, in the absence of overt toxicity. Differential blood cell counts showed that the drug did not affect the total number of relative percentages of the various white blood cell types present in peripheral blood. Ex vivo immune function assays established that SK&F 105685 did not cause generalized immunosuppression. The ability of PBL or spleen cells from drug-treated animals to produce IL-2 (Fig. 6) or proliferate in response to mitogenic stimulation (Figs 3 - 5) was not significantly different from that of control dogs indicating that SK&F 105685 did not globally
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Fig. 9. Dose-dependent inhibition of the anti-SRBC response of normal spleen cells by SK&F 105685 in vitro. Different concentrations of SK&F 105685 were added to cultures of normal dog spleen cells stimulated with SRBC. The anti-SRBC PFC response was measured after 5 days of incubation.
Immune Function and Anti-arthritic Agent
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Fig. 10. Time dependence of inhibition of the anti-SRBC response of normal spleen cells by SK&F 105685. SK&F 105685 (50 nM) was added to cultures of normal spleen cells and SRBC at different time points. The antiSRBC response was measured after 5 days of incubation. suppress lymphocyte function. The candidacidal activity of PMN and spleen cells was also unaffected by treatment with SK&F 105685 (Fig. 7) suggesting that the compound is unlikely to compromise nonspecific resistance to infection. In contrast to its lack of effect on responses to non-specific stimuli, SK&F 105685 was able to significantly inhibit the generation of a specific immune response. SK&F 105685 strongly suppressed the ability of spleen cells from treated dogs to mount a primary antibody response against SRBC (Fig. 8). The inhibitory effect of SK&F 105685 on the generation of an antiSRBC response was also observed in vitro upon addition of the drug to normal spleen cells (Fig. 9). The development of an anti-SRBC PFC response involves a complex interplay between antigen-presenting cells, T-helper lymphocytes and B-lymphocytes. The identity of the cell population(s) whose activity is inhibited by SK&F 105685 or SK&F 105685-induced SC remains to be determined. Addition of the drug at different time points during the 5 days in vitro culture period indicated that SK&F 105685 was interfering with an event(s)
121
occurring during the first 72 h of culture (Fig. 10). IL-2 production by T-helper lymphocytes, an early event in the development of humoral responses, is not likely to be a target for suppression by SK&F 105685 as drug treatment did not inhibit IL-2 production by PBL and spleen cells in this study (Fig. 6). In separate studies, SK&F 105685 also failed to inhibit IL-2 production by rat splenocytes and human Jurkat cells (results submitted for publication). Accessory cell function, production of lymphokines other than IL-2, as well as responsiveness of B- and T-cells to lymphokines represent other possible targets for suppression of the anti-SRBC response. B-cell differentiation as well as the synthesis and secretion of antibodies are not likely to be inhibited by SK&F 105685 as these events occur late in the development of an antibody response. Moreover, addition of the drug at the time of the PFC assay showed that SK&F 105685 did not interfere with the secretion of preformed antibodies (not shown). The ability of SK&F 105685 to selectively inhibit an antigen-specific immune response ex vivo without affecting circulating levels of lymphoid cells or their functional activity in response to non-specific stimulation is in sharp contrast with the effect of most anti-rheumatic drugs. The immunomodulatory drugs currently used in the treatment of rheumatoid arthritis tend to induce a global, non-specific immunosuppression as a result of their cytotoxic activity. The 3 main groups of immunosuppressive anti-rheumatic drugs; folic acid analogs (e.g. methotrexate), purine analogs (e.g. azathioprine) and alkylating agents (e.g. cyclophosphamide), all exert anti-proliferative activity and careful monitoring of blood cell counts during the course of treatment is required to reduce the risk of bone marrow suppression (Erhardt, 1984; Stroehmann, 1986; Littler, 1990). For example, leukopenia is a common side-effect of treatment with cyclophosphamide, azathioprine or methotrexate and has been associated with an increased susceptibility to infection (Erhardt, 1984; Gross, 1986; Stroehman, 1986; Littler, 1990). In addition to depleting leucocytes from the peripheral blood and tissues, anti-proliferative drugs can also affect the functional activity of leucocytes, at least in part, by interfering with their ability to divide. Methotrexate is capable of non-specifically impairing both humoral and cellmediated immune responses while cyclophosphamide favors the inhibition of humoral responses and azathioprine shows more effectiveness against cell-mediated immune reactions (Leppner & Calabresi, 1976; Otterness & Chang, 1976; Elion, 1977).
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Other disease-modifying anti-rheumatic drugs such as gold, sulfasalazine and penicillamine whose mode of action is less defined, also display bone marrow toxicity leading to blood dyscrasias such as thrombocytopenia and neutropenia (Scott, 1984; Littler, 1990). Finally, glucocorticoids which exert potent anti-inflammatory activity have been shown to cause depletion of monocytes and lymphocytes, in particular T-cells, from peripheral blood in humans and experimental animals (Haynes & Fauci, 1978; Rees & Lockwood, 1982). The lymphopenia appears to be due to a change in the migratory pattern of lymphocytes leading to their accumulation in the bone marrow (Fauci & Dale, 1975; Fauci, 1975). Glucocorticoids also have profound immunomodulatory effects and treatment in humans has been associated with decreased proliferative responses of lymphocytes to mitogens (Fauci & Dale, 1974; Webel, Ritts, Taswell, Donadio & Woods, 1974) and decreased bactericidal and fungicidal activity of monocytes (Rinehart, Sagone, Balcerzak, Ackerman & LoBuglio, 1975). In addition to other manifestations of toxicity, the potential risk for increased frequency of infection and malignancy associated with the generalized immunosuppression caused by cytotoxic drugs has generated interest in the development of more "selective" immunosuppressive agents. For example, cyclosporin A, which selectively inhibits T-cell function, has been tested in the treatment of rheumatoid arthritis but, despite encouraging results, its associated nephrotoxicity makes cyclosporin A a poor candidate (Kingsley, Panayi &
Lanchbury, 1991). In this study, SK&F 105685 has displayed many desirable characteristics as a selective immunomodulator. In contrast to cytotoxic drugs, SK&F 105685 did not have any significant effect on the level of circulating leucocytes. It also preserved the functional activity of PMN and lymphocytes in response to non-specific stimulation. Finally, SK&F 105685 was able to inhibit the generation of an antibody response to the T-dependent antigen SRBC. The significance of SK&F 105685-mediated suppression of splenic antibody responses as it relates to the inhibitory activity of the drug against autoimmune responses remains to be determined. In the rat, a species in which SK&F 105685 effectively downregulates autoimmunity, oral dosing under different regimens failed to inhibit systemic antibody production as assessed by the measurement of serum levels of antigen-specific antibodies (unpublished results). The observation of suppressed antibody production by spleen cells in this study may reflect the presence of suppressor cells in that organ as opposed to the peripheral blood. Indeed, SC activity can be detected in the spleen but not the peripheral blood of SK&F 105685-treated dogs (Thiem et al., 1992) and rats (unpublished observation). Studies are under way to resolve this issue and clarify the mechanism of action of SK&F 105685. Acknowledgements - - We wish to thank Gary Gries and
Denise Fennelly for the dosing and care of the animals and we are grateful to Terry Sellers and Cherry Gunning for the collection of hematology parameters.
REFERENCES
ALBRIGHTSON-WINSLOW,C. R., BRICKSON,B., KING, A., OLIVERA, D., SHORT, B., SAUNDERS,C. & BADGER,A. M. (1990). Beneficial effects of long-term treatment with SK&F 105685 in murine lupus nephritis. J. Pharmac. exp. Ther., 255, 382-387. BADGER,A. M., DIMARTINO,M. J., TALMADGE,J. E., PICKER,D. H., SCHWARTZ,D. A., DORMAN,J. W., MIRABELLI, C. K. & HANNA,N. (1989). Inhibition of animal models of autoimmune disease and the induction of non-specific suppressor cells by SK&F 105685 and related azaspiranes. Int. J. Immunopharmac., 11, 839- 846. BADGER, A. M., KING, A. G., TALMADGE,J. E., SCHWARTZ,D. A., PICKER, D. H., MIRABELLI,C. K. & HANNA, N. (1990a). Induction of non-specific suppressor cells in normal Lewis rats by a novel azaspirane SK&F 105685. J. Autoimmunity, 3, 485- 500. BADGER,A. M., MIRABELLI,C. K. & DIMARTINO,M. (1985). Generation of suppressor cells in normal rats by treatment with Spirogermanium, a novel heterocyclic anticancer drug. Immunopharmacology, 10, 207- 210. BADGER, A. M., SCHWARTZ,D. A., PICKER,D. H., DORMAN,J. W., BRADLEY,F. C., CHESEMAN,E. N., DIMARTINO, M. J., HANNA,N. & MIRABELLI,C. K. (1990b). Antiarthritic and suppressor cell inducing activity of azaspiranes: structure-function relationships of a novel class of immunomodulatory agents. J. med. Chem., 33, 2963- 2970. BOYUM,A. (1968). Isolation of mononuclear cells and granulocytes from human peripheral blood. Scand. J. clin. Lab. Invest., 21, 77-89. ELION, G. B. (1977). Immunosuppressive agents. Transplant. Proc., 9, 975- 979.
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ERHARDT, C. C. (1984). Immunosuppressive therapy for rheumatoid arthritis. In Rheumatoid Arthritis: the Treatment Controversy (eds Goddard, D. and Butler, R.), pp. 9 7 - 1 1 8 . Sheridan House, New York. FAUCI, A. S. (1975). Mechanisms of corticosteroid on lymphocyte subpopulations. I. Redistribution of circulating T and B lymphocytes to the bone marrow. Immunology, 28, 6 6 9 - 680. FAUCI, A. S. & DALE, D. C. (1974). The effect of in vivo hydrocortisone on subpopulations of human lymphocytes. J. clin. invest., 53, 240-246. FAUCI, A. S. & DALE, D. C. (1975). The effect of hydrocortisone on the kinetics of normal human lymphocytes. Blood, 46, 235 - 243. GROSS, D. (1986). Methotrexate therapy in patients with rheumatoid arthritis. Rheumatology, 9, 113- 115. HASIMOTO, T. (1983). A micromethod for the quantitative determination of the viability of Candida albicans hyphae. J. microbiol, Meth., 1, 8 9 - 9 8 . HAYNES, B. F. & FAUC1, A. S. (1978). The differential effect of in vivo hydrocortisone on the kinetics of subpopulations of human peripheral blood thymus derived lymphocytes. J. clin. Invest., 61, 7 0 3 - 707. KARBASSI, A., BECKER, J. M., FOSTER, J. S. & MOORE, R. N. (1987). Enhanced killing of Candida albicans by murine macrophage colony-stimulating factor: evidence for augmented expression of mannose receptors. J. Immun., 139, 417 - 421. KINGSLEY, G., PANAYI, G. & LANCHBURY, J. (1991). Immunotherapy of rheumatic diseases - - practice and prospects. Immunology Today, 12, 177- 179. LEPPNER, G. H. & CALABRESI,P. (1976). Selective suppression of humoral immunity by antineoplastic drugs. Ann. Rev. Pharmac, Toxic., 16, 3 6 7 - 379. LITTLER, T. R. (1990). Anti-rheumatic drugs. In Anti-Rheumatic Drugs (ed. Orme, M.C.L.'E.), pp. 189- 216. Pergamon Press, New York. MISHELL, R. I. & DUTTON, R. W. (1967). Immunization of dissociated spleen cell cultures from normal mice. J. exp. Med., 126, 423 - 442. OTTERNESS, I. G. & CHANG, Y. H. (1976). Cooperative study of cyclophosphamide, 6-mercaptopurine, azathioprine and methotrexate. Clin. exp. Immun., 26, 346-354. REES, A. J. & LOCrWOOD, C. M. (1982). Immunosuppressive drugs in clinical practice. In ClinicalAspects of Immunology (eds Lachman, P. J. and Peters, D. K.), pp. 5 0 7 - 564. Blackwell Scientific, Oxford. RINEHART, J . J . , SAGONE, A . L . , BALCERZAK, S . P . , ACKERMAN, G . A . & LoBUGLIO, A . F . (1975). Effect of corticosteroid therapy on human monocyte function. New Engl. J. Med., 292, 236 - 241. SCOTT, D. L. (1984). Penicillamine. In Rheumatoid Arthritis: the Treatment Controversy (eds Goddard, D. and Butler, R.), pp. 3 7 - 55. Sheridan House, New York. STROEHMANN,I. (1986). Immunosuppressive therapy of rheumatic diseases. Rheumatology, 9, 4 2 - 4 5 . THIEM, P. A., KAPLAN, J. M., BUGELSKI, P. J., RUGGIERI, E. V. & BADGER, A. M. (1992). Induction of suppressor cell activity in vivo and suppression of lymphoproliferative responses in vitro by SK&F 105685 in the dog. Immunopharmacology, 23, 6 7 - 74. WEBEL, M . L . , RITTS, R. E., TASWELL, H . E . , DONAD10, J. V, & WOODS, J . E . (1974). Cellular immunity after intravenous administration of methyl prednisolone. J. Lab. clin. Med., 83, 3 8 3 - 392. WORTIS, H. H., TAYLOR, R. B. & DRESSER, D. W. (1968). Antibody production studied by means of the localized haemolysis in gel (LHG) assay. II. Assay procedure. Immunology, 14, 6 9 - 79.
Int. J. lmmunopharmac., Vol. 15, No. 2, pp. 113-123, 1993. Printed in Great Britain.
EFFECTS OF SK&F 105685, A NOVEL ANTI-ARTHRITIC AGENT, ON IMMUNE FUNCTION IN THE DOG JOHANNE M. KAPLAN, ALISON M . BADGER, ELIZABETH V. RUGGIERI, BARBARA A. SWIFT a n d PETER J. BUGELSKI* Departments of Toxicology and Cell Sciences, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406-0939, U.S.A. (Received 21 July 1992 and in final forrn 19 October 1992)
Abstract - - SK&F 105685 (N,N-dimethyl-8,8-dipropyl-2-azaspiro[4,5]decane-2-propauamine dihydrochlo-
ride) is a novel azaspirane with beneficial activity in animal models of autoimmune disease such as adjuvantinduced arthritis and experimental encephalomyelitis in the Lewis rat and lupus-like disease in the MRL mouse. The activity of SK&F 105685 in these models is associated with the induction of non-specific suppressor cell (SC) activity as defined by the ability of cells from drug-treated animals to inhibit the proliferative response of lymphocytes from control animals to concanavalin A. To evaluate the immunotoxicologic potential of SK&F 105685, the effect on immune function of one month of dosing with 1 mg/kg/day of SK&F 105685 was examined in the dog. Differential blood cell counts and ex vivo immune function assays were performed using blood collected before dosing on days 1 (baseline), 15 and 29, of the study. Immune function assays were performed on spleen cells on day 30. Under the conditions of the study, SK&F 105685 displayed pharmacological activity as demonstrated by the induction of splenic SC activity. The drug did not affect the total number or relative percentages of the various white blood cell types present in peripheral blood and did not cause generalized immunosuppression. The ability of peripheral blood lymphocytes or spleen cells to produce IL-2 or proliferate in response to mitogenic stimulation was not affected by drug treatment. SK&F 105685 also failed to affect the candidacidal activity of polymorphonuclear leucocytes and spleen cells indicating that it is unlikely to compromise nonspecific resistance to infection. SK&F 105685 however, was able to inhibit the generation of a specific in vitro antibody response to sheep red blood cells (SRBC) by splenocytes from treated animals. Inhibition of the anti-SRBC antibody response was also observed upon addition of the drug to normal spleen cells. Addition of the drug at different time points during the culture period indicated that SK&F 105685 was interfering with an event(s) occurring during the first 72 h of culture. Taken together, these results suggest that, in a therapeutic setting, SK&F 105685 is unlikely to compromise the immune status of the host as it can down-regulate a specific immune response without causing generalized immunosuppression.
SK&F 105685 (N,N-dimethyl-8,8-dipropyl-2-azaspiro[4,5]decane-2-propanamine dihydrochloride) (Fig. 1) is a novel azaspirane with beneficial activity in a n i m a l models o f a u t o i m m u n e disease such as adjuvant-induced arthritis and experimental encephalomyelitis in the Lewis rat a n d lupus-like disease in the M R L m o u s e (Badger et al., 1989; A I b r i g h t s o n - W i n s l o w et al., 1990). T h e activity of SK&F 105685 a n d related analogs in these models was f o u n d to correlate with their ability to induce non-specific splenic suppressor cell (SC) activity as defined by the ability o f splenocytes f r o m drug-
treated animals to suppress the c o n c a n a v a l i n A (Con A)-induced p r o l i f e r a t i o n o f n o r m a l spleen cells (Badger et al., 1989; A l b r i g h t s o n - W i n s l o w et al., 1990; Badger et al., 1990b). In the rat, the SC activity appears to reside within a n o n - T , n o n - B , n o n - N K , s e m i - a d h e r e n t spleen cell p o p u l a t i o n with a density ~ 1 . 0 7 g / m l (Badger et al., 1990a). While the i n d u c t i o n o f SC activity by SK&F 105685 m a y be beneficial in the suppression of a u t o i m m u n e responses, the i m m u n o m o d u l a t o r y activity o f the c o m p o u n d also raises concerns a b o u t the potential i n d u c t i o n o f generalized i m m u n o s u p p r e s s i o n . T o
*Author to whom correspondence should be addressed: SmithKline Beecham Pharmaceuticals, P.O. Box 1539, Department of Toxicology, L-60, King of Prussia, PA 19406-0939, U.S.A. 113
J. M. KAPLANel aL
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address this issue in a non-rodent species, the effects of SK&F 105685 on immune function were examined in male dogs, a species commonly used in toxicology studies. SK&F 105685 has recently been shown to induce splenic SC activity in the dog thereby supporting the biological, in addition to the toxicological, relevance of this species (Thiem, Kaplan, Bugelski, Ruggieri & Badger, 1992). Dogs were treated orally with a non-toxic dose of SK&F 105685 (1 m g / k g / d a y ) or vehicle for one month. To assess the immune status of the animals, differential blood cell counts and various parameters of immune function were evaluated ex vivo using blood collected before closing on days 1 (baseline), 15 and 29 of the study. Immune function assays were performed on spleen cells on day 30.
placed in ice-cold phosphate-buffered saline (PBS). The spleen was then minced and forced through a 60-mesh sieve to form a single cell suspension. For the suppressor cell assay, spleen cells were washed 3 times in PBS and resuspended in culture medium. For all other assays involving spleen cells, red blood cells were first lyzed with 0.16 M Tris-ammonium chloride buffer, pH 7.4. Blood cell counts
Automated differential blood cell counts were performed on samples of whole blood using a Technicon H*I instrument (Technicon Instrument Corporation, Tarrytown, NY). Suppressor cell assay
EXPERIMENTAL PROCEDURES Animals
Purebred male beagle dogs (Marshall Animal Farms, Inc., North Rose, NY), 9.5 - 10.5 months old were housed individually in stainless steel cages. A physical examination, as well as hematologic and serum biochemical evaluations, were performed to ensure that only healthy animals were included in the study. The dihydrochloride salt of SK&F 105685 (MW 381.473; supplied by Johnson Matthey, West Chester, PA) was administered orally in gelatin capsules, once daily for 29 consecutive days, to 3 dogs at a dosage of 1 m g / k g / d a y . A control group, consisting of 3 dogs, received empty capsules. Cell preparation
Heparinized blood was collected from each dog before dosing on days 1 (baseline), 15 and 29 of the study. Peripheral blood lymphocytes (PBL) were isolated by density gradient centrifugation on a F i c o l l - Hypaque gradient (1.077 g/ml density) and polymorphonuclear leucocytes (PMN) were obtained by dextran sedimentation (Boyum, 1968). Cytospin preparations of the PBL and PMN fractions from each dog were made for differential analysis. Varying amounts of contaminating P M N were found to be present in the PBL preparations and therefore, values obtained in experiments using PBL were adjusted for the actual number of lymphocytes present in the assay. PMN preparations were 91 - 99070 pure. On day 30 of the study, a portion of spleen (approximately 25 g) was collected aseptically from each dog as soon as the abdomen was opened and
Suppressor cell activity was evaluated using a coculture system which measures the ability of irradiated effector (E) cells to suppress the proliferative response of normal responder (R) cells to the mitogen concanavalin A (Con A) (Badger, Mirabelli & DiMartino, 1985). Briefly, 100/A of effector spleen cells from individual drug-treated or control animals were seeded into 6 replicate wells of a round bottom microtiter plate and serially diluted 2-fold ( 2 . 5 - 0 . 0 8 x 10~ cells/well) in RPMI-1640 medium supplemented with 2 mM glutamine, I00 U/ml penicillin, 100/~g/ml streptomycin and 10070 heat-inactivated fetal calf serum (FCS, Hyclone, Logan, UT). Plated cells were irradiated (3000 Rads) using a Gammacell 40J37Cs irradiation unit (Atomic Energy of Canada Ltd, Ottawa, Canada). Fifty microliters (2.5 x 10~) of responder spleen cells from control animals and 50 ~1 of Con A (5 ~g/ml final concentration) were added to each well. Effector spleen cells from each of the 6 study dogs were tested for suppressor cell activity against responder cells from each of the 3 control dogs thus giving rise to 18 co-culture combinations. The cultures were incubated at 37°C/5% CO2 for 72 h, pulsed with 0.5/~Ci of tritiated thymidine (specific activity 6.7 Ci/mmol, New England Nuclear, Boston, MA) for the final 18 h of incubation and harvested onto glass fiber filters with a 96 well plate harvester (Skatron Inc., Sterling, VA). Cellassociated radioactivity was measured by liquid scintillation counting (Beckman Instruments, Irvine, CA). The percent suppression at each E : R ratio was calculated according to the following equation: °70 suppression= 1 counts/min of heterologous co-culture counts/rain of autologous co-culture x 100.
Immune Function and Anti-arthritic Agent Total "units of suppression" for each co-culture combination were derived from the area under the curve (AUC) of a plot of the percent suppresssion versus the log of the number of effector cells (Badger et al., 1989). Results shown are the mean units of suppression +_ standard error of the mean (S.E.M.) of the control and drug-treated groups tested against each of the control dogs.
Mitogen responses of peripheral blood lymphocytes and spleen cells The mitogenic responsiveness of spleen cells and of PBL after isolation or as present in whole blood was determined. In the whole blood assay, heparinized blood from each dog was diluted 5-fold in serum-free Iscove's Modified Dulbecco's culture medium (Gibco, Grand Island, NY) supplemented with 100 U / m l penicillin, 100/ag/ml streptomycin and 5 × 10 -5 M 2-mercaptoethanol (2-ME). Twenty five microliters of the diluted blood was added in triplicate to the wells of a 96 well round bottom plate with 100/A of serial 5-fold dilutions of mitogen. For isolated PBL and spleen cells, 100/A of cell suspension was added in triplicate to the wells of a 96 well round bottom plate (2.5 x 105 cells/well) with 100 ~1 of mitogen in Iscove's culture medium supplemented with 10°70 heat-inactivated FCS. The mitogens used were: phytohemagglutinin (PHA, Difco Laboratories, Detroit, MI), pokeweed mitogen (PWM, Gibco Laboratories) and Con A (Pharmacia, Piscataway, N J). Cultures were incubated for 72 h at 37°C/5% CO2, pulsed with 0.5/aCi tritiated thymidine for the final 18 h of incubation and tritium incorporation measured.
IL-2 production by peripheral blood lymphocytes and spleen cells To evaluate IL-2 production, 2 × 106 isolated PBL or 4 × 106 spleen cells from each dog were added to the wells of a 24 well plate in a 1 ml volume with 10/al of Con A (10/ag/ml final) or 10/A of medium as a control. Cultures were done in triplicate and the culture medium consisted of RPMI-1640 medium supplemented with 10% heat-inactivated FCS. Culture supernatants were collected after 24 h of incubation at 37°C/5% COy The IL-2 content of the supernatants was determined using the IL-2dependent CT20 murine cytotoxic T-cell line. Triplicate assay cultures in the wells of a 96 well flat bottom plate contained 100~1 of CT20 cells (1 × 104/well) and 100/al of either diluted test supernatant, serial 2-fold dilutions of recombinant mouse IL-2 (Genzyme Corporation, Cambridge,
115
MA) or culture medium as a control. The cultures were incubated overnight at 37°C/5°70 CO2, pulsed with 0.5/aCi of tritiated thymidine for 6 h and cellassociated radioactivity measured. The number o f IL-2 units/ml of supernatant was derived by linear regression analysis from a standard curve of the counts/rain versus recombinant IL-2 units/ml.
Candidacidal activity of polymorphonuclear leucocytes and spleen cells Constitutive microbicidal activity against the facultative intracellular pathogen Candida albicans was measured by a micro-colony formation assay adapted from Karbassi, Becker, Foster & Moore (1987) and Hasimoto (1983). Briefly, C. albicans strain B792 (gift of Dr G. Livi, Biopharmaceutical Sciences, SmithKline Beecham) was maintained on Sabouraud's dextrose (YEPD) agar at 28°C. Yeasts were opsonized by incubating for 2 h at 4°C in RPMI-1640 with 20°70 dog serum. PMN or spleen cells from each dog were resuspended in RPMI-1640 with 10% heat-inactivated FCS and added to quadruplicate wells of a 24 well plate in a 1 ml volume (1 × 10 6 cells/well). The cells were allowed to adhere for approximately 90 min at 37°C/5% CO2 and the supernatant removed. Each well then received 0.2 ml of culture medium and 0.2 ml of opsonized C. albicans (approximately 100 colonyforming units). Control quadruplicate wells contained yeasts without phagocytes. The plates were incubated at 37°C/5% CO2 for 2 h and candidacidal activity was stopped by the addition of 1 ml of YEPD agar to each well. The plates were then incubated overnight to permit the growth of surviving yeasts and subsequent enumeration of the number of colonies/well under a dissecting microscope. The " % kill" was derived from the following formula:
~No.
colonies in experimental well~ o70 kill= 1 - ~ h/~..c--~-~oni'~n~ w e l ~ s J × 100.
Primary antibody response against sheep red blood cells The ability of spleen cells from individual control and drug-treated dogs to mount a primary antibody response against the antigen sheep red blood cell (SRBC) was measured after 29 days of dosing. In in vitro studies, SK&F 105685 ( 0 . 5 - 2 0 0 nM) was added directly to cultures of normal dog spleen cells at different time points during the incubation period. Immunization with SRBC was performed according
116
J. M. KAPLANet at.
to a modification of the method of Mishell & Dutton (1967). Spleen cells were resuspended in Iscove's Modified Dulbecco's culture medium with 10070heatinactivated FCS. Two milliliters of the spleen cell suspensions were added to the wells of a 24-well plate (1 × 107 cells/well) and stimulated with 50/~1 of 1070 SRBC or medium as a background control. Cultures were done in quadruplicate. The plates were incubated for 5 days at 37°C/5% CO, to allow for the development of an anti-SRBC response. The cultures were fed on the second day of incubation with 1 ml of fresh medium containing the appropriate concentration of SK&F 105685 in the case of drug-containing wells. The anti-SRBC response was assessed on the fifth day of culture by the localized hemolysis in gel technique (plaque assay; Wortis, Taylor & Dresser, 1968). Cells harvested from each individual well were resuspended in 0.5 ml of RPMI-1640 medium. A 100/~1 aliquot of cell suspension was mixed with 100/A of a 20070 suspension of SRBC and 0.8 ml of 0.625070 agarose (FMC Bioproducts, Rockland, ME) at 45°C. At this point, SK&F 105685 was also added to aliquots of control cells to look at its effect on antibody secretion. The mixture was then poured into 35 × 10 mm petri dishes pre-coated with 1 ml of 0.5°70 agarose and the dishes were incubated at 37°C for 2 h. At the end of this incubation period, 0.5 ml of guinea pig complement (Cedarlane Laboratories Inc., Ontario, Canada) diluted 1 : 10 in RPMI-1640 medium was poured onto the surface of the gel and the dishes were incubated for an additional hour to allow for the development of IgM plaques.
Stat&tical analysis Results for the control and dosed groups are expressed as the mean _+ S.E.M. of the values obtained for each individual dog. Statistical significance between the 2 groups was determined using the 2-sample Student's t-test. A P value equal to or lower than 0.05 was considered significant.
RESULTS
Splenic suppressor cell activity Splenic suppressor cell (SC) activity was measured after 29 days of dosing, to confirm the pharmacological activity of SK&F 105685 administered at 1 mg/kg/day. SC activity was defined by the ability of irradiated effector spleen cells to suppress the proliferative response of control responder spleen
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117
Immune Function and Anti-arthritic Agent Table 1. Hematology parameters in control dogs and dogs treated with SK&F 105685 Time point
Cell type
Control
SK&F 105685 (1 mg/kg)
WBC Neut Lymph Mono Eos Baso
9.2 5.6 2.6 0.4 0.4 0.06
+0.1" ± 0.1 ± 0.1 ± 0.02 ± 0.1 +_ 0.01
9.4 5.6 2.7 0.5 0.4 0.05
± 1.1 +_ 0.7 +_ 0.3 ± 0.1 ± 0.1 ± 0.01
Day 15
WBC Neut Lymph Mono Eos Baso
9.1 5.5 2.6 0.5 0.4 0.06
+ 0.3 +_0.2 +_ 0.1 ± 0.1 ± 0.1 ± 0.01
8.6 4.8 2.7 0.6 0.5 0.05
± 1.5 ± 1.1 ± 0.2 +- 0.1 +- 0.1 ± 0.01
Day 29
WBC Neut Lymph Mono Eos Baso
10.0 6.3 2.7 0.6 0.3 0.06
_+ 0.8 ± 0.8 _+ 0.3 _+ 0.2 +0.1 ± 0.01
8.8 5.1 2.7 0.4 0.5 0.05
± 0.9 _ 0.8 ± 0.2 +_ 0.1 _+0.1 ± 0.01
Day 1 Baseline
* × 103//al
Neut = neutrophil; Lymph = lymphocyte; Mono = monocyte; Eos = eosinophil; Baso = basophil. the 3 control dogs, cells f r o m drug-treated animals showed greater SC activity than spleen cells from control animals. Statistical significance however, was achieved against only 2 of the control dogs (responders 1,2), the third one being a poor target for suppression. The SC activity observed nonetheless confirmed that SK&F 105685 was pharmacologically active under the conditions of the study.
Blood cell counts Differential blood cell counts were performed on blood collected prior to dosing on days 1 (baseline), 15 and 29 o f the study. Results shown in Table 1 indicate that SK&F 105685 did not affect the total number or relative percentages of the various white blood cell types present in peripheral blood.
Mitogen responses Mitogen-induced proliferation of blood lymphocytes collected before dosing on days 1 (baseline), 15 and 29 of the study was measured to provide a gross index of lymphocyte function. Results f r o m these experiments indicated that SK&F 105685 did not affect the response of unfractionated blood cells or isolated peripheral blood lymphocytes (PBL) to varying concentrations o f the B- and T-cell mitogen
pokeweed mitogen (PWM) and the T-cell mitogens phytohemagglutinin ( P H A ) and Con A. There was no statistically significant difference in the proliferative responses of whole blood [Figs 3(A), (B), (C)] and isolated P B L [Figs 4 (A), (B), (C)] from drug-treated and control animals to optimal concentrations of mitogens. Similar results were obtained with suboptimal and supraoptimal concentrations of mitogens (results not shown). SK&F 105685 also failed te affect the mitogen responses o f spleen cells after 29 days o f dosing (Fig. 5). No significant difference was observed between the proliferative responses of splenocytes from control and drug-treated animals after stimulation with optimal (Fig. 5), suboptimal and supraoptimal (not shown) concentrations of mitogens.
IL-2 production IL-2 production by P B L and spleen cells was evaluated as a parameter of T-cell function. SK&F 105685 did not inhibit the ability of T-cells from the spleen or peripheral blood to produce IL-2 upon stimulation with Con A. There was no significant difference in the a m o u n t of IL-2 produced by PBL from control and drug-treated animals on days 1 (baseline value), 15 and 29 of the study [Fig. 6 (A)]. Similarly, SK&F 105685 had no significant effect on
118
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Candidacidal activity
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DAY 15
DAY 29
Fig. 4. Mitogenic responses of isolated PBL. Isolated PBL from control ([~) and drug-treated dogs (g'A)were stimulated with optimal concentrations of mitogens. (A) PWM (1/250 dilution), (B) PHA (1/7500 dilution), (C) Con A (1.6/~g/ml). Proliferative responses were measured after 72 h of incubation.
microorganisms. To examine the effect o f SK&F 105685 on this aspect of immune defense, the ability of peripheral blood P M N and spleen cells to kill the yeast form o f C. albicans was measured. As depicted in Fig. 7 (A), the same high levels o f candidacidal activity were displayed by P M N from the control and drug-treated groups on days 1 (baseline), 15 and
119
Immune Function and Anti-arthritic Agent PBL
A
1 lq
II 0
DAY 15
DAY1
DAY 29
SPLEEN
B
16 PWM 1/250
PHA 1/7500
CON A 1.6 uglml
Fig. 5. Mitogenic responses of spleen cells on day 30. Spleen cells from control (1-1) and drug-treated (~) dogs were stimulated with optimal concentrations of the mitogens PWM, PHA and Con A. Proliferative responses were measured after 72 h of incubation. 29 of the study. The candidacidal activity of spleen cells (mediated by PMN and/or macrophages) also remained unaffected after 29 days of dosing [Fig. 7 (B)].
._1
10
co
8
6
Primary antibody response of spleen cells to sheep erythrocytes The ability of spleen cells from control and drugtreated animals to mount an antibody response to the antigen sheep red blood cells (SRBC) was measured ex vivo in a hemolytic plaque-forming cell (PFC) assay after 29 days of dosing. As shown in Fig. 8, there was a significant reduction in the ability of spleen cells from SK&F 105685-treated animals to mount an anti-SRBC PFC response indicating that the drug was able to selectively suppress a specific immune response. The inhibitory effect of SK&F 105685 on the generation of an anti-SRBC response was also observed in vitro. Addition of SK&F 105685 to normal dog spleen cells at the beginning of culture resulted in a dose-dependent inhibition of the antiSRBC PFC response (Fig. 9). The suppression was not due to drug cytotoxicity since there was no significant difference in the number of viable cells recovered at the end of the culture period in control and drug-treated wells (not shown). Addition of SK&F 105685 at different time points showed that
DAY 30
Fig. 6. IL-2 production by PBL and spleen cells. PBL (A) and spleen cells (B) from control (to) and drug-treated (~) dogs were stimulated with Con A for 24 h and the amount of IL-2 released in the supernatant measured. the suppressive effect was no longer observed when addition of the drug was delayed by 72 h indicating that SK&F 105685 was affecting an event(s) occurring during the first 72 h of culture (Fig. 10). Addition of SK&F 105685 at the time of the hemolytic PFC assay indicated that the drug did not interfere with secretion of preformed antibodies (not shown). DISCUSSION SK&F 105685 administered for one month at a dosage of 1 mg/kg/day displayed pharmacological activity as defined by the induction of splenic
120
J. M. KAPLANet al. 1000
PMN
100'
A
900
800
+
80' 700
60'
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t,Z l.iJ nUJ 0...
40"
•
600
500
@
(~ ~L
20'
400
30O
2OO
DAY 1
DAY ~5
DAY 29 100
SPLEEN 100'
B G
ANTI. ] R B C
BACKGROUND
Fig. 8. Primary antibody response of spleen cells to SRBC on day 30. Spleen cells from control (0) and drug-treated (~A) dogs were stimulated with SRBC in vitro and the antiSRBC PFC response measured after 5 days of incubation. _~
60"
bZ
o U./ n
40"
G
DAY 30
Fig. 7. Candidacidal activity of peripheral blood PMN and spleen ceils. The candidacidal activity of peripheral blood PMN (A) and spleen cells (B) from control (gl) and drugtreated (VI) dogs was assessed before dosing on days 1, 15, 29 and on day 30 of the study, respectively.
suppressor cell activity, in the absence of overt toxicity. Differential blood cell counts showed that the drug did not affect the total number of relative percentages of the various white blood cell types present in peripheral blood. Ex vivo immune function assays established that SK&F 105685 did not cause generalized immunosuppression. The ability of PBL or spleen cells from drug-treated animals to produce IL-2 (Fig. 6) or proliferate in response to mitogenic stimulation (Figs 3 - 5) was not significantly different from that of control dogs indicating that SK&F 105685 did not globally
t2J t~
?o 12.
v
CONTROL
0.5 nM
2 nM
10 nM
50 nM
200 nM Background
105685 CONC ENrRATION
Fig. 9. Dose-dependent inhibition of the anti-SRBC response of normal spleen cells by SK&F 105685 in vitro. Different concentrations of SK&F 105685 were added to cultures of normal dog spleen cells stimulated with SRBC. The anti-SRBC PFC response was measured after 5 days of incubation.
Immune Function and Anti-arthritic Agent
UJ ¢r bO tl. t~
0H
24 H
48 H
7;' H
96 H
CONTROL
TIME OF ADDITION
Fig. 10. Time dependence of inhibition of the anti-SRBC response of normal spleen cells by SK&F 105685. SK&F 105685 (50 nM) was added to cultures of normal spleen cells and SRBC at different time points. The antiSRBC response was measured after 5 days of incubation. suppress lymphocyte function. The candidacidal activity of PMN and spleen cells was also unaffected by treatment with SK&F 105685 (Fig. 7) suggesting that the compound is unlikely to compromise nonspecific resistance to infection. In contrast to its lack of effect on responses to non-specific stimuli, SK&F 105685 was able to significantly inhibit the generation of a specific immune response. SK&F 105685 strongly suppressed the ability of spleen cells from treated dogs to mount a primary antibody response against SRBC (Fig. 8). The inhibitory effect of SK&F 105685 on the generation of an antiSRBC response was also observed in vitro upon addition of the drug to normal spleen cells (Fig. 9). The development of an anti-SRBC PFC response involves a complex interplay between antigen-presenting cells, T-helper lymphocytes and B-lymphocytes. The identity of the cell population(s) whose activity is inhibited by SK&F 105685 or SK&F 105685-induced SC remains to be determined. Addition of the drug at different time points during the 5 days in vitro culture period indicated that SK&F 105685 was interfering with an event(s)
121
occurring during the first 72 h of culture (Fig. 10). IL-2 production by T-helper lymphocytes, an early event in the development of humoral responses, is not likely to be a target for suppression by SK&F 105685 as drug treatment did not inhibit IL-2 production by PBL and spleen cells in this study (Fig. 6). In separate studies, SK&F 105685 also failed to inhibit IL-2 production by rat splenocytes and human Jurkat cells (results submitted for publication). Accessory cell function, production of lymphokines other than IL-2, as well as responsiveness of B- and T-cells to lymphokines represent other possible targets for suppression of the anti-SRBC response. B-cell differentiation as well as the synthesis and secretion of antibodies are not likely to be inhibited by SK&F 105685 as these events occur late in the development of an antibody response. Moreover, addition of the drug at the time of the PFC assay showed that SK&F 105685 did not interfere with the secretion of preformed antibodies (not shown). The ability of SK&F 105685 to selectively inhibit an antigen-specific immune response ex vivo without affecting circulating levels of lymphoid cells or their functional activity in response to non-specific stimulation is in sharp contrast with the effect of most anti-rheumatic drugs. The immunomodulatory drugs currently used in the treatment of rheumatoid arthritis tend to induce a global, non-specific immunosuppression as a result of their cytotoxic activity. The 3 main groups of immunosuppressive anti-rheumatic drugs; folic acid analogs (e.g. methotrexate), purine analogs (e.g. azathioprine) and alkylating agents (e.g. cyclophosphamide), all exert anti-proliferative activity and careful monitoring of blood cell counts during the course of treatment is required to reduce the risk of bone marrow suppression (Erhardt, 1984; Stroehmann, 1986; Littler, 1990). For example, leukopenia is a common side-effect of treatment with cyclophosphamide, azathioprine or methotrexate and has been associated with an increased susceptibility to infection (Erhardt, 1984; Gross, 1986; Stroehman, 1986; Littler, 1990). In addition to depleting leucocytes from the peripheral blood and tissues, anti-proliferative drugs can also affect the functional activity of leucocytes, at least in part, by interfering with their ability to divide. Methotrexate is capable of non-specifically impairing both humoral and cellmediated immune responses while cyclophosphamide favors the inhibition of humoral responses and azathioprine shows more effectiveness against cell-mediated immune reactions (Leppner & Calabresi, 1976; Otterness & Chang, 1976; Elion, 1977).
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J. M. KAPLANet aL
Other disease-modifying anti-rheumatic drugs such as gold, sulfasalazine and penicillamine whose mode of action is less defined, also display bone marrow toxicity leading to blood dyscrasias such as thrombocytopenia and neutropenia (Scott, 1984; Littler, 1990). Finally, glucocorticoids which exert potent anti-inflammatory activity have been shown to cause depletion of monocytes and lymphocytes, in particular T-cells, from peripheral blood in humans and experimental animals (Haynes & Fauci, 1978; Rees & Lockwood, 1982). The lymphopenia appears to be due to a change in the migratory pattern of lymphocytes leading to their accumulation in the bone marrow (Fauci & Dale, 1975; Fauci, 1975). Glucocorticoids also have profound immunomodulatory effects and treatment in humans has been associated with decreased proliferative responses of lymphocytes to mitogens (Fauci & Dale, 1974; Webel, Ritts, Taswell, Donadio & Woods, 1974) and decreased bactericidal and fungicidal activity of monocytes (Rinehart, Sagone, Balcerzak, Ackerman & LoBuglio, 1975). In addition to other manifestations of toxicity, the potential risk for increased frequency of infection and malignancy associated with the generalized immunosuppression caused by cytotoxic drugs has generated interest in the development of more "selective" immunosuppressive agents. For example, cyclosporin A, which selectively inhibits T-cell function, has been tested in the treatment of rheumatoid arthritis but, despite encouraging results, its associated nephrotoxicity makes cyclosporin A a poor candidate (Kingsley, Panayi &
Lanchbury, 1991). In this study, SK&F 105685 has displayed many desirable characteristics as a selective immunomodulator. In contrast to cytotoxic drugs, SK&F 105685 did not have any significant effect on the level of circulating leucocytes. It also preserved the functional activity of PMN and lymphocytes in response to non-specific stimulation. Finally, SK&F 105685 was able to inhibit the generation of an antibody response to the T-dependent antigen SRBC. The significance of SK&F 105685-mediated suppression of splenic antibody responses as it relates to the inhibitory activity of the drug against autoimmune responses remains to be determined. In the rat, a species in which SK&F 105685 effectively downregulates autoimmunity, oral dosing under different regimens failed to inhibit systemic antibody production as assessed by the measurement of serum levels of antigen-specific antibodies (unpublished results). The observation of suppressed antibody production by spleen cells in this study may reflect the presence of suppressor cells in that organ as opposed to the peripheral blood. Indeed, SC activity can be detected in the spleen but not the peripheral blood of SK&F 105685-treated dogs (Thiem et al., 1992) and rats (unpublished observation). Studies are under way to resolve this issue and clarify the mechanism of action of SK&F 105685. Acknowledgements - - We wish to thank Gary Gries and
Denise Fennelly for the dosing and care of the animals and we are grateful to Terry Sellers and Cherry Gunning for the collection of hematology parameters.
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