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Techniques for assessing canine mononuclear phagocyte function as part of an immunotoxicologic evaluation
Int. J. Immunopharmac., Vol. 10 No. 6, pp. 765 Printed in Great Britain.
771, 1988.
0 1 9 2 - 0 5 6 1 / 8 8 $3.00+ .00 International Society for l m m u n o p h a r m a c o l o g y .
TECHNIQUES FOR ASSESSING CANINE MONONUCLEAR PHAGOCYTE FUNCTION AS PART OF AN IMMUNOTOXICOLOGIC EVALUATION PATRICIA A. THIEM, LEE K. HALPER and JOHN C. BLOOM Department of Experimental Pathology, Smith Kline and French Laboratories, King of Prussia, PA 19406-0939, U.S.A. (Received 14 July 1987 and in final form 2 March 1988)
Abstract -- Although the dog is used extensively as a model for toxicologic investigation, few practical
techniques for assessing immunotoxicity in this species have been reported. We have adapted techniques for assessing peripheral blood monocyte (PBM) function in the dog. Methods for isolating PBM were compared; a technique employing hypertonic conditioning followed by density gradient centrifugation, which resulted in > 90% purity and a greater yield of PBM than that obtained with other previously reported methods is described, along with methods for measuring antibody-dependent cellular cytotoxicity (ADCC) and the production of prostaglandin E2 (PGE2) and superoxide anion (O2). An assay for the measurement of interleukin-1 (IL-1), based on its ability to induce interleukin-2 (IL-2) production by the EL-4 mouse thymoma line in the presence of the calcium ionophore A23187, was examined. Together, these assays provide the tools to better define drug or chemical effects on the mononuclear phagocyte system (MPS) of this important animal model.
The MPS plays an important role in immune regulation and host defense that includes the processing and presentation of antigen, opsonization and phagocytosis of microorganisms, cytolysis of tumor cells, chemotaxis and the synthesis and secretion of various immunoregulatory products (Neveu, 1986; van Furth, Cohn, Hirsch, Humphrey, Spector & Langevoort, 1972). This system is therefore an important target for the pharmacologic and toxicologic actions of immunomodulating and immunotoxic drugs and chemicals. While the dog has been used extensively as a model for safety and toxicity studies in the pharmaceutical industry, our understanding of immune function in this species is limited (Bloom, Thiem & Morgan, 1987; Colgrove & Shifrine, 1980). Moreover, many of the assays useful in defining these parameters have not been validated in the dog or are impractical for monitoring drug or chemical effects. Assays which can be applied to dogs over the course of acute, subacute or chronic toxicity and safety studies must be minimally invasive, use sample volumes which are uncompromising to the animal and accomodate the processing of several samples at one time. Previous reports on the assessment of immune function in the dog have either not addressed MPS function specifically, or include
techniques that are unacceptably invasive for interim monitoring (Oghiso, Kubota, Fukuda & Iida, 1985). We report here assays for afferent (IL-1 and PGE2 production) and efferent (ADCC and 02 production) mononuclear functions that employ canine PBM and have been adapted to meet the aforementioned requirements.
EXPERIMENTAL PROCEDURES
Animals
Purebred beagles obtained from White Eagle Laboratories (Doylestown, PA) were maintained at Smith Kline & French Laboratories under conventional conditions. Males and females 1 - 7 years old were included in each group. The dogs were serving, at the time of evaluation, as controls for one of two long-term toxicity studies and receiving either 0.3 ml/kg 0.5o70 tragacanth, N.F., (gum tragacanth, a demulcent used as vehicle for administration of water-insoluble compounds) per os per day or intramuscular injections of 0.04 ml sterile water once every 3 days. Physical examinations, as well as hematologic, clinical biochemical and uroanalytic evaluations were performed periodically throughout the study; animals showing positive findings on these 765
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health screens were disqualified from the study. Additional normal naive dogs were also examined. P B M &olation
Peripheral blood mononuclear cells were isolated by a modified method of Recalde (1984). Briefly, blood collected in 3.8°7o sodium citrate was spun at 1000 × g for 5 rain at 25°C, and the plasma and buffy coat removed. Bovine thrombin (American Scientific Products, McGaw Park, IL) was added to the p l a s m a - b u f f y coat to provide a final concentration of 1 U/ml. The suspension was incubated for 15 min at 25°C with gentle mixing. The cells were then incubated at 37°C for 15 min following each of three additions of 9.007o saline (10, 20 and 20 ~1 per ml p l a s m a - b u f f y coat). The suspension was then diluted with 2 ml 0.907o saline and 54 /A 9.007o saline per ml p l a s m a - b u f f y coat, layered on Ficoll-PaqueTM (Pharmacia Fine Chemicals, Piscataway, N J), 0 = 1.077, adjusted to 3 9 0 - 4 1 0 m o s m / k g with the addition of crystalline sodium chloride, as determined by the freezing-point depression method), and centrifuged at 700 × g for 30 min at 25°C in 15 ml conical tubes. The PBM band was removed and washed three times with Dulbecco's phosphate-buffered saline (PBS), pH 7.4; the leukocyte concentration was determined and adjusted as required in the appropriate assay buffer. In all assays, PBM preparations were > 90070 pure as defined by non-specific a-naphthyl butyrate esterase staining (Sigma Chemical Co., St Louis, MO). Sterility was maintained during the separation procedure when PBM were to be cultured for IL-1 or PGE2 production. 0 2 assay
Superoxide anion production was measured by an adaptation of a procedure described by Babior, Kipnes & Curnutte (1973), based on the principle that PBM stimulated with phorbol myristate acetate (PMA) produces 0 2 which can be measured spectrophotometrically by the reduction of ferricytochrome c in a superoxide dismutase (SOD) inhibitable system. All reagents were obtained from Sigma. The assay buffer was PBS containing 0.1% glucose, 0.6 mM CaCI2 and 1.0 mM MgC12. Cytochrome c was prepared immediately before use. Superoxide dismutase, dissolved in buffer, was stored at 4°C for up to 1 week. Phorbol myristate acetate was dissolved in dimethylsulfoxide, stored at - 7 0 ° C and diluted in buffer. The test was carried out in 96-well flat-bottom plates. Wells contained 40 /A (26 nmoles) cytochrome c (0.32 mg), 10/A SOD
(0.04 mg) or buffer, 10/al PMA (0.2 #g) or buffer, and 40/A PBM (1.6 x 105 cells), which were added last. Plates were incubated at 37°C and read at 1 and 2 h intervals on a MR600 plate reader (Dynatech Laboratories, Inc., Alexandria, VA) at 550 nm vs a 490 nm reference. Data are reported as nmoles cytochrome c reduced, which was corrected for reduction in the presence of SOD, and in the absence of PMA. A D C C assay
Antibody-dependent cellular cytotoxicity was measured according to the method of Perlman & Holm (1969), by the release of ~Cr from labelled antibody-coated chicken erythrocyte (ChE) target cells after incubation with PBM. Assay medium was RPMI-1640 containing 10% fetal calf serum (FCS; Hazleton Dutchland Inc., Denver, PA), which was heat-inactivated at 56°C for 60 rain. Chicken erythrocytes (Colorado Serum Co., Denver, CO) were stored for up to 2 weeks in Alsever's solution at 4°C. Washed ChE (1 × 107 cells) were incubated with 250 ~Ci 5~Cr (sodium chromate in normal saline, pH 8, 200-900 Ci/g; New England Nuclear, Boston, MA) and RPMI-1640 in a total volume of 2 ml at 37°C for 2 h with occasional mixing, washed three times, and resuspended in 10 ml RPMI-1640. The ChE were incubated for an additional 30 min to remove excess chromium, washed five times, and resuspended to 1 × 10 5/ml in medium. The assay was performed in triplicate in 96-well U-bottom plates. Wells containing 100/A 5JCr-ChE suspension and 10 /A optimally diluted heat-inactivated rabbit anti-ChE antiserum (Organon Teknika, Jessup, MD) or medium alone, were incubated at 4°C for 30 min. Monocytes (100 ~1 at 5, 2.5 or 1.25 × 105/ml) were added and the plates spun at 80 × g for 3 min, followed by incubation at 37°C in a humidified chamber of 5% CO2 in air for 3 h. The plates were then spun at 400 × g for 10 min, and 100 /al of supernatant were transferred to a 12 × 75 mm tube. Radioactivity was counted for 1 rain using a System 5500 gamma counter (Beckman Instruments, Irvine, CA). Replicate counts/min were corrected for spontaneous release (in the absence of antibody and PBM); the % 5~Cr-release was determined based on total release using 1% Triton X-100 (Sigma). Data are reported as specific % 5'Cr-release adjusted for that due to antibody or PBM alone. P G E 2 production and assay
Prostaglandin E2 produced by stimulation of PBM with lipopolysaccharide (LPS, E. coli 055.B5; Difco Laboratories, Detroit, MI) was measured using a
Assessing PBM Function in Dogs radioimmunoassay kit (New England Nuclear) according to the method of Maclouf, Praedel, Pradelles & Dray (1976) based on competitive inhibition of binding. The culture medium for PGE2 production, as well as IL-1 production and assay was RPMI-1640 containing 5% heat-inactivated FCS, 10 mM Hepes, 2 mM L-glutamine, 100 U/ml penicillin and 100 /ag/ml streptomycin. All culture reagents were supplied by Gibco Laboratories, Grand Island, NY. Cultures containing 500/al PBM suspension (5 × 105 cells) and 5/~1 LPS (2.5/ag) or medium were incubated at 37°C in a humidified chamber of 5o70 CO2 in air for 24 h in 48-well plates. Supernatants were harvested through a 0.22 /am filter, stored at - 70°C and assayed according to the manufacturer's directions. Briefly, dilutions of PGE2 standard or supernatant, 125I-PGE2tracer, and rabbit anti-human PGE2 were incubated at 4°C overnight. The tubes were incubated on ice for 30 min with PEG-6000, to precipitate the immune complexes, and then centrifuged. The supernatants were aspirated and the pellets counted for 1 min. PGE2 concentrations were determined from the standard curve and corrected for dilution. In one experiment, indomethacin (0.25 /ag; Sigma) was added to the cultures to block PGE2 production.
IL-1 production and assay Interleukin-1 was produced under identical conditions to PGE2, except that the cultures were incubated for 48 h. Interleukin-I in the supernatants was measured according to the method of Simon, Laydon & Lee (1985). Briefly, EL-4 murine lymphoma cells, in the presence of IL-1 and the calcium ionophore A23187, produce IL-2, which is assayed by the proliferation of CT-20 murine IL-2dependent cytotoxic T-cells. EL-4 and CT-20 cells and supernatant from LPS-stimulated human placental tissue or recombinant human IL-1 (which served as positive IL-1 controls) were kindly provided by Dr Philip Simon, Department of Immunology and Antiinfectives Therapy, Smith Kline and French Laboratories. Rat T-cell growth factor (TCGF), supernatant from rat spleen cells (5 × 106) incubated with concanavalin A (5 /ag; Pharmacia) for 48 h at 37°C in a humidified chamber of 5o7o CO2 in air, served as an IL-2 positive control. Cells were maintained in vitro in assay medium supplemented with (CT-20) or without (EL-4) 20°7o TCGF. Duplicate serial dilutions (100 /al) of PBM supernatants, IL-I positive control or medium alone, 50/al A23187 (2 x 10-7M final concentration; Sigma) and 100/al EL-4 cells (1.5 × 105 viable cells)
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were prepared in 96-well flat-bottom plates. Additional plates containing dilutions of the TCGF or test supernatants, A23187 and medium were run in parallel to test for the presence of IL-2. The plates were incubated at 37°C as above overnight. Following the transfer of 100/al of supernatant from each well to a new plate, 100 /al CT-20 (1 × 104 viable cells) were added and the plate was incubated at 37°C overnight. Cultures were pulsed with 10/al (1.0/aCi) 3H-thymidine (2 Ci/mmole; New England Nuclear), incubated again for 4 - 6 h, and harvested on glass fiber filters using a PHD cell harvester (Cambridge Technology, Cambridge, MA). Radioactivity was measured for 1 min on an LS 3800 beta counter (Beckman). IL-1 U / m l were calculated by parallel line analysis, using a computer program, in comparison to the standard. In one experiment, indomethacin (0.25/ag) was added to the cultures to determine whether prostaglandin production could be inhibiting that of IL-1. Statistics Statistical analysis was performed on all data using the least squares method to fit general linear models. Data points that were < or > 3 S.D.s from the mean of all observations were excluded. Significance (P <0.05) was determined by Tukey's studentized range test comparing each group to the others (SAS, 1985).
RESULTS
P B M &olation Monocyte preparations isolated by hypertonic conditioning contained 9 8 - 99°7o mononuclear cells and 1 -2°7o granulocytes and exhibited > 90°7o nonspecific esterase positivity. Viability was routinely > 95%, as determined by trypan blue dye exclusion. Preparations contained an average of five erythrocytes and 25 platelets per PBM. Yields ranged from 5 to 30%, based on absolute monocyte count. Table 1. Of production by peripheral blood monocytes* Group Tragacanth Water Naive Total
Age (years) 6-7 6-7 1-7
1 hour 1.9 + 0.3 1.4 _+0.3 1.0 _ 0.3 1.5 _+0.2
2 hours
(12) 2.9 + 0.3 (14) (13) 1.9 _+0.4 (13) (4) 1.1 ___0.3 (4) (29) 2.2_+0.3 (31)
*Data are reported as nmoles cytochrome c reduced by 1.6 × 105 PBM stimulated with 0.2/ag PMA and represent the mean _+S.E.M. with the number tested in parentheses.
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Table 2. Monocyte antibody-dependent cellular cytotoxicity*
Group Tragacanth Water Naive Total
Age (years) 6-7 6-7 1-7
Effector (PBM): target (ChE) ratio 5:1 2.5:1 1.25:1 56_+4 59_+4 50_+4 56_+ 3
(14) (14) (9) (37)
42+5 44_+6 45 +_7 43_+3
(14) 31 _+ 5 (14) (14) 30_+6 (14) (7) (35) 30_+4 (28)
*Data are reported as specific °7o 5~Cr release from antibody-coated target cells after incubation with PBM effector cells for 3 h, and represent the mean + S.E.M. with the number tested in parentheses. P e r i p h e r a l b l o o d samples of 2 0 - 4 0 ml p r o v i d e d sufficient n u m b e r s o f m o n o c y t e s ( 0 . 5 - 2 × 10 6) for any o f the assays described in this report.
to minimize the n u m b e r of cells required. The o p t i m a l dilution o f a n t i b o d y for sensitization of C h E was previously d e t e r m i n e d by titration. Ten-fold serial dilutions o f a n t i b o d y were tested; a dilution of 1:1000 was f o u n d to be optimal. The 5~Cr-labelled cells were p r e p a r e d o n the day o f the assay. To increase the sensitivity o f the system, 5~Cr-ChE were pre-incubated with a n t i b o d y in the microtiter plates at 4°C prior to the a d d i t i o n of P B M . This was followed by low speed c e n t r i f u g a t i o n to increase effector: target interaction. The total % S'Cr-release from all the controls was < 5%. A decrease in the % 5~Cr-release was observed with decreasing effector: target ratios (Table 2). There was n o significant statistical difference between the groups at any effector: target ratio.
02production The o p t i m a l c o n c e n t r a t i o n s o f b o t h P B M a n d P M A for stimulating 0 2 p r o d u c t i o n were studied over time. P B M were tested at 0 . 5 - 2 × 105/weU; 1.6 × 105 was f o u n d to be o p t i m a l a n d employed in the study. P h o r b o l myristate acetate was tested at final doses r a n g i n g f r o m 0.1 to 1.0 /ag; 0.2 /ag stimulated m a x i m u m 0 2 g e n e r a t i o n by P B M . Plates were read at intervals up to 4 h. Superoxide generation was negligible at 30 min, maximal at 2 - 3 h a n d decreased by 4 h. One a n d 2 h i n c u b a t i o n s were selected for this study; n a n o m o l e s of c y t o c h r o m e c reduced are s h o w n in Table 1. U p to 2 0 % o f the total a m o u n t o f c y t o c h r o m e c present in the system was reduced. This was completely i n h i b i t e d by SOD. T h e reduction o f c y t o c h r o m e c by P B M alone in the absence o f P M A was < 1 nmole. T h e r e was a 2 - 10 fold increase in O~ p r o d u c t i o n w h e n cells were stimulated with P M A . There was n o statistically significant difference between the groups.
A D C C assay The effect o f varying effector: target ratios was studied. Since the release o f 51Cr at 5 a n d 10:1 was c o m p a r a b l e , ratios o f 5, 2.5 a n d 1.25:1 were chosen
PGE2 production Three two-fold serial dilutions o f s u p e r n a t a n t s f r o m control a n d L P S - s t i m u l a t e d cultures were assayed for PGE2 content in duplicate. C o u n t s that did n o t fall in the linear p o r t i o n o f the curve were discarded. Results o f the assay are s h o w n in Table 3. The kit employs a r a b b i t a n t i - h u m a n PGE2 antibody, which has been s h o w n to recognize PGE2 from several species, including m o u s e a n d rat ( J o h n s o n , D i M a r t i n o & H a n n a , 1986). To show cross-reactivity o f the a n t i b o d y with canine PGE2, L P S - s t i m u l a t e d PGE2 p r o d u c t i o n by P B M cultured in the presence of i n d o m e t h a c i n was examined. P r o s t a g l a n d i n E2 synthesis was effectively blocked by this inhibitor as s h o w n in Table 3. There was n o statistically significant difference between any o f the groups.
IL-1 production Eight t w o - f o l d serial dilutions of s u p e r n a t a n t s f r o m control a n d L P S - s t i m u l a t e d cultures were assayed for b o t h IL-1 a n d IL-2. The optimal c o n c e n t r a t i o n o f L P S (5/ag/ml) h a d been previously determined from dose-response experiments.
Table 3. PGE2 production by peripheral blood monocytes* Group Tragacanth Water Naive Total
Age (years) 6-7 6-7 1-7
+ LPS 202 170 181 184
+ 20 + 22 _+ 64 +_ 17
(11) (13) (6) (30)
-LPS
+ LPS + indomethacin
21 + 11 (4) 130 _+ 47 (4) 5 +_ 1 (5) 75 __. 30 (8)
*Data are reported as ng/ml PGEz in supernatants from PBM incubated with or without LPS in the presence or absence of indomethacin for 24 h, and represent the mean -!-_S.E.M. with the number tested in parentheses.
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Table 4. IL-1 production by peripheral blood monocytes* Group Tragacanth Water Naive Total
Age (years) 6- 7 6-7 1-7
+ LPS 899 ± 263 459 ± 66 919±405 746 ± 114
- LPS
(13) (13) (10) (36)
211 ± 180 ± 154± 181 ±
126 36 56 35
+ LPS + indomethacin (5) (12) (5) (22)
2020 ± 462 (6)
*Data are reported as IL-1 U/ml in supernatants from PBM incubated with or without LPS in the presence or absence of indomethacin for 48 h, and represent the mean ± S.E.M. with the number tested in parentheses. Results are shown in Table 4. No IL-2 was detected in these supernatants that could artifactually influence the IL-1 activity measured. Associated with the addition of indomethacin to LPS-stimulated cultures, and the subsequent inhibition of PGE2 production by PBM from normal dogs, was a moderate increase in IL-1 production, which was not statistically significant. This could represent inhibition of IL-1 production by prostaglandin synthesis in vitro. There was no statistically significant difference between any of the groups.
DISCUSSION In this study, we examined a panel of assays for canine PBM that represent efferent (O; generation and ADCC) and afferent (IL-I and PGE2 production) immune function. The selection of assays was based on the importance of these aspects of MPS function to the integrity of the immune system and the fact that they entail endpoints that are quantifiable with reasonable precision using practical techniques that conform to the aforementioned constraints. They have been widely applied in studies in man and other species on immunomodulatory agents such as gold compounds (Barrett & Lewis, 1986; Griswold, Lee, Poste & Hanna, 1985; Bray & Gordon, 1978). These assays have been used recently in our laboratory to study the effect of long-term chrysotherapy on immune function in the dog (Bloom, Thiem, Halper, Saunders & Morgan, 1988). Some of the control animals from that study were used to establish the normal parameters reported here. The treatment of these dogs, as described above, produced no statistical differences in immune parameters examined. Monocytes stimulated by non-specific membrane activators undergo a respiratory burst associated with increased oxygen consumption and secretion of superoxide anion. Commonly used stimuli are PMA,
opsonized zymosan and formylated oligopeptides, such as N-formyl-methionyl-leucyl-phenylalanine (FMLP). Canine granulocytes, however, are reported to lack receptors for FMLP (Redl, Flynn, Lamche, Schiesser, Schlag & Hammerschmidt 1983). In our assay, PMA-stimulated PBM reduced up to 20°70 of the cytochrome c in the system. Similar concentrations of neutrophils, stimulated with approximately one-tenth the amount of PMA for 30 min, reduced up to 55°70 of comparable concentrations of cytochrome c (data not shown). Antibody-dependent cellular cytotoxicity is dependent on three distinct steps: binding of antibody (IgG) to the target cell, binding of IgG on the target cell to the Fcy receptor of the effector cell and, finally, lysis of the target by the monocyte. Because both of the latter mechanisms must be intact for this to occur, ADCC is a useful "screen" for these important aspects of monocyte/macrophage effector function. Despite the variability inherent in the preparation and sensitization of 5~Cr-labelled target cells, the percent 51Cr release observed in this study remained fairly constant over a period of 6 months, with the amount of 5JCr release correlating well with the effector: target ratios. Prostaglandin E2 and IL-1 are monokines that mediate inflammation and a variety of other functions. Their production following LPSstimulation in these studies was variable. Similar variability has been observed using PBM from other animal species and man (Simon & Willoughby, 1982; Newton, 1986). Whether this phenomenon represents experimental artifact or normal biologic variability, it serves to obscure subtle changes in these parameters thus limiting the sensitivity of these tests. The EL-4 assay for the quantitation of IL-1 offers many advantages over the conventional murine thymocyte co-stimulator assay. The system is 1 0 - 2 5 times more sensitive (Simon et al., 1985), is less timeconsuming, requires less sample and obviates the need for maintaining a supply of weanling mice. There is a wide inter-animal variation in IL-1
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production by PBM, possibly due to the phenomenon of spontaneous production discussed below and/or the day-to-day variability common to assays employing cultured cells. In addition, canine IL-1 does not withstand repeated freeze-thaw cycles. Because of these problems, changes in IL-1 production in a test animal, whether iatrogenic or due to spontaneous disease, should be compared to a baseline value for that animal, as well as that of control dogs. It is therefore highly desirable that samples from a given study be assayed in a single experiment. One of the major problems previously encountered in assessing PBM function has been obtaining sufficient numbers of PBM from a volume of blood sample that would neither compromise the animal nor influence other parameters monitored. This was accomplished through the adaptation of a separation technique employing hypertonic conditioning previously described for isolating human PBM, which provides isolates of comparable purity and greater yield than those obtained by variations of the conventional adherence techniques (Mantovani, 1981). The isolation procedure does not require special equipment and can usually be completed within 5 h, thus allowing completion of most function assays within a normal working day. In addition, it has been reported that human cells isolated by this procedure retain functions characteristic of monocytes, including rosette formation, phagocytosis of antibody-coated erythrocytes and bactericidal activity (Recalde, 1984). Of major concern, regarding the isolation technique, was the possibility that the physical stress imposed by the isolation treatment employing hypertonic Ficoll-PaqueT M centrifugation contributed to the spontaneous PGE2 and IL-1 production in unstimulated cultures observed in this study. This was explored by comparing these activities in cultures containing PBM isolated as described with cells isolated using conventional Ficoll-Hypaque centrifugation (Boyum, 1968). Although higher titers were measured in supernatants from cells isolated by the hypertonic conditioning method, the amount of IL-1 produced in the presence of LPS relative to that in the unstimulated cultures was comparable. Another possible explanation for the spontaneous PGE2 and IL-1 observed is endotoxin contamination of reagents. Although FCS containing endotoxin concentrations of < 0.003 to 0.4 ng/ml could have contributed to this effect, pretreatment with polymyxin B produced similar results as those observed using untreated medium (data not shown).
This measure, however, does not rule out the possibility that significant levels of LPS were present, as polymyxin B has been reported to bind some, but not all, endotoxins (Kluger, Singer & Eiger, 1985). I n vivo activation of PBM with subsequent IL-1 release has been previously reported from canine peripheral blood mononuclear cells after three hours, whether or not LPS was added to the culture medium (Cerruti-Sola, Kristensen, Vandevelde & de Weck, 1984). Whether the "spontaneous" release in both circumstances is a consequence of LPS contamination of reagents is problematic. In addition to the possible contribution of experimental conditions relating to the cell separation and assay procedures, the variability observed in these parameters of mononuclear phagocyte function could also reflect the range of immunostimulation or immunoreactivity inherent in these dogs. Whether or not this variation is large enough to obscure biologically significant druginduced changes is uncertain and clarification awaits more experience with these tests in immunotoxicologic studies. Studies with agents known to affect these parameters are also indicated before such tests can be considered fully validated for use in experimental toxicology. These studies are in progress. Finally, caution is also warranted when interpreting positive results of these tests on PBM as effects on the MPS in general. It is well established that mononuclear phagocytes from different tissue compartments will vary substantially regarding both affector and effector immune function (Hearst, Warr & Jakab, 1980; Hopper, Wood & Nelson, 1979; Knop, 1980). While the use of PBM has the considerable advantage of accessibility, making possible interim monitoring, these are the least differentiated and specialized cells of the MPS. The relative sensitivity with which the PBM reflects drug and chemical effects on this important target organ system has yet to be determined. With the increased efforts by the pharmaceutical industry to develop immunomodulators, as well as other immunoactive biotechnology-derived candidate drugs, reliable, validated and practical assays for immune function will play an increasingly important role in the safety evaluation of these compounds. The assays we have examined, together with other more well established assays, such as tests for bactericidal/phagocytic activity or surface receptor expression, should prove useful in allowing us to better define the effects of drugs and chemicals on the MPS of the dog.
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REFERENCES
BABIOR,B. M., KIPNES,R. S. & CURNUTTE,J. T. (1973). Biological defense mechanisms. The production by leukocytes of superoxide, a potential bactericidal agent. J. clin. Invest., 52, 741- 744. BARRETT, M. L. & LEWIS, G. P. (1986). Unique properties of auranofin as a potential anti-rheumatic drug. Agents Actions, 19, 109- 115. BLOOM, J. C., TH1EM, P. A. & MORGAN,D. G. (1987). The role of conventional pathology and toxicology in evaluating the immunotoxic potential of xenobiotics. Toxicol. Path., 15, 283-293. BLOOM, J. C., THIEM, P. A., HALPER, L. K., MAUNDERS,L. Z. & MORGAN, D. G. (1988). The effects of long-term treatment with auranofin and gold sodium thiomalate on immune function in the dog. J. Rheumat., 15, 409-417. BoYuM, A. (1968). Isolation of mononuclear cells and granulocytes from human blood. Scand. J. clin. Lab. Invest., 21 (suppl. 97), 7 7 - 8 9 . BRAY, M. A. & GORDON, D. (1978). Prostaglandin production by macrophages and the effects of anti-inflammatory drugs. Br. J. Pharmac., 63, 635-642. CERRUTI-SOLA,S., KRISTENSEN,F., VANDEVELDE,M. & DE WECK, A. L. (1984). lnterleukin 1- and 2-like activities in the dog. Vet. Immun. Immunopath., 6, 261-271. COLGROVE, G. S. & SHIFRXNE,M. (1980). Canine immunology: current status. In The Canine as a Biomedical Research Mode# Immunological, Hematological, and Oncological Aspects (eds Shifrine, M. and Wilson, F. D.) pp. 43 - 66. U.S. Department of Energy, Washington, DC. HEARST, J. E., WARR, G. A. & JAKAB, G. J. (1980). Characterization of murine lung and peritoneal macrophages. J. Reticuloendothel. Soc., 27, 443 - 454. HOPPER, K. E., WOOD, P. R. & NELSON, D. S. (1979). Macrophage heterogeneity. Vox Sang., 36, 257-274. GRISWOLD, D. E., LEE, J. C., POSTE, G. & HANNA, N. (1985). Modulation of macrophage - lymphocyte interactions by the antiarthritic gold compound, auranofin. J. Rheumat., 12, 490-497. JOHNSON, W. J., DIMARTINO, M. J. & HANNA, N. (1986). Macrophage activation in rat models of inflammation and arthritis: determination of markers of stages of activation. Cell. Immun., 103, 54-64. KLUGER, M. J., SINGER, R. & EIGER, S. M. (1985). Polymyxin B use does not ensure endotoxin-free solution. J. Immun. Meth., 83, 201- 207. KNOP, J. (1980). Influence of various macrophage populations on Con-A induced T-cell proliferation. Immunology, 41, 379-385. MACLOUF, J., PRAEDEL, M., PRADELLES, P. & DRAY, F. (1976). ~251derivatives of prostaglandins. A novel approach in prostaglandin analysis by radioimmunoassay. Biochem. biophys. Acta, 431, 139- 146. MANTOVANI, A. (1981). Adherence to microexudate-coated plastic. In Manual o f Macrophage Methodology (eds Herscowitz, H. B., Holden, H. T., Bellanti, J. A. and Ghaffar, A.) pp. 69-73. Marcel Dekker, New York. NEVEU, P. J. (1986). The mononuclear phagocyte system. Bull. Inst. Pasteur, 84, 23-66. NEWTON, R. C. (1986). Human monocyte production of interleukin-l: parameters of the induction of interleukin-I secretion of lipopolysaccharides. J. leuk. Biol., 39, 2 9 9 - 311. OGHISO, Y., KUBOTA,Y., FUKUDA,S. & hDA, H. (1985). lnterleukin 1 released from beagle alveolar macrophages exposed to dust particles. Jap. J. Vet. Sci., 47, 851-854. PERLMAN, P. & HOLM, G. (1969). Cytotoxic effect of lymphoid cells in vitro. Adv. Immun., 11, 117-193. RECALDE, H. R. (1984). A simple method of obtaining monocytes in suspension. J. Immun. Meth., 69, 71-77. REDL, H., FLYNN, P. J., LAMCHE, H., SCHIESSER, A., SCHLAG, G. & HAMMERSCHMIDT, D. E. (1983). Aggregation, chemotaxis, and chemiluminescence of canine granulocytes. Inflammation, 7, 67 - 80. SAM Institute Inc. (1985). SAM User's Guide: Statistics, (Sth Edn). SAM Institute Inc., Cary, NC. S~MON, P. L., LAYDON,J. T. & LEE, J. C. (1985). A modified assay for interleukin-1 (IL-I). J. Immun. Meth., 84, 85 - 9 4 . SIMON, P. L. & WILLOUGHBY, W. F. (1982). Biochemical and biological characterization of rabbit interleukin-I (IL-1). Lymphokines, 6, 4 7 - 64. VAN FURTH, R., COHN, Z. A., HIRSCn, J. G. HUMPHREY, J. H., SPECTOR, W. G. & LANGEVOORT, H. L. (1972). The mononuclear phagocyte system: a new classification of macrophages, monocytes and their precursor cells. Bull. World Hlth Organiz., 46, 845- 852.
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0 1 9 2 - 0 5 6 1 / 8 8 $3.00+ .00 International Society for l m m u n o p h a r m a c o l o g y .
TECHNIQUES FOR ASSESSING CANINE MONONUCLEAR PHAGOCYTE FUNCTION AS PART OF AN IMMUNOTOXICOLOGIC EVALUATION PATRICIA A. THIEM, LEE K. HALPER and JOHN C. BLOOM Department of Experimental Pathology, Smith Kline and French Laboratories, King of Prussia, PA 19406-0939, U.S.A. (Received 14 July 1987 and in final form 2 March 1988)
Abstract -- Although the dog is used extensively as a model for toxicologic investigation, few practical
techniques for assessing immunotoxicity in this species have been reported. We have adapted techniques for assessing peripheral blood monocyte (PBM) function in the dog. Methods for isolating PBM were compared; a technique employing hypertonic conditioning followed by density gradient centrifugation, which resulted in > 90% purity and a greater yield of PBM than that obtained with other previously reported methods is described, along with methods for measuring antibody-dependent cellular cytotoxicity (ADCC) and the production of prostaglandin E2 (PGE2) and superoxide anion (O2). An assay for the measurement of interleukin-1 (IL-1), based on its ability to induce interleukin-2 (IL-2) production by the EL-4 mouse thymoma line in the presence of the calcium ionophore A23187, was examined. Together, these assays provide the tools to better define drug or chemical effects on the mononuclear phagocyte system (MPS) of this important animal model.
The MPS plays an important role in immune regulation and host defense that includes the processing and presentation of antigen, opsonization and phagocytosis of microorganisms, cytolysis of tumor cells, chemotaxis and the synthesis and secretion of various immunoregulatory products (Neveu, 1986; van Furth, Cohn, Hirsch, Humphrey, Spector & Langevoort, 1972). This system is therefore an important target for the pharmacologic and toxicologic actions of immunomodulating and immunotoxic drugs and chemicals. While the dog has been used extensively as a model for safety and toxicity studies in the pharmaceutical industry, our understanding of immune function in this species is limited (Bloom, Thiem & Morgan, 1987; Colgrove & Shifrine, 1980). Moreover, many of the assays useful in defining these parameters have not been validated in the dog or are impractical for monitoring drug or chemical effects. Assays which can be applied to dogs over the course of acute, subacute or chronic toxicity and safety studies must be minimally invasive, use sample volumes which are uncompromising to the animal and accomodate the processing of several samples at one time. Previous reports on the assessment of immune function in the dog have either not addressed MPS function specifically, or include
techniques that are unacceptably invasive for interim monitoring (Oghiso, Kubota, Fukuda & Iida, 1985). We report here assays for afferent (IL-1 and PGE2 production) and efferent (ADCC and 02 production) mononuclear functions that employ canine PBM and have been adapted to meet the aforementioned requirements.
EXPERIMENTAL PROCEDURES
Animals
Purebred beagles obtained from White Eagle Laboratories (Doylestown, PA) were maintained at Smith Kline & French Laboratories under conventional conditions. Males and females 1 - 7 years old were included in each group. The dogs were serving, at the time of evaluation, as controls for one of two long-term toxicity studies and receiving either 0.3 ml/kg 0.5o70 tragacanth, N.F., (gum tragacanth, a demulcent used as vehicle for administration of water-insoluble compounds) per os per day or intramuscular injections of 0.04 ml sterile water once every 3 days. Physical examinations, as well as hematologic, clinical biochemical and uroanalytic evaluations were performed periodically throughout the study; animals showing positive findings on these 765
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health screens were disqualified from the study. Additional normal naive dogs were also examined. P B M &olation
Peripheral blood mononuclear cells were isolated by a modified method of Recalde (1984). Briefly, blood collected in 3.8°7o sodium citrate was spun at 1000 × g for 5 rain at 25°C, and the plasma and buffy coat removed. Bovine thrombin (American Scientific Products, McGaw Park, IL) was added to the p l a s m a - b u f f y coat to provide a final concentration of 1 U/ml. The suspension was incubated for 15 min at 25°C with gentle mixing. The cells were then incubated at 37°C for 15 min following each of three additions of 9.007o saline (10, 20 and 20 ~1 per ml p l a s m a - b u f f y coat). The suspension was then diluted with 2 ml 0.907o saline and 54 /A 9.007o saline per ml p l a s m a - b u f f y coat, layered on Ficoll-PaqueTM (Pharmacia Fine Chemicals, Piscataway, N J), 0 = 1.077, adjusted to 3 9 0 - 4 1 0 m o s m / k g with the addition of crystalline sodium chloride, as determined by the freezing-point depression method), and centrifuged at 700 × g for 30 min at 25°C in 15 ml conical tubes. The PBM band was removed and washed three times with Dulbecco's phosphate-buffered saline (PBS), pH 7.4; the leukocyte concentration was determined and adjusted as required in the appropriate assay buffer. In all assays, PBM preparations were > 90070 pure as defined by non-specific a-naphthyl butyrate esterase staining (Sigma Chemical Co., St Louis, MO). Sterility was maintained during the separation procedure when PBM were to be cultured for IL-1 or PGE2 production. 0 2 assay
Superoxide anion production was measured by an adaptation of a procedure described by Babior, Kipnes & Curnutte (1973), based on the principle that PBM stimulated with phorbol myristate acetate (PMA) produces 0 2 which can be measured spectrophotometrically by the reduction of ferricytochrome c in a superoxide dismutase (SOD) inhibitable system. All reagents were obtained from Sigma. The assay buffer was PBS containing 0.1% glucose, 0.6 mM CaCI2 and 1.0 mM MgC12. Cytochrome c was prepared immediately before use. Superoxide dismutase, dissolved in buffer, was stored at 4°C for up to 1 week. Phorbol myristate acetate was dissolved in dimethylsulfoxide, stored at - 7 0 ° C and diluted in buffer. The test was carried out in 96-well flat-bottom plates. Wells contained 40 /A (26 nmoles) cytochrome c (0.32 mg), 10/A SOD
(0.04 mg) or buffer, 10/al PMA (0.2 #g) or buffer, and 40/A PBM (1.6 x 105 cells), which were added last. Plates were incubated at 37°C and read at 1 and 2 h intervals on a MR600 plate reader (Dynatech Laboratories, Inc., Alexandria, VA) at 550 nm vs a 490 nm reference. Data are reported as nmoles cytochrome c reduced, which was corrected for reduction in the presence of SOD, and in the absence of PMA. A D C C assay
Antibody-dependent cellular cytotoxicity was measured according to the method of Perlman & Holm (1969), by the release of ~Cr from labelled antibody-coated chicken erythrocyte (ChE) target cells after incubation with PBM. Assay medium was RPMI-1640 containing 10% fetal calf serum (FCS; Hazleton Dutchland Inc., Denver, PA), which was heat-inactivated at 56°C for 60 rain. Chicken erythrocytes (Colorado Serum Co., Denver, CO) were stored for up to 2 weeks in Alsever's solution at 4°C. Washed ChE (1 × 107 cells) were incubated with 250 ~Ci 5~Cr (sodium chromate in normal saline, pH 8, 200-900 Ci/g; New England Nuclear, Boston, MA) and RPMI-1640 in a total volume of 2 ml at 37°C for 2 h with occasional mixing, washed three times, and resuspended in 10 ml RPMI-1640. The ChE were incubated for an additional 30 min to remove excess chromium, washed five times, and resuspended to 1 × 10 5/ml in medium. The assay was performed in triplicate in 96-well U-bottom plates. Wells containing 100/A 5JCr-ChE suspension and 10 /A optimally diluted heat-inactivated rabbit anti-ChE antiserum (Organon Teknika, Jessup, MD) or medium alone, were incubated at 4°C for 30 min. Monocytes (100 ~1 at 5, 2.5 or 1.25 × 105/ml) were added and the plates spun at 80 × g for 3 min, followed by incubation at 37°C in a humidified chamber of 5% CO2 in air for 3 h. The plates were then spun at 400 × g for 10 min, and 100 /al of supernatant were transferred to a 12 × 75 mm tube. Radioactivity was counted for 1 rain using a System 5500 gamma counter (Beckman Instruments, Irvine, CA). Replicate counts/min were corrected for spontaneous release (in the absence of antibody and PBM); the % 5~Cr-release was determined based on total release using 1% Triton X-100 (Sigma). Data are reported as specific % 5'Cr-release adjusted for that due to antibody or PBM alone. P G E 2 production and assay
Prostaglandin E2 produced by stimulation of PBM with lipopolysaccharide (LPS, E. coli 055.B5; Difco Laboratories, Detroit, MI) was measured using a
Assessing PBM Function in Dogs radioimmunoassay kit (New England Nuclear) according to the method of Maclouf, Praedel, Pradelles & Dray (1976) based on competitive inhibition of binding. The culture medium for PGE2 production, as well as IL-1 production and assay was RPMI-1640 containing 5% heat-inactivated FCS, 10 mM Hepes, 2 mM L-glutamine, 100 U/ml penicillin and 100 /ag/ml streptomycin. All culture reagents were supplied by Gibco Laboratories, Grand Island, NY. Cultures containing 500/al PBM suspension (5 × 105 cells) and 5/~1 LPS (2.5/ag) or medium were incubated at 37°C in a humidified chamber of 5o70 CO2 in air for 24 h in 48-well plates. Supernatants were harvested through a 0.22 /am filter, stored at - 70°C and assayed according to the manufacturer's directions. Briefly, dilutions of PGE2 standard or supernatant, 125I-PGE2tracer, and rabbit anti-human PGE2 were incubated at 4°C overnight. The tubes were incubated on ice for 30 min with PEG-6000, to precipitate the immune complexes, and then centrifuged. The supernatants were aspirated and the pellets counted for 1 min. PGE2 concentrations were determined from the standard curve and corrected for dilution. In one experiment, indomethacin (0.25 /ag; Sigma) was added to the cultures to block PGE2 production.
IL-1 production and assay Interleukin-1 was produced under identical conditions to PGE2, except that the cultures were incubated for 48 h. Interleukin-I in the supernatants was measured according to the method of Simon, Laydon & Lee (1985). Briefly, EL-4 murine lymphoma cells, in the presence of IL-1 and the calcium ionophore A23187, produce IL-2, which is assayed by the proliferation of CT-20 murine IL-2dependent cytotoxic T-cells. EL-4 and CT-20 cells and supernatant from LPS-stimulated human placental tissue or recombinant human IL-1 (which served as positive IL-1 controls) were kindly provided by Dr Philip Simon, Department of Immunology and Antiinfectives Therapy, Smith Kline and French Laboratories. Rat T-cell growth factor (TCGF), supernatant from rat spleen cells (5 × 106) incubated with concanavalin A (5 /ag; Pharmacia) for 48 h at 37°C in a humidified chamber of 5o7o CO2 in air, served as an IL-2 positive control. Cells were maintained in vitro in assay medium supplemented with (CT-20) or without (EL-4) 20°7o TCGF. Duplicate serial dilutions (100 /al) of PBM supernatants, IL-I positive control or medium alone, 50/al A23187 (2 x 10-7M final concentration; Sigma) and 100/al EL-4 cells (1.5 × 105 viable cells)
767
were prepared in 96-well flat-bottom plates. Additional plates containing dilutions of the TCGF or test supernatants, A23187 and medium were run in parallel to test for the presence of IL-2. The plates were incubated at 37°C as above overnight. Following the transfer of 100/al of supernatant from each well to a new plate, 100 /al CT-20 (1 × 104 viable cells) were added and the plate was incubated at 37°C overnight. Cultures were pulsed with 10/al (1.0/aCi) 3H-thymidine (2 Ci/mmole; New England Nuclear), incubated again for 4 - 6 h, and harvested on glass fiber filters using a PHD cell harvester (Cambridge Technology, Cambridge, MA). Radioactivity was measured for 1 min on an LS 3800 beta counter (Beckman). IL-1 U / m l were calculated by parallel line analysis, using a computer program, in comparison to the standard. In one experiment, indomethacin (0.25/ag) was added to the cultures to determine whether prostaglandin production could be inhibiting that of IL-1. Statistics Statistical analysis was performed on all data using the least squares method to fit general linear models. Data points that were < or > 3 S.D.s from the mean of all observations were excluded. Significance (P <0.05) was determined by Tukey's studentized range test comparing each group to the others (SAS, 1985).
RESULTS
P B M &olation Monocyte preparations isolated by hypertonic conditioning contained 9 8 - 99°7o mononuclear cells and 1 -2°7o granulocytes and exhibited > 90°7o nonspecific esterase positivity. Viability was routinely > 95%, as determined by trypan blue dye exclusion. Preparations contained an average of five erythrocytes and 25 platelets per PBM. Yields ranged from 5 to 30%, based on absolute monocyte count. Table 1. Of production by peripheral blood monocytes* Group Tragacanth Water Naive Total
Age (years) 6-7 6-7 1-7
1 hour 1.9 + 0.3 1.4 _+0.3 1.0 _ 0.3 1.5 _+0.2
2 hours
(12) 2.9 + 0.3 (14) (13) 1.9 _+0.4 (13) (4) 1.1 ___0.3 (4) (29) 2.2_+0.3 (31)
*Data are reported as nmoles cytochrome c reduced by 1.6 × 105 PBM stimulated with 0.2/ag PMA and represent the mean _+S.E.M. with the number tested in parentheses.
P. A. THIEM et aL
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Table 2. Monocyte antibody-dependent cellular cytotoxicity*
Group Tragacanth Water Naive Total
Age (years) 6-7 6-7 1-7
Effector (PBM): target (ChE) ratio 5:1 2.5:1 1.25:1 56_+4 59_+4 50_+4 56_+ 3
(14) (14) (9) (37)
42+5 44_+6 45 +_7 43_+3
(14) 31 _+ 5 (14) (14) 30_+6 (14) (7) (35) 30_+4 (28)
*Data are reported as specific °7o 5~Cr release from antibody-coated target cells after incubation with PBM effector cells for 3 h, and represent the mean + S.E.M. with the number tested in parentheses. P e r i p h e r a l b l o o d samples of 2 0 - 4 0 ml p r o v i d e d sufficient n u m b e r s o f m o n o c y t e s ( 0 . 5 - 2 × 10 6) for any o f the assays described in this report.
to minimize the n u m b e r of cells required. The o p t i m a l dilution o f a n t i b o d y for sensitization of C h E was previously d e t e r m i n e d by titration. Ten-fold serial dilutions o f a n t i b o d y were tested; a dilution of 1:1000 was f o u n d to be optimal. The 5~Cr-labelled cells were p r e p a r e d o n the day o f the assay. To increase the sensitivity o f the system, 5~Cr-ChE were pre-incubated with a n t i b o d y in the microtiter plates at 4°C prior to the a d d i t i o n of P B M . This was followed by low speed c e n t r i f u g a t i o n to increase effector: target interaction. The total % S'Cr-release from all the controls was < 5%. A decrease in the % 5~Cr-release was observed with decreasing effector: target ratios (Table 2). There was n o significant statistical difference between the groups at any effector: target ratio.
02production The o p t i m a l c o n c e n t r a t i o n s o f b o t h P B M a n d P M A for stimulating 0 2 p r o d u c t i o n were studied over time. P B M were tested at 0 . 5 - 2 × 105/weU; 1.6 × 105 was f o u n d to be o p t i m a l a n d employed in the study. P h o r b o l myristate acetate was tested at final doses r a n g i n g f r o m 0.1 to 1.0 /ag; 0.2 /ag stimulated m a x i m u m 0 2 g e n e r a t i o n by P B M . Plates were read at intervals up to 4 h. Superoxide generation was negligible at 30 min, maximal at 2 - 3 h a n d decreased by 4 h. One a n d 2 h i n c u b a t i o n s were selected for this study; n a n o m o l e s of c y t o c h r o m e c reduced are s h o w n in Table 1. U p to 2 0 % o f the total a m o u n t o f c y t o c h r o m e c present in the system was reduced. This was completely i n h i b i t e d by SOD. T h e reduction o f c y t o c h r o m e c by P B M alone in the absence o f P M A was < 1 nmole. T h e r e was a 2 - 10 fold increase in O~ p r o d u c t i o n w h e n cells were stimulated with P M A . There was n o statistically significant difference between the groups.
A D C C assay The effect o f varying effector: target ratios was studied. Since the release o f 51Cr at 5 a n d 10:1 was c o m p a r a b l e , ratios o f 5, 2.5 a n d 1.25:1 were chosen
PGE2 production Three two-fold serial dilutions o f s u p e r n a t a n t s f r o m control a n d L P S - s t i m u l a t e d cultures were assayed for PGE2 content in duplicate. C o u n t s that did n o t fall in the linear p o r t i o n o f the curve were discarded. Results o f the assay are s h o w n in Table 3. The kit employs a r a b b i t a n t i - h u m a n PGE2 antibody, which has been s h o w n to recognize PGE2 from several species, including m o u s e a n d rat ( J o h n s o n , D i M a r t i n o & H a n n a , 1986). To show cross-reactivity o f the a n t i b o d y with canine PGE2, L P S - s t i m u l a t e d PGE2 p r o d u c t i o n by P B M cultured in the presence of i n d o m e t h a c i n was examined. P r o s t a g l a n d i n E2 synthesis was effectively blocked by this inhibitor as s h o w n in Table 3. There was n o statistically significant difference between any o f the groups.
IL-1 production Eight t w o - f o l d serial dilutions of s u p e r n a t a n t s f r o m control a n d L P S - s t i m u l a t e d cultures were assayed for b o t h IL-1 a n d IL-2. The optimal c o n c e n t r a t i o n o f L P S (5/ag/ml) h a d been previously determined from dose-response experiments.
Table 3. PGE2 production by peripheral blood monocytes* Group Tragacanth Water Naive Total
Age (years) 6-7 6-7 1-7
+ LPS 202 170 181 184
+ 20 + 22 _+ 64 +_ 17
(11) (13) (6) (30)
-LPS
+ LPS + indomethacin
21 + 11 (4) 130 _+ 47 (4) 5 +_ 1 (5) 75 __. 30 (8)
*Data are reported as ng/ml PGEz in supernatants from PBM incubated with or without LPS in the presence or absence of indomethacin for 24 h, and represent the mean -!-_S.E.M. with the number tested in parentheses.
Assessing PBM Function in Dogs
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Table 4. IL-1 production by peripheral blood monocytes* Group Tragacanth Water Naive Total
Age (years) 6- 7 6-7 1-7
+ LPS 899 ± 263 459 ± 66 919±405 746 ± 114
- LPS
(13) (13) (10) (36)
211 ± 180 ± 154± 181 ±
126 36 56 35
+ LPS + indomethacin (5) (12) (5) (22)
2020 ± 462 (6)
*Data are reported as IL-1 U/ml in supernatants from PBM incubated with or without LPS in the presence or absence of indomethacin for 48 h, and represent the mean ± S.E.M. with the number tested in parentheses. Results are shown in Table 4. No IL-2 was detected in these supernatants that could artifactually influence the IL-1 activity measured. Associated with the addition of indomethacin to LPS-stimulated cultures, and the subsequent inhibition of PGE2 production by PBM from normal dogs, was a moderate increase in IL-1 production, which was not statistically significant. This could represent inhibition of IL-1 production by prostaglandin synthesis in vitro. There was no statistically significant difference between any of the groups.
DISCUSSION In this study, we examined a panel of assays for canine PBM that represent efferent (O; generation and ADCC) and afferent (IL-I and PGE2 production) immune function. The selection of assays was based on the importance of these aspects of MPS function to the integrity of the immune system and the fact that they entail endpoints that are quantifiable with reasonable precision using practical techniques that conform to the aforementioned constraints. They have been widely applied in studies in man and other species on immunomodulatory agents such as gold compounds (Barrett & Lewis, 1986; Griswold, Lee, Poste & Hanna, 1985; Bray & Gordon, 1978). These assays have been used recently in our laboratory to study the effect of long-term chrysotherapy on immune function in the dog (Bloom, Thiem, Halper, Saunders & Morgan, 1988). Some of the control animals from that study were used to establish the normal parameters reported here. The treatment of these dogs, as described above, produced no statistical differences in immune parameters examined. Monocytes stimulated by non-specific membrane activators undergo a respiratory burst associated with increased oxygen consumption and secretion of superoxide anion. Commonly used stimuli are PMA,
opsonized zymosan and formylated oligopeptides, such as N-formyl-methionyl-leucyl-phenylalanine (FMLP). Canine granulocytes, however, are reported to lack receptors for FMLP (Redl, Flynn, Lamche, Schiesser, Schlag & Hammerschmidt 1983). In our assay, PMA-stimulated PBM reduced up to 20°70 of the cytochrome c in the system. Similar concentrations of neutrophils, stimulated with approximately one-tenth the amount of PMA for 30 min, reduced up to 55°70 of comparable concentrations of cytochrome c (data not shown). Antibody-dependent cellular cytotoxicity is dependent on three distinct steps: binding of antibody (IgG) to the target cell, binding of IgG on the target cell to the Fcy receptor of the effector cell and, finally, lysis of the target by the monocyte. Because both of the latter mechanisms must be intact for this to occur, ADCC is a useful "screen" for these important aspects of monocyte/macrophage effector function. Despite the variability inherent in the preparation and sensitization of 5~Cr-labelled target cells, the percent 51Cr release observed in this study remained fairly constant over a period of 6 months, with the amount of 5JCr release correlating well with the effector: target ratios. Prostaglandin E2 and IL-1 are monokines that mediate inflammation and a variety of other functions. Their production following LPSstimulation in these studies was variable. Similar variability has been observed using PBM from other animal species and man (Simon & Willoughby, 1982; Newton, 1986). Whether this phenomenon represents experimental artifact or normal biologic variability, it serves to obscure subtle changes in these parameters thus limiting the sensitivity of these tests. The EL-4 assay for the quantitation of IL-1 offers many advantages over the conventional murine thymocyte co-stimulator assay. The system is 1 0 - 2 5 times more sensitive (Simon et al., 1985), is less timeconsuming, requires less sample and obviates the need for maintaining a supply of weanling mice. There is a wide inter-animal variation in IL-1
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P. A. THIEMet al.
production by PBM, possibly due to the phenomenon of spontaneous production discussed below and/or the day-to-day variability common to assays employing cultured cells. In addition, canine IL-1 does not withstand repeated freeze-thaw cycles. Because of these problems, changes in IL-1 production in a test animal, whether iatrogenic or due to spontaneous disease, should be compared to a baseline value for that animal, as well as that of control dogs. It is therefore highly desirable that samples from a given study be assayed in a single experiment. One of the major problems previously encountered in assessing PBM function has been obtaining sufficient numbers of PBM from a volume of blood sample that would neither compromise the animal nor influence other parameters monitored. This was accomplished through the adaptation of a separation technique employing hypertonic conditioning previously described for isolating human PBM, which provides isolates of comparable purity and greater yield than those obtained by variations of the conventional adherence techniques (Mantovani, 1981). The isolation procedure does not require special equipment and can usually be completed within 5 h, thus allowing completion of most function assays within a normal working day. In addition, it has been reported that human cells isolated by this procedure retain functions characteristic of monocytes, including rosette formation, phagocytosis of antibody-coated erythrocytes and bactericidal activity (Recalde, 1984). Of major concern, regarding the isolation technique, was the possibility that the physical stress imposed by the isolation treatment employing hypertonic Ficoll-PaqueT M centrifugation contributed to the spontaneous PGE2 and IL-1 production in unstimulated cultures observed in this study. This was explored by comparing these activities in cultures containing PBM isolated as described with cells isolated using conventional Ficoll-Hypaque centrifugation (Boyum, 1968). Although higher titers were measured in supernatants from cells isolated by the hypertonic conditioning method, the amount of IL-1 produced in the presence of LPS relative to that in the unstimulated cultures was comparable. Another possible explanation for the spontaneous PGE2 and IL-1 observed is endotoxin contamination of reagents. Although FCS containing endotoxin concentrations of < 0.003 to 0.4 ng/ml could have contributed to this effect, pretreatment with polymyxin B produced similar results as those observed using untreated medium (data not shown).
This measure, however, does not rule out the possibility that significant levels of LPS were present, as polymyxin B has been reported to bind some, but not all, endotoxins (Kluger, Singer & Eiger, 1985). I n vivo activation of PBM with subsequent IL-1 release has been previously reported from canine peripheral blood mononuclear cells after three hours, whether or not LPS was added to the culture medium (Cerruti-Sola, Kristensen, Vandevelde & de Weck, 1984). Whether the "spontaneous" release in both circumstances is a consequence of LPS contamination of reagents is problematic. In addition to the possible contribution of experimental conditions relating to the cell separation and assay procedures, the variability observed in these parameters of mononuclear phagocyte function could also reflect the range of immunostimulation or immunoreactivity inherent in these dogs. Whether or not this variation is large enough to obscure biologically significant druginduced changes is uncertain and clarification awaits more experience with these tests in immunotoxicologic studies. Studies with agents known to affect these parameters are also indicated before such tests can be considered fully validated for use in experimental toxicology. These studies are in progress. Finally, caution is also warranted when interpreting positive results of these tests on PBM as effects on the MPS in general. It is well established that mononuclear phagocytes from different tissue compartments will vary substantially regarding both affector and effector immune function (Hearst, Warr & Jakab, 1980; Hopper, Wood & Nelson, 1979; Knop, 1980). While the use of PBM has the considerable advantage of accessibility, making possible interim monitoring, these are the least differentiated and specialized cells of the MPS. The relative sensitivity with which the PBM reflects drug and chemical effects on this important target organ system has yet to be determined. With the increased efforts by the pharmaceutical industry to develop immunomodulators, as well as other immunoactive biotechnology-derived candidate drugs, reliable, validated and practical assays for immune function will play an increasingly important role in the safety evaluation of these compounds. The assays we have examined, together with other more well established assays, such as tests for bactericidal/phagocytic activity or surface receptor expression, should prove useful in allowing us to better define the effects of drugs and chemicals on the MPS of the dog.
Assessing PBM Function in Dogs
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