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Immunotoxicity testing: An economical multiple-assay approach
FUNDAMENTAL
AND
APPLIED
lmmunotoxicity
TOXICOL.OGY
7,387-397 ( 1986)
Testing: An Economical
Multiple-Assay
Approach
JERRY H. EXON,* LOREN D. KoLLER,t PATRICIA A. TALCOTT,* CONNIE A. O’REILLY,* AND GERRY M. HENNINGSEN* *Department Idaho
of Veterinary Medicine, University 83843, and 7 College of Veterinary
of Idaho, Medicine,
WOI Regional Program Oregon State University,
of Veterinary Medicine, Moscow, Corvallis, Oregon 97330
Immunotoxicity Testing: An Economical Multiple-Assay Approach. EXON, J. H., KOLLER, L. D., TALCOTT, P. A., O’REILLY, C. A., AND HENNINGSEN, G. M. (1986). Fundam. Appl. Toxicol. 7, 387-397. A model for assessing immunotoxicologic effects of chemicals and drugs was developed in the Sprague-Dawley rat whereby multiple concomitant immunoassays were performed in a single animal. The multiple parameters of immunity assessedin each rat included T cell-dependent IgG antibody production, delayed hypersensitivity, natural killer cell cytotoxicity, and production of three potent immune regulating immunocytokines: macrophage-derived interleukin 1 and prostaglandin E2, and lymphocyte-derived interleukin 2. Splenocyte and resident peritoneal macrophage numbers were also quantitated and spleen and thymus weights recorded. The sensitivity of this animal model was tested by treating rats with the immunepotentiating drugs, NPT 15392 (erythro-9-[2-hydroxy,3-nonyllhypoxanthine) and avridine (N,N-dioctadecyl-N’,N’-bis-[2-hydroxyethyl]propan~amine, or the immune-suppressive cyclophosphamide (N,iV-bis[2-chloroethyl]tetrahydro-2H1,3,2-oxazaphosphorin-2dws, amine-2-oxide) and dexamethasone. Rats treated with NF’T 15392 or avridine generally had enhanced immune responses, while those treated with cyclophosphamide or dexamethasone had decreased immune responses. Differential responsiveness of various immunocyte populations within individual rats to different drugs, or to doses of the same drug, indicates the efficacy of measuring multiple responses within the same animal. The multiassay-single animal ap preach represents an economical, versatile, sensitive, and relatively comprehensive paradigm for assessing immunotoxicologic/pharmacologic properties of chemicals and drugs. The ap preach is extremely economical since multiple immune responses are evaluated in each animal. The approach is versatile because it is amenable to incorporation of a variety of in vitro and in viva assaysand could be applied to almost any species. The model is relatively comprehensive because major types of immune response.s/immunocyte populations and immunoregulatory pathways are tested. Finally, the model is sensitive for detecting immunosuppression as well as immunoenhancement, as validated by the use of known immune response modifiers in this study. 0 1986 SocietyofToticalogy.
It has been well-documented during the past decade that certain environmental chemicals, at relatively low doses, adversely affect normal functions of the mammalian immune system (reviews by Dean et al., 1982; Koller, 1979; Vos, 1977). It has also been demonstrated that chemical-induced immune dysfunction is associated with increased incidence of infectious disease and cancer in laboratory animals (Exon et al., 1975, 1979; Dean et al., 1980, 1982; Ward et al., 1984). Based on these data, the question has arisen as to whether government regula-
tory agencies should require immunotoxicologic testing within the general toxicity profiles generated for clearance/registration of chemicals for which significant exposure to humans and animals is anticipated. Although not presently required, many private companies have begun developing methods for detection of immunotoxicologic effects of chemicals in anticipation that the requirement will be implemented in the near future by regulatory agencies. Most experts in the field of immunotoxicology agree that no single immunoassay is 387
0272-059Of86 $3.00 Copyright 0 1986 by the Society of Toxicology. AU rights of reproduction in any form resewed.
388
EXON ET AL.
adequate to comprehensively assess overall immune competence of an individual, mainly due to the complexity of the immune system. The immune system is composed of a variety of cell types which differ in ontogeny and mediate different types of immune responses (reviewed by Klein, 1982). Each separate class or subset of immunocyte is capable of reacting individually to elicit a particular type of immune reaction, or can act in an integrated manner with other types of immunocytes to augment or suppress their functions. Four major classes of immunocytes are T and B lymphocytes (cells), macrophages, and natural killer cells (NKCs). The B cells are primarily the effector cells of humoral immunity (i.e., antibody production), but require the “help” of T cells and macrophages for optimal response to most antigens (Kung and Paul, 1983; Julius, 1982). The T cells are composed of both effector and regulator subpopulations (Reinherz and Schlossman, 1980). The effector T cells are responsible for specific cell-mediated immunity (SCMI) and can be divided into at least two subsets based on function and phenotype. These are the cytotoxic T cells and the T cells that mediate delayed hypersensitivity reactions (Berke, 1983; Liew, 1982). The regulatory T cells can be divided into helper (HT) and suppressor (ST) subsets, based on phenotype and regulatory activity, and mediate their actions mainly via production of soluble lymphokines (Reinherz and Schlossman, 198 1; Geha and Rosen, 1983). Macrophages are also composed of effector and regulator subsets. Macrophages are phagocytic cells which can act directly to ingest, kill, or inactivate foreign agents, or to “process” the antigen so that it is recognizable as foreign to other types of immunocytes (Kende, 1982; Grey el al., 1984). Natural killer cells are a population of large granular immunocytes which have some phenotypic characteristics of both lymphocytes and macrophages (reviewed by Herberman, 1982). The NKCs and effector macrophages are considered an important first-line defense mechanism (i.e., natural immunity) since,
unlike T cells, they react immediately with certain foreign agents without prior sensitization. The NKCs are believed to be especially important in immune surveillance to virusinfected and neoplastic cells. Cytotoxic response of macrophages and NKCs is generally termed nonspecific cell-mediated immunity. In addition to direct effector actions of different classes of immunocytes, these cells also produce a variety of immunoregulatory cytokines (reviews by Goldstein and Chirigos, 198 1; Oppenheim and Cohen, 1983). Prominent among these cytokines are interleukin 1 (ILl) and interleukin 2 (IL2). These immunocytokines act on activated effector immunocytes in an interrelated manner to regulate the magnitude of ongoing immune responses. Due to the apparent complexity of the immune system, no single immunoassay is adequate to assessoverall immune competence. Therefore, a battery of immunoassays is required which is designed to monitor separate segments of the immune system. Myriad in vivo and in vitro assays are available by which to assessthe integrity of the immune system in different species of animals. A comprehensive tier 1 panel of immunoassays should include, as a minimum, assessment of humoral immunity (i.e., antibody production by B cells), specific cell-mediated immunity (i.e., T-cell function), nonspecific cell-mediated immunity (i.e., NKC and macrophage function), and qualitative and quantitative pathotoxicologic examination of lymphoid tissues (i.e., spleen, thymus, major lymph nodes). Research in our laboratory during the past several years has been directed toward developing a representative immune function assay for each major segment of the immune system. Furthermore, a major goal has been to develop a sensitive, versatile, and comprehensive panel of immunoassays which can be performed concomitantly in a single animal in order to reduce animal numbers, experimentation costs, and variation between assays. The immunoprofile obtained from each animal consists of IgG antibody production
MULTIASSAY
IMMUNOTOXICOLOGY
to a specific T-dependent protein antigen; delayed hypersensitivity reaction to a second protein antigen; NKC cytotoxicity response; and the production of three potent immune response-regulating cytokines, namely macrophage-derived prostaglandin E2 (PGE2) and IL 1 and lymphocyte-derived IL2. Pathotoxicologic parameters include quantitation of immunocyte numbers, major lymphoid organ weights, and histopathologic changes. The purpose of this study is to validate the sensitivity and reproducibility of this multiple-immunoassay model to exogenous influence using known immune-modulating drugs. We have previously reported the sensitivity of parameters of this model to certain environmental contaminants (Exon et al., 1984a, 1985; Exon and Koller, 1983, 1984). The drugs tested were avridine (AVD) and NPT 15392, both of which are known immunopotentiators (Faanes et al., 1980; Niblack et al., 1979), and cyclophosphamide (CY) and dexamethasone (DEX), both of which are known immunosuppressors (Dean et al., 1979; Rosenberg and Lysz, 1980). A detailed discussion of the effects of these drugs in this rat model has been published elsewhere (Exon et al., 1986). The rat was chosen for use in this animal model because of the prominent use and familiarity of this species in other types of toxicologic/pharmacologic research.
METHODS
AND
MATERIALS
Male Sprague-Dawley rats were obtained from Washington State University, Laboratory Animal Resources, at 7 weeks of age and randomly divided into treatment groups of 12 rats each. The rats were housed four/cage in stainless-steel hanging wire cages and given deionized water and commercial rodent chow ad libitum. Animal quarters were maintained within recommended limits of temperature and humidity in rooms equipped with a 12hr on-off lighting cycle. Following 1 week of acclimation, each rat was injected subcutaneously (SC)twice at 7- or S-day intervals with the antigens, bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH), to induce either a delayed-type hypersensitivity reaction (BSA) or
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a humoral immune response (KLH; Table 1). Animals in each group were also injected SC with various dosage regimens of the immunomodulating drugs NPT 15392,’ avridine,2 cyclophosphamide,3 or dexamethasone.4 Groups of rats received drug treatments consisting of either 0.1 or 1 mg/kg NPT 15392,l or 25 mg/kg AVD, 25 or 75 mg/kg CY, or 0.1 or 1 m&kg DEX (Table 2). NPT 15392 was injected twice concomitant with each BSA injection and a third time 3 days before termination. Avridine was injected 18 hr prior to each BSA injection and again 18 hr before the animals were terminated. Cyclophosphamide and DEX were injected twice, initially 1 day before the second BSA injection and then 3 days before the animals were terminated (Table 2). All rats were sacrificed by CO1 asphyxiation 14 days after the initial BSA and KLH injections at Day 0 (note that the footpad DTH response was measured on Day 8). At the time of sacrifice, serum samples were collected by cardiac puncture for analysis of antibody levels to KLH (Table 1). Spleens were used as the cell source to monitor for NKC cytotoxicity and IL2 production. Resident peritoneal macrophages were harvested for determination of PGE2 and IL 1 production. All assayswere performed on each rat on experiment. The effect of multiple-antigen treatment and the use of FCA on individual immune functions in the panel have previously been reported to be minimal and not statistically significant (Exon et al., 1984b). A brief description of the separate assaysis provided below. Delayed-type hypersensitivity (DTH) assay. Delayedtype hypersensitivity reactions, an in vivo indicator of specific CM1 responsiveness by T cells, was measured as footpad swelling in rats by a method described by Henningsen et at. (1984). Rats were sensitized (Day 0) sc with BSA in Freund’s complete adjuvant (FCA) and challenged 7 days later in the footpad with heat-aggregated BSA (see Table 1). Briefly, equal volumes of FCA and BSA in physiological saline (2 &ml) were emulsified and 0.1 ml, containing 100 gg of BSA, was injected sc at the base of the tail of each rat. Seven days later, the let% hind footpad was challenged by injection of 75 ~1 of a 2% BSA heat-aggregated solution. The right hind footpad was sham-injected with saline, and 24 hr later (Day 8) footpad swelling in both hind footpads was measured using an electronic digital micrometer. The thickness of the saline-injected footpad was subtracted from the BSA-in-
’ Erythro-9-(2-hydroxy,3-nonyl)hypoxanthine, Newport Pharmaceuticals Int., Newport Beach, Calif. ’ CP-20,96 1 or N,Ndioctadecyl-N’,N’-bis-(2-hydroxyethyl)propanediamine, Pfizer Central Research Inc., Groton, Conn. 3 2H- 1,2,3-oxazaphosphorine, Sigma Chemical Company, St. Louis, MO. 4 Azium, Schering Veterinary, Schering Corporation, Kenilworth, N.J., 2 mg/ml.
EXON
390
ET AL.
TABLE 1 TIME AND ANTIGEN TREATMENT SCHEDULEFOR MULTIPLE-IMMUNOASSAY ANALYSIS” Dosage
Antigens
Days
Routes SC(lumbar) SC(base of tail)
KLH 1 mg BSA + FCA 100 Pg BSA 75 @l (2% solution) (heat-aggregated) Measure DTH response KLH 1 mg Collect peritoneal macrophages for PGE, and IL 1 analysis Collect spleen cells for IL2 and NKC analysis Collect serum sample for ELISA analysis
Footpad Footpad SC
Schematic of the multiple immunoassay” RAT (antigen-injected)
Blood
Peritoneal macrophages
Spleen
Footpad
Serum ELISA W-H)
NKC
IL2
PGEz
IL1
DTH (BSA)
a KLH = keyhole limpet hemocyanin; BSA = bovine serum albumin; FCA = Freund’s complete adjuvant; DTH = delayed-type hypersensitivity; PGEz = prostaglandin E2; IL1 and IL2 = interleukins 1 and 2; NKC = natural killer cell; ELISA = enzyme-linked immunosorbent assay.
jetted footpad to determine the DTH reaction. The data are reported as mean millimeter difference in swelling between the two footpads. Enzyme-linked immunosorbent assay (ELBA). Serum samples of rats injected SCin the lumbar region with 1 mg KLH twice at an S-day interval (Days 0 and 8) were collected by cardiac puncture 6 days after the last KLH injection (see Table 1). The amount of anti-KLH IgG antibody contained in the serum was analyzed by an indirect enzyme-linked immunosorbent assay as previously reported in detail (Exon et al., 1985). Briefly, antibody-containing serum samples were diluted in a phosphate-buffered saline (PBS)-Tween 20 solution and added in 100~~1 aliquots to wells of a 96-well microtiter plate which had been coated with 2.0 mg/ml KLH overnight at 4°C and rinsed. The serum-containing plates were incubated at 37’C for 1 hr and rinsed in PBS-Tween 20. Alkaline phosphatase, conjugated to goat anti-rat IgG (I:500 dilution), was added to each well in 100~~1 aliquots, incubated for 1 hr at 37°C and then rinsed with PBS-Tween 20. The substrate for alkaline phosphatase, p-nitrophenylphosphate, diluted to 1 mg/ml in dietha-
nolamine buffer, was added to each well in 100~~1 aliquots. The color reaction was allowed to develop for 30 min in the dark at room temperature and quantitated on a spectrophotometer (Titertek Multiskan MC) at 405 nm. The results are expressed as mean absorbance at a 1: 1000 dilution of the serum. Collection of spleen cells. Briefly, rats were sacrificed via CO2 asphyxiation and the spleens removed aseptically and minced with scissors. Single-cell suspensions were obtained by forcing the minced spleen through a stainless-steel mesh screen into complete medium (CM) consisting of RPM1 1640, 10% heat-inactivated fetal bovine serum, 25 mM Hepes buffer, 100 units/ml penicillin, and 100 pg/rnl streptomycin. The cells were centrifuged and the erythrocytes lysed by hypotonic shock for 6 sec. An equal volume of Hank’s balanced salt solution (2X) was added and the cells were centrifuged and resuspended in 6 ml of CM. A l-ml aliquot (or less) of cells was removed at this point for the IL2 assay.The remaining cells were depleted of B cells and adherent phagocytic cells by incubating on columns of nylon wool for 45 min and then in plastic culture plates for 1 hr at 37°C 10%
MULTIASSAY
IMMUNOTOXICOLGGY
CO2 The nonadherent semipurified NKCs were eluted in 25 ml of CM, centrifuged, and resuspended to 1 X 10’ cells/ml for use in the NKC assay. Assay for natural killer cell cytotoxicity. The method for analyzing the function of NKCs is to test their capacity to lyse tumor cells in vitro. The method used was a modification of that by Ristow et al. (1982). The YAC- 1 (target) cells, a murine Moloney leukemia virus-induced T cell lymphoma of A-strain mice, were grown as stationary suspension cultures in CM. These target cells were labeled with 400 PCi of ‘ichromium (as sodium chromate) per 10’ cells at 37°C 10% CO2 , for 1 hr with gentle shaking. The target cells were then washed and resuspended to 1 X 10’ cells/ml. Viability for both the target and the NKC, collected as above, was determined by trypan blue exclusion (usually >95%). The YAC-1 cells, prepared as above, were added in 100-p aliquots to the appropriate wells of a 96well flatbottomed microtest plate containing 100 ~1 of varying concentrations of NKC (effector). After a 4-hr incubation at 37°C in an atmosphere of the 10% CO2 in air, the plates were centrifuged at 200g for 10 min and 100 ~1 of the cell-free supematants was collected from each well and counted in a gamma counter. Specific 5’Cr release was calculated by the formula cpm experimental release - cpm spontaneous release cpm maximum release by 2% SDS - cpm spontaneous release X
100% = % cytotoxicity.
Interleukin 2 assay. The common method for analysis of IL2 is the ability to maintain growth of an IL2dependent T-cell clone in culture. The method is a modification of that reported by Gillis et al. (1978). Briefly, spleens were removed aseptically from each animal, forced through sterile, stainless-steel screens into IscoveMelchers modified Dulbecco’s medium (supplemented with 100 U/ml penicillin, 100 &ml streptomycin, 5 X 10m5M 2-mercaptoethanol (2-ME), human transferrin, and 400 pg/ml fatty acid-free BSA) to obtain singlecell suspensions. The cells were then counted on a Coulter counter and diluted to 1 X lo6 cells/ml. Onemilliliter ahquots were incubated 24 hr with 1.O&ml of concanavalin A (Con A), and the cell-free supematant was harvested and tested for IL2 activity. Briefly, 4 X IO3 ILZ-dependent murine cytotoxic T cells (CTLL) were cultured in replicate 200~~1 volumes in flat-bottomed 96-well culture plates in the presence of a log-2 dilution series of putative ILZ-containing supematants. After 18 hr, the cells were pulsed with 1 pCi [‘H]TdR (6.7 Ci/ mmol) for 4 hr, harvested onto glass fiber filters, and counted on a scintillation counter. IL2 activity was quantified by probit analysis of the [3H]TdR incorporation data as described by Gillis et al. (1978). The standard
391
preparation of human IL25 contained 1119.75 units of IL2 activity per milliliter. The results are expressed as units of IL2 per milliliter of culture supematant. Interleukin I assay. Macrophage function was measured by their capacity to produce IL1 and PGE2 in vitro. Following asphyxiation with CO*, 30 ml of sterile HBSS was injected ip into each rat. The abdominal area was gently massaged for approximately 1 min to dislodge resident peritoneal cells. A small (2 mm) slit was made in the abdominal wall and the medium containing resident peritoneal cells was allowed to drain into a 50-ml siliconized centrifuge tube. The peritoneal cells were pelleted and washed twice and cell numbers determined by Coulter counting. The cells were diluted to 1.5 X 106/ml for use in the IL1 and PGE2 assays.IL1 synthesis by rat resident peritoneal cells was induced in 72-hr lipopolysaccharide (LPS)-stimulated cultures as previously reported (Gery et al., 1981). Indomethacin was added to prevent prostaglandin synthesis in the IL1 assay. The ILl-containing supematants were stored at -20°C and later assayedfor activity by the capacity to induce proliferation of thymocytes in culture from 6-week-old C3H/ HeJ mice. Serial dilutions of ILlcontaining supematants were cultured for 68 hr with 1.5 X lo6 thymocytes in the presence of 2-mercaptoethanol and phytohemagglutinin (PHA) followed by a 4-hr incubation with [‘H]TdR. The cells were harvested onto glass filter paper and [3H]TdR uptake was measured by scintillation counting. Results are expressed as mean cpm/ 1.5 X 1O6 adherent cells. Assay for prostaglandin E2. Resident peritoneal macrophages were collected by lavage as for the IL1 assay. The macrophages were washed and plated in 24-well cluster plates. After 1 to 2 hr, the wells were rinsed three times to remove nonadherent cells. One-millimeter culture medium (Dulbecco’s MEM, 5% fetal bovine serum, 100 units penicillin/ml, and 100 rg streptomycin/ml) was added to each well. Three cultures were assayed for each rat. One was a control culture (no additives) and two were cultured with 0.1 &ml LPS (lipopolysaccharide, Escherichia coli, 055:B5). Media controls were run with each assay. Cluster plates were incubated at 37°C 10% CO* for 18 hr at which time supematants were removed and centrifuged. The cell-free supematants were assayed for PGEl using a commercially available radioimmunoassaykit6 The kit offers a sensitive method of measuring PGE2 by monitoring the competitive binding of [3H]TdR-labeled prostaglandin and unlabeled PGEz with antibody specific for PGEz Separation of antibody-bound PGE, from free PGEl was achieved by precipitating the antibody-PGE, complex with a second antibody. The assay is sensitive to approximately 0.03 ng per 1 ml medium. The medium controls allowed correction for any
5 Cellular Products, Inc., Buffalo, N.Y. 6 Clinical Assays, Cambridge, Mass., CA-50 1.
392
EXON ET AL. TABLE 2 TREATMENTSCHEDULEFORTESTINGIMMUNEMODULATINGDRUGS"
Time
Dose
Antigens/drugs
Routes
-18hr
Avridineb
1 or 25 mg/kg
SC
Day 0
KLH BSA-FCA NPT 15392
1 mg 100 & 0.1 or 1.O mg/kg
SC SC SC
Day 6
Cyclophosphamide Avridineb
25
or 15 mg/kg 1or 25 mg/kg
SC SC
Day I
BSA-HA NPT 15392
100 /lI (2%) 0.1 or 1 .O mg/kg
FP SC
1 mg
SC
Day 8
Assay: DTH-footpad
swelling KLH
Day 11
NPT 15392 Cyclophosphamide
0.1 or 1.Omg/kg 25 or 75 mg/kg
SC SC
Day 13
Avridineb
1 or 25 mg/kg
SC
Day 14
Assay: PGE and ILl-resident peritoneal cells Assay: IL2 and NKC-splenocytes Assay: anti-KLH antibody-serum
’ KLH = keyhole limpet hemocyanin; BSA = bovine serum albumin; BSA-HA = heat-aggregated BSA; FCA = Freund’s complete adjuvant; DTH = delayed-type hypersensitivity; PGE2 = prostaglandin EZ; IL1 and IL2 = interleukins 1 and 2; NKC = natural killer cell; SC= subcutaneous; FP = footpad. b Avridine was given 18 hr prior to antigen (i.e., KLH or BSA) or termination.
interference from MEM, FBS, or LPS. Samples were counted on a liquid scintillation counter, unknowns were compared to the standard curve, and PGEz concentrations are reported as nanograms per milliliter. These values were corrected for the number of adherent cells and final PGEz levels are reported as ng/ 1 X 1OSadherent cells. Statistical analysis. All &ta were analyzed by a conventional analysis of variance technique and leastsquares mean comparisons to respective controls or by probit analysis.
RESULTS A significant (p < 0.05) dose-dependent decrease in serum antibody to KLH, DTH reactions to BSA, NKC cytotoxic responses, spleen and thymus weights, and numbers of splenocytes and resident peritoneal cells was observed in all CY-treated rats (Tables 3 and 4). The production of IL2 by splenocytes was significantly (p < 0.05) reduced in rats treated
with 75 mg/kg CY (Table 5). Macrophagederived PGEz production was significantly (p f 0.05) elevated in both groups of CY-treated rats, while IL1 synthesis was significantly (p < 0.05) greater in the 75 mg/kg group (Table 5). Rats treated with DEX had dose-dependent significant (p < 0.05) decreases in DTH reactions, NKC cytotoxicity responses (Table 4), thymus weights, and numbers of splenocytes and resident peritoneal cells (Table 3). Rats treated with 1 rng/kg DEX also had significantly (p f 0.05) reduced IL1 and IL2 production (Table 5) and spleen weights (Table 3). No significant effect of DEX treatment was observed on serum antibody levels to KLH or PGE2 production. Rats injected with either 1 or 25 mg/kg AVD had significantly (p < 0.05) augmented DTH reactions inverse to the AVD dose (Table 4). The NKC cytotoxic responses were
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393
IMMUNOTOXICOLOGY TABLE 3
THE EFFECTSOF IMMUNE Treatment”
R!ZSFQNSE MODIFTERS
Spleen weight g (k SE)b
ON ORGAN
Thymus weight g (+SE)b
WEIGHTS
AND IMMUN~CYTE
Splenocytes X lO’/ml (HE)
NUMBERS
Peritoneal cells X lO’/ml (*SE)
Avridine (AVD) and NPT 15392 (NPT) Control
0.75 + 0.06
0.48 + 0.04
5.0 -t 0.7
1.4-t0.2
1 mg AVD/kg 25 mg AVD/kg
0.86 + 0.05 0.92 + 0.03d
0.43 f 0.03 0.48 + 0.06
5.1 +- 0.6 5.4 * 0.5
1.3 f 0.2 1.6 kO.2
0.1 mg NPT/kg 1.O mg NPT/kg
0.80 f 0.05 0.84 + 0.06
0.50 f 0.05 0.55 + 0.06
5.2 f 0.3 5.8 f 0.4
1.3 kO.2 1.4-t0.2
Control
0.75 rt 0.06
0.48 f 0.04
4.8 + 0.4
1.3 i 0.1
25 mg CY/kg 75 mg CY/kg
0.55 f 0.05d 0.37 f 0.06d
0.3 1 f 0.04d 0.20 + 0.04d
1.4 + 0.2d 0.7 f 0.2d
0.8 -t O.ld 0.6 k 0. Id
0.1 mg DEX/kg 1.O mg DEX/kg
0.64 + 0.06 0.39 f 0.05d
0.20 + 0.03d 0.20 2 0.02d
2.1 *0.1d 0.7+0.1”
0.9 t O.ld 0.7 I!z0. Id
Cyclophosphamide (CY) and Dexamethasone (DEX)
’ See Table 2 for treatment times. b Unadjusted for body weight; no significant differences between body weight compared to controls. ’ Inclusive of adherent and nonadherent fractions. dp 6 0.05 by analysis of variance and least-squares mean comparison to respective control.
significantly (p < 0.05) enhanced in the 25 mg/kg treatment group. The synthesis of IL2 and IL1 (Table 5) was significantly (p < 0.05) increased in rats injected with 1 mg/kg AVD, and spleen weights were significantly (p < 0.05) greater in rats exposed to 25 mg/kg AVD (Table 3). Synthesis of PGE2 was significantly suppressed in the 25 mg/kg group (Table 5). No significant effects of AVD treatment were observed in regard to antibody synthesis, thymus weights, or numbers of spleen cells and resident peritoneal cells. Rats treated with 0.1 mg/kg NPT 15392 had significantly (p & 0.05) enhanced DTH reactions (Table 4). Animals treated with the 1-mg/kg dose of NPT 15 392 had significantly increased NKC cytotoxic activity (Table 4) and suppressed PGE2 synthesis (Table 5). No significant effects of NPT 15392 treatment were observed in regard to antibody production, IL1 and IL2 synthesis, or organ weights and cell numbers.
DISCUSSION Alterations of immune functions observed in rats exposed to the known immune response-modifying drugs in this study indicate that this animal model is sensitive to exogenously induced immunomodulation by xenobiotics. Rats treated with the immunosuppressive drugs, CY and DEX, generally had reduced immune functions, while animals treated with the immune stimulating compounds, AVD and NPT 15392, generally had enhanced immune responsiveness. These results suggest that other immune responsemodifying agents, such as environmental toxicants, could also be detected by these methods and thereby may represent a practical immunotoxicological screen. Immunomodulating agents which potentially could be detected include those which either augment immune responsiveness and thereby predispose to development of autoimmune-related dis-
394
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ET AL.
TABLE 4
Treatment’
Antibody productionb (Meanabsorbance at 405 mm (GE))
Delayed hypersensitivity’ (mean mm swelling (GE))
Natural killer cytotoxicityd (% killing (*SE) ratio 50: 1)
Avridine (AVD) and NPT 5 1392 (NPT) Control
1.41 + 0.18
3.4OkO.16
26.4 + 1.6
1 mg AVD/kg 25 mg AVD/kg
1.63 *0.15 1.57 + 0.22
4.46 +- 0.49’ 4.21 f 0.21’
32.6 + 2.0 37.8 f 2.0’
0.1 mg NPT/kg 1.Omg NPT/kg
1.62kO.13 1.4450.16
4.92 + 0.41’ 3.95 + 0.39
26.5 2 2.0 33.9 k l.3e
Cyclophosphamide (CY) and Dexamethasone (DEX) Control
1.40 f 0.21
4.10 + 0.20
23.6 + 1.9
25 mg CY/kg 15 mg CY/kg
0.42 f 0.06’ 0.26 -c 0.10’
2.32 f 0.87’ 0.86 f 0.2 le
6.3 + 0.8e 5.2 + 1.4’
0.1 mg DEX/kg 1.Omg DEX/kg
1.40 + 0.26 1.31 +0.13
2.88 + 0.10’ 1.35 + 0.17’
13.0 f 1.3’ 7.9 f 1.7’
’ See Table 2 for treatment times. b By an indirect ELISA to KLH; 1:1000 serum dilution. ’ By a footpad-swelling assayto BSA. d By a 4-hr 5’Cr release assayto YAC- 1 target cells; ratio 50: 1 = NKC:target cells. ‘p G 0.05 by analysis of variance and least-squares mean comparison to respective control.
eases, or those which depress the immune system and render the host more susceptible to immunodeficiency-related disorders. Furthermore, since all of the immunoassays are performed in each animal, it is possible to compare the relative overall alteration of different types of immune responses in the same rat following exposure to a chemical or drug. This multiple-immunoassay model is an extremely economical method of assessing immune competence, since all immunoassays are performed concomitantly in a single animal. Consequently, a minimal number of animals and space is required, thereby reducing the overall cost of purchasing and maintaining experimental animals. Furthermore, certain types of experimental variation/error are minimized since all assays are performed at approximately the same time in the same animal. Also, multiple responses can be cor-
related in individual animals. A limitation of the multiple immunoassay approach is the availability of adequate numbers of personnel to perform assays and the numbers of immunocytes available from each animal. Cell numbers available for use in each assay could, however, be increased by pooling cells from inbred strains. The multiple-immunoassay model can be very versatile. Although the ELISA, DTH, NKC cytotoxicity, immunocytokine production, and pathotoxicologic techniques were utilized in this study, any suitable representative immunoassay could be substituted, depending on the investigator’s preference or the chemical tested. Also, additional assays could be easily incorporated into the model (e.g., bone marrow progenitor, helper/suppressor T-cell ratios, complement fixation, macrophage and T-cell cytotoxicity). This immunotoxicologic testing model is
MULTIASSAY
IMMUNOTOXICOLOGY
395
TABLE 5 THE
EFFECTSOFIMMUNERESPONSEMODIL~ERSONIM~OREGULATORYCYTOK~NEPRODUCTION
Treatment’
Interleukin 2 productionb (mean units/ml (+SE))
Interleukin 1 production’ (mean CPM/l.5 X lo6 cells @SE))
Prostaglandin E2 productiond (mean ng PGEr/ 10’ cells (+SE))
Avridine (AVD) and NPT 15392 (NPT) Control
73.7 * 6.5
11,950 f 2,798
54.0 + 4.0
1 mg AVD/kg 25 mg AVD/kg
93.4 f 8.0e 76.1 f 5.2
25,394 f 3,678’ 10,278 & 3,873
58.4 f 4.0 32.5 f 4.6’
0.1 mg NPT/kg 1.O mg NPT/kg
88.4 f 9.0 73.6 k 5.5
22,900 f 5,906 19,633 + 3,900
53.0 + 4.3 22.4 + 4.0’
Cyclophosphamide (CY) and Dexamethasone (DEX) Control
70.3 f 2.3
9,858 + 2,113
59.9 f 2.8
25 mg CY/kg 75 mg CY/kg
53.3 -t 1.6 36.3 -t 2.0’
13,440 + 2,040 22,821 t 1,904e
84.4 f 3.5’ 85.7 2 3.5’
0.1 mg DEX/kg 1.O mg DEX/kg
45.7 e 2.3 29.7 f 1.5’
8,874 + 1,815 2,665 + 503e
55.6 + 2.8 52.6 1?I2.8
’ See Table 2 for treatment times. b By Con A-induced splenocytes; CTLL cells were used as the IL2-dependent T-cell clone. ’ By LPS-stimulated resident plastic adherent peritoneal cells; IL1 levels determined by stimulation of PHA-induced mice thymocytes. d By LPS-stimulated plastic adherent peritoneal cells; PGE, levels determined by a double antibody radioimmunoaSSay. ep < 0.05 by analysis of variance and least-squares mean comparison to respective control.
relatively inclusive in that the functions of the prevalent populations of effecter/regulator immunocytes are assessed. The immunoprofile obtained from each rat includes assessment of major types of immune responses, immunocyte populations, and immunoregulatory pathways. For instance, the production of antibodies to a T cell-dependent antigen such as KLH requires the interaction of B cells, regulator T cells, and macrophages (Julius, 1982). Any effect on antibody production, as measured by the ELISA or any other suitable method, could result from altered reactivity of any one of these immunocyte populations. Macrophage function is further tested by their capacity to produce the immune response-regulating monokines, IL 1 and PGE2 . The function of T effector cells in-
volved in specific CM1 responses is assessed by their capacity to elicit a DTH response. Regulatory T-cell populations are further tested by their capacity to produce IL2, a potent immunoregulatory lymphokine which is required for optimal cytotoxicity functions of NKC and antigen-specific clonal expansion of T cells mediating the DTH response and cytotoxicity reactions. The NKC cytotoxicity assay assessesthe function of these first-line host defense immunocytes to kill tumor cells. Therefore, the assays composing the multiple-immunoassay model collectively test for the competence of B cells, effecter/regulator T cells, effecter/regulator macrophages, and NKCs. The functions tested relate to humoral immunity, specific acquired CM1 immunity, natural nonspecific CM1 (i.e., mac-
396
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ET AL.
rophages and NKCs), and production of relevant immune assays for use in immunoassessment must potent endogenous immunoregulatory cy- toxicologic/pharmacologic tokines. The model does, however, lack an be determined by extensive testing and valiadequate assay for hypersensitivity reactions, dation, especially before any recommendawhich should be included in any tier 1 screen. tions can be made with regard to an appropriThe model also lacks a host resistance assay ate battery of immunoassays which may be although, due to the in vivo immunoselectivrequired by regulatory agencies in general tier ity of many of the pathogens used in these 1 or tier 2 toxicologic testing protocols. types of assays, their feasibility for inclusion REFERENCES in tier 1 testing is questionable and they may be more appropriately included in the tier 2 panel. BERKE, G. (1983). Cytotoxic T-lymphocytes. How do they function? Immunol. Rev. 12,5-4 1. This general model could be adapted to alDEAN, J. H., LUSTER, M. I., AND BOORMAN, G. A. most any species, depending on preference. (1982). Immunotoxicology. In Research Monographs The rat was chosen for use in this model since in Immunology. Immunopharmacology (P. Sirois and it is often the species of choice in toxicologic/ M. Rola-Pleszczynski, eds.), Vol. 4, pp. 349-398. pharmacologic research, and thus facilitates DEAN, J. H., LUSTER, M. I., BOORMAN, G. A., LAUER, a direct comparison of immunotoxic effects L. D., AND LUEBKE, R. W. (1980). The effect of adult exposure to diethylstilbestrol in the mouse: Alterations with other toxicologic parameters and could in tumor susceptibility and host resistance parameters. be readily incorporated into the standard 90J. Reticuloendothel. Sot. 28,57 l-583. day subchronic toxicity testing protocols cur- DEAN, J. H., PADARATHSINGH, M. L., JERRELLLS, T. R., rently in use. Furthermore, a substantial KEYS, L., AND NORTHING, J. W. (1979). Assessment amount of baseline toxicologic data has been of immunobiological effects induced by chemicals, drugs or food additives. II. Studies with cyclophosphacompiled for this species. Therefore, there is mid& Drug Chem. Toxicol. 2,133- 153. a need to be able to assess immunotoxicity J. H., AND KOLLER, L. D. (1983). Effects of chloin the rat. The immune system of the rat is EXON, rinated phenols on immunity in rats. Int. J. Immunorelatively well-characterized, and state-ofpharmacoi. 5, I 3 I- 136. the-art reagents and inbred strains are availEXON, J. H., AND KOLLER, L. D. (1984). Toxicity of 2chlorophenol, 2,4-dichlorophenol and 2,4,6-trichloroable for detailed study of the immunologic phenol. In Water Chlorination: Chemistry, Environfunction. Greater numbers of immunocyte mental Impact and Health E&cts (R. L. Jolley, ed.), subpopulations can be collected from the rat Vol. 5, pp. 307-330. Lewis Publishers, Chelsea. as compared, for instance, to the mouse. This EXON, J. H., HENNINGSEN, G. M., KOLLER, L. D., AND facilitates the multiple-assay approach, TALCOTT, P. A. ( 1986). The selectivity of isoprinosine, NPT 15392, avridine and cyclophosphamide on mulThe rat model described in this report for tiple immune responses in the rat. Int. J. Immunopharuse in immunotoxicity testing of drugs and macol. 8,53-62. chemicals is comprehensive, economical, EXON, J. H., HENNINGSEN, G. M., OSBORNE, C. A., AND versatile, and apparently sensitive to immuKOLLER, L. D. (1984a). Toxicologic, pathologic and nomodulation. However, it is not the intent immunotoxic effects of 2,4-dichlorophenol in rats. J. Toxicol. Environ. Health 14,723-730. of this report to recommend that this particular model system is the best for use in the de- EXON, J. H., KOLLER, L. D., AND KJZRKVLIET, N. I. (1979). Lead-cadmium interaction: Effects on viral-intection of immune-modulating xenobiotics. duced mortality and tissue residues in mice. Arch. EnThe species and specific immunoassays utiviron. Health 34,469-475. lized in the model are secondary, in many re- EXON, J. H., KOLLER, L. D., HENNINGSEN, G. M., AND OSBORNE, C. A. (1984b). Multiple immunoassay in a spects, to the main concept of assessing mutisingle animal: A practical approach to immunotoxicople concomitant immune responses in each logic testing. Fundam. Appl. Toxicol. 4,278-283. animal. Also, several of the assays may be EXON, J. H., PATTON, N. M., AND KOLLER, L. D. more appropriate for tier 2 testing (e.g., IL1 , (1975). Hexamitiasis in cadmium-exposed mice. Arch. IL2, PGE2). The optimal species and most Environ. Health 30,463-464.
MULTIASSAY
IMMUNOTOXICOLOGY
EXON, J. H., TALCOTT, P. A., AND KOLLER, L. D. ( 1985). Effect of lead, polychlorinated biphenyls and cyclophosphamide on rat natural killer cells, interleukin 2 and antibody synthesis. Fundam. Appl. Toxicol. 5, 158-164. FAANES, R. B., MEFUUZZI, V. J., WALKER, M., WILLIAMS, N., RALPH, P., AND HADDEN, J. W. ( 1980). Immunoenhancing activity of NPT 15392: A potential immune response modifier. Int. 1. Immunopharmacol. 2, 197. GEHA, R. S., AND ROSEN, F. S. (1983). Immunoregulatory T-cell defects. Immunol. Today4,233-236. GERY, I., DAVIES, P., DERR, J., BRETT, N., AND BARRANGER, J. A. (198 1). Relationship between production and release of lymphocyte-activating factor (interleukin 1) by murine macrophages. Cell. Immunol. 64, 293-299.
GILLIS, S., FERM, M. M., WINNY, O., ANDSMITH, K. A. ( 1978). T cell growth factor: Parameters of production and a quantitative microassay for activity. J. Immunol. 120,2027-2032.
GOLDSTEIN, A. L., AND CHIRIGOS, M. A. (198 1). Lymphokines and Thymic Hormones. Raven Press, New York. GOODWIN, J. S., AND CEUPPENS, J. (1983). Regulation of immune responses by prostaglandins. J. Clin. Immunol. 3,295-3 15. GREY, H. M., CHESTNUT, R. W., SHIMONKEVITZ, R., MARRACK, P., AND KAPPLER, J. (1984). Mechanisms of antigen processing and presentation. Zmmunobiol. 168,202-212.
HENNINGSEN, G. M., KOLLER, L. D., EXON, J. H., TALcon, P. A., AND OSBORNE, C. A. ( 1984). A sensitive delayed-type hypersensitivity model in the rat for assessing in vivo cell-mediated immunity. J. Immunol. Meth. 70,53-165. HERBERMAN, R. B. (1982). NK Cells and other Natural Effector Cells. Academic Press, New York.
JULIUS, M. H. (1982). Cellular interactions involved in T-dependent B-cell activation. Zmmunol. Today 3, 295-299.
KAMPSCHMIDT, R. F. (1984). The numerous postulated manifestations of interleukin 1. J. Leucocyte Biol. 36, 341-355.
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KENDE, M. (1982). Role of macrophages in expression of immune responses. J. Amer. Vet. Med. Assoc. 181, 1038-1042. KLEIN, J. (1982). Immunology: TheScience ofSe&NonselfDiscrimination. Wiley, New York. KOLLER, L. D. (1979). Effects of environmental chemicals on the immune system.Adv. Vet. Sci. Comp. Med. 23,267-295.
KUNG, J. T., AND PAUL, W. E. (I 983). B-lymphocyte subpopulations. Immunol. Today 4,37-4 1. LIEW, F. Y. (1982). Regulation ofdelayed-type hypersensitivity to pathogens and alloantigens. Immunol. Today 3,18-23.
NIBLACK, J. F., O~RNESS, I. G., HEMSWORTH, G. R., WOLFF, J. S., III, HOF’FMAN, W. W., AND KRASKA, A. R. (1979). CP-20,961: A structurally novel, synthetic adjuvant. J. Reticuloendothel. Sot. 26,655-663. OPPENHEIM, J. J., AND COHEN, S. (1983). Interteukins, Lymphokines and Cytokines. Academic Press, New York. REINHERZ, E. L., AND SCHLOSSMAN, S. F. (1980). The differentiation and function of human T lymphocytes. Cell19,821-827.
REINHERZ, E. L., AND SCHLOSSMAN, S. F. (198 1). The characterization and function ofhuman immunoregulatory T lymphocyte subsets. Immunol. Today 2,6971. RISTOW, S. S., STARKEY, J. R., AND HASS, G. M. (1982). Inhibition of natural killer cell activity in vitro by alcohols. Biochem. Biophys. Res. Commmun. 105, 13 151321. ROSENBERG,J. C., AND LYSZ, K. (1980). Suppression of the immune response by steroids. Comparative potency of hydrocortisone, methylprednisolone and dexamethasone. Transplantation 29,425-428. Vos, J. G. (1977). Immune suppression as related to toxicology. CRC Crit. Rev. Toxicol. 5,67- 10 1. WARD, E. C., MURRAY, M. J., LAUER, L. D., HOUSE, R. V., IRONS, R., AND DEAN, J. H. (1984). Immunosuppression following 7,12-dimethylbenz[a]anthracene exposure in B6C3FI mine. Effects on humoral immunity and host resistance. Toxicol. Appl. PharmacoL75,299-308.
AND
APPLIED
lmmunotoxicity
TOXICOL.OGY
7,387-397 ( 1986)
Testing: An Economical
Multiple-Assay
Approach
JERRY H. EXON,* LOREN D. KoLLER,t PATRICIA A. TALCOTT,* CONNIE A. O’REILLY,* AND GERRY M. HENNINGSEN* *Department Idaho
of Veterinary Medicine, University 83843, and 7 College of Veterinary
of Idaho, Medicine,
WOI Regional Program Oregon State University,
of Veterinary Medicine, Moscow, Corvallis, Oregon 97330
Immunotoxicity Testing: An Economical Multiple-Assay Approach. EXON, J. H., KOLLER, L. D., TALCOTT, P. A., O’REILLY, C. A., AND HENNINGSEN, G. M. (1986). Fundam. Appl. Toxicol. 7, 387-397. A model for assessing immunotoxicologic effects of chemicals and drugs was developed in the Sprague-Dawley rat whereby multiple concomitant immunoassays were performed in a single animal. The multiple parameters of immunity assessedin each rat included T cell-dependent IgG antibody production, delayed hypersensitivity, natural killer cell cytotoxicity, and production of three potent immune regulating immunocytokines: macrophage-derived interleukin 1 and prostaglandin E2, and lymphocyte-derived interleukin 2. Splenocyte and resident peritoneal macrophage numbers were also quantitated and spleen and thymus weights recorded. The sensitivity of this animal model was tested by treating rats with the immunepotentiating drugs, NPT 15392 (erythro-9-[2-hydroxy,3-nonyllhypoxanthine) and avridine (N,N-dioctadecyl-N’,N’-bis-[2-hydroxyethyl]propan~amine, or the immune-suppressive cyclophosphamide (N,iV-bis[2-chloroethyl]tetrahydro-2H1,3,2-oxazaphosphorin-2dws, amine-2-oxide) and dexamethasone. Rats treated with NF’T 15392 or avridine generally had enhanced immune responses, while those treated with cyclophosphamide or dexamethasone had decreased immune responses. Differential responsiveness of various immunocyte populations within individual rats to different drugs, or to doses of the same drug, indicates the efficacy of measuring multiple responses within the same animal. The multiassay-single animal ap preach represents an economical, versatile, sensitive, and relatively comprehensive paradigm for assessing immunotoxicologic/pharmacologic properties of chemicals and drugs. The ap preach is extremely economical since multiple immune responses are evaluated in each animal. The approach is versatile because it is amenable to incorporation of a variety of in vitro and in viva assaysand could be applied to almost any species. The model is relatively comprehensive because major types of immune response.s/immunocyte populations and immunoregulatory pathways are tested. Finally, the model is sensitive for detecting immunosuppression as well as immunoenhancement, as validated by the use of known immune response modifiers in this study. 0 1986 SocietyofToticalogy.
It has been well-documented during the past decade that certain environmental chemicals, at relatively low doses, adversely affect normal functions of the mammalian immune system (reviews by Dean et al., 1982; Koller, 1979; Vos, 1977). It has also been demonstrated that chemical-induced immune dysfunction is associated with increased incidence of infectious disease and cancer in laboratory animals (Exon et al., 1975, 1979; Dean et al., 1980, 1982; Ward et al., 1984). Based on these data, the question has arisen as to whether government regula-
tory agencies should require immunotoxicologic testing within the general toxicity profiles generated for clearance/registration of chemicals for which significant exposure to humans and animals is anticipated. Although not presently required, many private companies have begun developing methods for detection of immunotoxicologic effects of chemicals in anticipation that the requirement will be implemented in the near future by regulatory agencies. Most experts in the field of immunotoxicology agree that no single immunoassay is 387
0272-059Of86 $3.00 Copyright 0 1986 by the Society of Toxicology. AU rights of reproduction in any form resewed.
388
EXON ET AL.
adequate to comprehensively assess overall immune competence of an individual, mainly due to the complexity of the immune system. The immune system is composed of a variety of cell types which differ in ontogeny and mediate different types of immune responses (reviewed by Klein, 1982). Each separate class or subset of immunocyte is capable of reacting individually to elicit a particular type of immune reaction, or can act in an integrated manner with other types of immunocytes to augment or suppress their functions. Four major classes of immunocytes are T and B lymphocytes (cells), macrophages, and natural killer cells (NKCs). The B cells are primarily the effector cells of humoral immunity (i.e., antibody production), but require the “help” of T cells and macrophages for optimal response to most antigens (Kung and Paul, 1983; Julius, 1982). The T cells are composed of both effector and regulator subpopulations (Reinherz and Schlossman, 1980). The effector T cells are responsible for specific cell-mediated immunity (SCMI) and can be divided into at least two subsets based on function and phenotype. These are the cytotoxic T cells and the T cells that mediate delayed hypersensitivity reactions (Berke, 1983; Liew, 1982). The regulatory T cells can be divided into helper (HT) and suppressor (ST) subsets, based on phenotype and regulatory activity, and mediate their actions mainly via production of soluble lymphokines (Reinherz and Schlossman, 198 1; Geha and Rosen, 1983). Macrophages are also composed of effector and regulator subsets. Macrophages are phagocytic cells which can act directly to ingest, kill, or inactivate foreign agents, or to “process” the antigen so that it is recognizable as foreign to other types of immunocytes (Kende, 1982; Grey el al., 1984). Natural killer cells are a population of large granular immunocytes which have some phenotypic characteristics of both lymphocytes and macrophages (reviewed by Herberman, 1982). The NKCs and effector macrophages are considered an important first-line defense mechanism (i.e., natural immunity) since,
unlike T cells, they react immediately with certain foreign agents without prior sensitization. The NKCs are believed to be especially important in immune surveillance to virusinfected and neoplastic cells. Cytotoxic response of macrophages and NKCs is generally termed nonspecific cell-mediated immunity. In addition to direct effector actions of different classes of immunocytes, these cells also produce a variety of immunoregulatory cytokines (reviews by Goldstein and Chirigos, 198 1; Oppenheim and Cohen, 1983). Prominent among these cytokines are interleukin 1 (ILl) and interleukin 2 (IL2). These immunocytokines act on activated effector immunocytes in an interrelated manner to regulate the magnitude of ongoing immune responses. Due to the apparent complexity of the immune system, no single immunoassay is adequate to assessoverall immune competence. Therefore, a battery of immunoassays is required which is designed to monitor separate segments of the immune system. Myriad in vivo and in vitro assays are available by which to assessthe integrity of the immune system in different species of animals. A comprehensive tier 1 panel of immunoassays should include, as a minimum, assessment of humoral immunity (i.e., antibody production by B cells), specific cell-mediated immunity (i.e., T-cell function), nonspecific cell-mediated immunity (i.e., NKC and macrophage function), and qualitative and quantitative pathotoxicologic examination of lymphoid tissues (i.e., spleen, thymus, major lymph nodes). Research in our laboratory during the past several years has been directed toward developing a representative immune function assay for each major segment of the immune system. Furthermore, a major goal has been to develop a sensitive, versatile, and comprehensive panel of immunoassays which can be performed concomitantly in a single animal in order to reduce animal numbers, experimentation costs, and variation between assays. The immunoprofile obtained from each animal consists of IgG antibody production
MULTIASSAY
IMMUNOTOXICOLOGY
to a specific T-dependent protein antigen; delayed hypersensitivity reaction to a second protein antigen; NKC cytotoxicity response; and the production of three potent immune response-regulating cytokines, namely macrophage-derived prostaglandin E2 (PGE2) and IL 1 and lymphocyte-derived IL2. Pathotoxicologic parameters include quantitation of immunocyte numbers, major lymphoid organ weights, and histopathologic changes. The purpose of this study is to validate the sensitivity and reproducibility of this multiple-immunoassay model to exogenous influence using known immune-modulating drugs. We have previously reported the sensitivity of parameters of this model to certain environmental contaminants (Exon et al., 1984a, 1985; Exon and Koller, 1983, 1984). The drugs tested were avridine (AVD) and NPT 15392, both of which are known immunopotentiators (Faanes et al., 1980; Niblack et al., 1979), and cyclophosphamide (CY) and dexamethasone (DEX), both of which are known immunosuppressors (Dean et al., 1979; Rosenberg and Lysz, 1980). A detailed discussion of the effects of these drugs in this rat model has been published elsewhere (Exon et al., 1986). The rat was chosen for use in this animal model because of the prominent use and familiarity of this species in other types of toxicologic/pharmacologic research.
METHODS
AND
MATERIALS
Male Sprague-Dawley rats were obtained from Washington State University, Laboratory Animal Resources, at 7 weeks of age and randomly divided into treatment groups of 12 rats each. The rats were housed four/cage in stainless-steel hanging wire cages and given deionized water and commercial rodent chow ad libitum. Animal quarters were maintained within recommended limits of temperature and humidity in rooms equipped with a 12hr on-off lighting cycle. Following 1 week of acclimation, each rat was injected subcutaneously (SC)twice at 7- or S-day intervals with the antigens, bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH), to induce either a delayed-type hypersensitivity reaction (BSA) or
389
a humoral immune response (KLH; Table 1). Animals in each group were also injected SC with various dosage regimens of the immunomodulating drugs NPT 15392,’ avridine,2 cyclophosphamide,3 or dexamethasone.4 Groups of rats received drug treatments consisting of either 0.1 or 1 mg/kg NPT 15392,l or 25 mg/kg AVD, 25 or 75 mg/kg CY, or 0.1 or 1 m&kg DEX (Table 2). NPT 15392 was injected twice concomitant with each BSA injection and a third time 3 days before termination. Avridine was injected 18 hr prior to each BSA injection and again 18 hr before the animals were terminated. Cyclophosphamide and DEX were injected twice, initially 1 day before the second BSA injection and then 3 days before the animals were terminated (Table 2). All rats were sacrificed by CO1 asphyxiation 14 days after the initial BSA and KLH injections at Day 0 (note that the footpad DTH response was measured on Day 8). At the time of sacrifice, serum samples were collected by cardiac puncture for analysis of antibody levels to KLH (Table 1). Spleens were used as the cell source to monitor for NKC cytotoxicity and IL2 production. Resident peritoneal macrophages were harvested for determination of PGE2 and IL 1 production. All assayswere performed on each rat on experiment. The effect of multiple-antigen treatment and the use of FCA on individual immune functions in the panel have previously been reported to be minimal and not statistically significant (Exon et al., 1984b). A brief description of the separate assaysis provided below. Delayed-type hypersensitivity (DTH) assay. Delayedtype hypersensitivity reactions, an in vivo indicator of specific CM1 responsiveness by T cells, was measured as footpad swelling in rats by a method described by Henningsen et at. (1984). Rats were sensitized (Day 0) sc with BSA in Freund’s complete adjuvant (FCA) and challenged 7 days later in the footpad with heat-aggregated BSA (see Table 1). Briefly, equal volumes of FCA and BSA in physiological saline (2 &ml) were emulsified and 0.1 ml, containing 100 gg of BSA, was injected sc at the base of the tail of each rat. Seven days later, the let% hind footpad was challenged by injection of 75 ~1 of a 2% BSA heat-aggregated solution. The right hind footpad was sham-injected with saline, and 24 hr later (Day 8) footpad swelling in both hind footpads was measured using an electronic digital micrometer. The thickness of the saline-injected footpad was subtracted from the BSA-in-
’ Erythro-9-(2-hydroxy,3-nonyl)hypoxanthine, Newport Pharmaceuticals Int., Newport Beach, Calif. ’ CP-20,96 1 or N,Ndioctadecyl-N’,N’-bis-(2-hydroxyethyl)propanediamine, Pfizer Central Research Inc., Groton, Conn. 3 2H- 1,2,3-oxazaphosphorine, Sigma Chemical Company, St. Louis, MO. 4 Azium, Schering Veterinary, Schering Corporation, Kenilworth, N.J., 2 mg/ml.
EXON
390
ET AL.
TABLE 1 TIME AND ANTIGEN TREATMENT SCHEDULEFOR MULTIPLE-IMMUNOASSAY ANALYSIS” Dosage
Antigens
Days
Routes SC(lumbar) SC(base of tail)
KLH 1 mg BSA + FCA 100 Pg BSA 75 @l (2% solution) (heat-aggregated) Measure DTH response KLH 1 mg Collect peritoneal macrophages for PGE, and IL 1 analysis Collect spleen cells for IL2 and NKC analysis Collect serum sample for ELISA analysis
Footpad Footpad SC
Schematic of the multiple immunoassay” RAT (antigen-injected)
Blood
Peritoneal macrophages
Spleen
Footpad
Serum ELISA W-H)
NKC
IL2
PGEz
IL1
DTH (BSA)
a KLH = keyhole limpet hemocyanin; BSA = bovine serum albumin; FCA = Freund’s complete adjuvant; DTH = delayed-type hypersensitivity; PGEz = prostaglandin E2; IL1 and IL2 = interleukins 1 and 2; NKC = natural killer cell; ELISA = enzyme-linked immunosorbent assay.
jetted footpad to determine the DTH reaction. The data are reported as mean millimeter difference in swelling between the two footpads. Enzyme-linked immunosorbent assay (ELBA). Serum samples of rats injected SCin the lumbar region with 1 mg KLH twice at an S-day interval (Days 0 and 8) were collected by cardiac puncture 6 days after the last KLH injection (see Table 1). The amount of anti-KLH IgG antibody contained in the serum was analyzed by an indirect enzyme-linked immunosorbent assay as previously reported in detail (Exon et al., 1985). Briefly, antibody-containing serum samples were diluted in a phosphate-buffered saline (PBS)-Tween 20 solution and added in 100~~1 aliquots to wells of a 96-well microtiter plate which had been coated with 2.0 mg/ml KLH overnight at 4°C and rinsed. The serum-containing plates were incubated at 37’C for 1 hr and rinsed in PBS-Tween 20. Alkaline phosphatase, conjugated to goat anti-rat IgG (I:500 dilution), was added to each well in 100~~1 aliquots, incubated for 1 hr at 37°C and then rinsed with PBS-Tween 20. The substrate for alkaline phosphatase, p-nitrophenylphosphate, diluted to 1 mg/ml in dietha-
nolamine buffer, was added to each well in 100~~1 aliquots. The color reaction was allowed to develop for 30 min in the dark at room temperature and quantitated on a spectrophotometer (Titertek Multiskan MC) at 405 nm. The results are expressed as mean absorbance at a 1: 1000 dilution of the serum. Collection of spleen cells. Briefly, rats were sacrificed via CO2 asphyxiation and the spleens removed aseptically and minced with scissors. Single-cell suspensions were obtained by forcing the minced spleen through a stainless-steel mesh screen into complete medium (CM) consisting of RPM1 1640, 10% heat-inactivated fetal bovine serum, 25 mM Hepes buffer, 100 units/ml penicillin, and 100 pg/rnl streptomycin. The cells were centrifuged and the erythrocytes lysed by hypotonic shock for 6 sec. An equal volume of Hank’s balanced salt solution (2X) was added and the cells were centrifuged and resuspended in 6 ml of CM. A l-ml aliquot (or less) of cells was removed at this point for the IL2 assay.The remaining cells were depleted of B cells and adherent phagocytic cells by incubating on columns of nylon wool for 45 min and then in plastic culture plates for 1 hr at 37°C 10%
MULTIASSAY
IMMUNOTOXICOLGGY
CO2 The nonadherent semipurified NKCs were eluted in 25 ml of CM, centrifuged, and resuspended to 1 X 10’ cells/ml for use in the NKC assay. Assay for natural killer cell cytotoxicity. The method for analyzing the function of NKCs is to test their capacity to lyse tumor cells in vitro. The method used was a modification of that by Ristow et al. (1982). The YAC- 1 (target) cells, a murine Moloney leukemia virus-induced T cell lymphoma of A-strain mice, were grown as stationary suspension cultures in CM. These target cells were labeled with 400 PCi of ‘ichromium (as sodium chromate) per 10’ cells at 37°C 10% CO2 , for 1 hr with gentle shaking. The target cells were then washed and resuspended to 1 X 10’ cells/ml. Viability for both the target and the NKC, collected as above, was determined by trypan blue exclusion (usually >95%). The YAC-1 cells, prepared as above, were added in 100-p aliquots to the appropriate wells of a 96well flatbottomed microtest plate containing 100 ~1 of varying concentrations of NKC (effector). After a 4-hr incubation at 37°C in an atmosphere of the 10% CO2 in air, the plates were centrifuged at 200g for 10 min and 100 ~1 of the cell-free supematants was collected from each well and counted in a gamma counter. Specific 5’Cr release was calculated by the formula cpm experimental release - cpm spontaneous release cpm maximum release by 2% SDS - cpm spontaneous release X
100% = % cytotoxicity.
Interleukin 2 assay. The common method for analysis of IL2 is the ability to maintain growth of an IL2dependent T-cell clone in culture. The method is a modification of that reported by Gillis et al. (1978). Briefly, spleens were removed aseptically from each animal, forced through sterile, stainless-steel screens into IscoveMelchers modified Dulbecco’s medium (supplemented with 100 U/ml penicillin, 100 &ml streptomycin, 5 X 10m5M 2-mercaptoethanol (2-ME), human transferrin, and 400 pg/ml fatty acid-free BSA) to obtain singlecell suspensions. The cells were then counted on a Coulter counter and diluted to 1 X lo6 cells/ml. Onemilliliter ahquots were incubated 24 hr with 1.O&ml of concanavalin A (Con A), and the cell-free supematant was harvested and tested for IL2 activity. Briefly, 4 X IO3 ILZ-dependent murine cytotoxic T cells (CTLL) were cultured in replicate 200~~1 volumes in flat-bottomed 96-well culture plates in the presence of a log-2 dilution series of putative ILZ-containing supematants. After 18 hr, the cells were pulsed with 1 pCi [‘H]TdR (6.7 Ci/ mmol) for 4 hr, harvested onto glass fiber filters, and counted on a scintillation counter. IL2 activity was quantified by probit analysis of the [3H]TdR incorporation data as described by Gillis et al. (1978). The standard
391
preparation of human IL25 contained 1119.75 units of IL2 activity per milliliter. The results are expressed as units of IL2 per milliliter of culture supematant. Interleukin I assay. Macrophage function was measured by their capacity to produce IL1 and PGE2 in vitro. Following asphyxiation with CO*, 30 ml of sterile HBSS was injected ip into each rat. The abdominal area was gently massaged for approximately 1 min to dislodge resident peritoneal cells. A small (2 mm) slit was made in the abdominal wall and the medium containing resident peritoneal cells was allowed to drain into a 50-ml siliconized centrifuge tube. The peritoneal cells were pelleted and washed twice and cell numbers determined by Coulter counting. The cells were diluted to 1.5 X 106/ml for use in the IL1 and PGE2 assays.IL1 synthesis by rat resident peritoneal cells was induced in 72-hr lipopolysaccharide (LPS)-stimulated cultures as previously reported (Gery et al., 1981). Indomethacin was added to prevent prostaglandin synthesis in the IL1 assay. The ILl-containing supematants were stored at -20°C and later assayedfor activity by the capacity to induce proliferation of thymocytes in culture from 6-week-old C3H/ HeJ mice. Serial dilutions of ILlcontaining supematants were cultured for 68 hr with 1.5 X lo6 thymocytes in the presence of 2-mercaptoethanol and phytohemagglutinin (PHA) followed by a 4-hr incubation with [‘H]TdR. The cells were harvested onto glass filter paper and [3H]TdR uptake was measured by scintillation counting. Results are expressed as mean cpm/ 1.5 X 1O6 adherent cells. Assay for prostaglandin E2. Resident peritoneal macrophages were collected by lavage as for the IL1 assay. The macrophages were washed and plated in 24-well cluster plates. After 1 to 2 hr, the wells were rinsed three times to remove nonadherent cells. One-millimeter culture medium (Dulbecco’s MEM, 5% fetal bovine serum, 100 units penicillin/ml, and 100 rg streptomycin/ml) was added to each well. Three cultures were assayed for each rat. One was a control culture (no additives) and two were cultured with 0.1 &ml LPS (lipopolysaccharide, Escherichia coli, 055:B5). Media controls were run with each assay. Cluster plates were incubated at 37°C 10% CO* for 18 hr at which time supematants were removed and centrifuged. The cell-free supematants were assayed for PGEl using a commercially available radioimmunoassaykit6 The kit offers a sensitive method of measuring PGE2 by monitoring the competitive binding of [3H]TdR-labeled prostaglandin and unlabeled PGEz with antibody specific for PGEz Separation of antibody-bound PGE, from free PGEl was achieved by precipitating the antibody-PGE, complex with a second antibody. The assay is sensitive to approximately 0.03 ng per 1 ml medium. The medium controls allowed correction for any
5 Cellular Products, Inc., Buffalo, N.Y. 6 Clinical Assays, Cambridge, Mass., CA-50 1.
392
EXON ET AL. TABLE 2 TREATMENTSCHEDULEFORTESTINGIMMUNEMODULATINGDRUGS"
Time
Dose
Antigens/drugs
Routes
-18hr
Avridineb
1 or 25 mg/kg
SC
Day 0
KLH BSA-FCA NPT 15392
1 mg 100 & 0.1 or 1.O mg/kg
SC SC SC
Day 6
Cyclophosphamide Avridineb
25
or 15 mg/kg 1or 25 mg/kg
SC SC
Day I
BSA-HA NPT 15392
100 /lI (2%) 0.1 or 1 .O mg/kg
FP SC
1 mg
SC
Day 8
Assay: DTH-footpad
swelling KLH
Day 11
NPT 15392 Cyclophosphamide
0.1 or 1.Omg/kg 25 or 75 mg/kg
SC SC
Day 13
Avridineb
1 or 25 mg/kg
SC
Day 14
Assay: PGE and ILl-resident peritoneal cells Assay: IL2 and NKC-splenocytes Assay: anti-KLH antibody-serum
’ KLH = keyhole limpet hemocyanin; BSA = bovine serum albumin; BSA-HA = heat-aggregated BSA; FCA = Freund’s complete adjuvant; DTH = delayed-type hypersensitivity; PGE2 = prostaglandin EZ; IL1 and IL2 = interleukins 1 and 2; NKC = natural killer cell; SC= subcutaneous; FP = footpad. b Avridine was given 18 hr prior to antigen (i.e., KLH or BSA) or termination.
interference from MEM, FBS, or LPS. Samples were counted on a liquid scintillation counter, unknowns were compared to the standard curve, and PGEz concentrations are reported as nanograms per milliliter. These values were corrected for the number of adherent cells and final PGEz levels are reported as ng/ 1 X 1OSadherent cells. Statistical analysis. All &ta were analyzed by a conventional analysis of variance technique and leastsquares mean comparisons to respective controls or by probit analysis.
RESULTS A significant (p < 0.05) dose-dependent decrease in serum antibody to KLH, DTH reactions to BSA, NKC cytotoxic responses, spleen and thymus weights, and numbers of splenocytes and resident peritoneal cells was observed in all CY-treated rats (Tables 3 and 4). The production of IL2 by splenocytes was significantly (p < 0.05) reduced in rats treated
with 75 mg/kg CY (Table 5). Macrophagederived PGEz production was significantly (p f 0.05) elevated in both groups of CY-treated rats, while IL1 synthesis was significantly (p < 0.05) greater in the 75 mg/kg group (Table 5). Rats treated with DEX had dose-dependent significant (p < 0.05) decreases in DTH reactions, NKC cytotoxicity responses (Table 4), thymus weights, and numbers of splenocytes and resident peritoneal cells (Table 3). Rats treated with 1 rng/kg DEX also had significantly (p f 0.05) reduced IL1 and IL2 production (Table 5) and spleen weights (Table 3). No significant effect of DEX treatment was observed on serum antibody levels to KLH or PGE2 production. Rats injected with either 1 or 25 mg/kg AVD had significantly (p < 0.05) augmented DTH reactions inverse to the AVD dose (Table 4). The NKC cytotoxic responses were
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393
IMMUNOTOXICOLOGY TABLE 3
THE EFFECTSOF IMMUNE Treatment”
R!ZSFQNSE MODIFTERS
Spleen weight g (k SE)b
ON ORGAN
Thymus weight g (+SE)b
WEIGHTS
AND IMMUN~CYTE
Splenocytes X lO’/ml (HE)
NUMBERS
Peritoneal cells X lO’/ml (*SE)
Avridine (AVD) and NPT 15392 (NPT) Control
0.75 + 0.06
0.48 + 0.04
5.0 -t 0.7
1.4-t0.2
1 mg AVD/kg 25 mg AVD/kg
0.86 + 0.05 0.92 + 0.03d
0.43 f 0.03 0.48 + 0.06
5.1 +- 0.6 5.4 * 0.5
1.3 f 0.2 1.6 kO.2
0.1 mg NPT/kg 1.O mg NPT/kg
0.80 f 0.05 0.84 + 0.06
0.50 f 0.05 0.55 + 0.06
5.2 f 0.3 5.8 f 0.4
1.3 kO.2 1.4-t0.2
Control
0.75 rt 0.06
0.48 f 0.04
4.8 + 0.4
1.3 i 0.1
25 mg CY/kg 75 mg CY/kg
0.55 f 0.05d 0.37 f 0.06d
0.3 1 f 0.04d 0.20 + 0.04d
1.4 + 0.2d 0.7 f 0.2d
0.8 -t O.ld 0.6 k 0. Id
0.1 mg DEX/kg 1.O mg DEX/kg
0.64 + 0.06 0.39 f 0.05d
0.20 + 0.03d 0.20 2 0.02d
2.1 *0.1d 0.7+0.1”
0.9 t O.ld 0.7 I!z0. Id
Cyclophosphamide (CY) and Dexamethasone (DEX)
’ See Table 2 for treatment times. b Unadjusted for body weight; no significant differences between body weight compared to controls. ’ Inclusive of adherent and nonadherent fractions. dp 6 0.05 by analysis of variance and least-squares mean comparison to respective control.
significantly (p < 0.05) enhanced in the 25 mg/kg treatment group. The synthesis of IL2 and IL1 (Table 5) was significantly (p < 0.05) increased in rats injected with 1 mg/kg AVD, and spleen weights were significantly (p < 0.05) greater in rats exposed to 25 mg/kg AVD (Table 3). Synthesis of PGE2 was significantly suppressed in the 25 mg/kg group (Table 5). No significant effects of AVD treatment were observed in regard to antibody synthesis, thymus weights, or numbers of spleen cells and resident peritoneal cells. Rats treated with 0.1 mg/kg NPT 15392 had significantly (p & 0.05) enhanced DTH reactions (Table 4). Animals treated with the 1-mg/kg dose of NPT 15 392 had significantly increased NKC cytotoxic activity (Table 4) and suppressed PGE2 synthesis (Table 5). No significant effects of NPT 15392 treatment were observed in regard to antibody production, IL1 and IL2 synthesis, or organ weights and cell numbers.
DISCUSSION Alterations of immune functions observed in rats exposed to the known immune response-modifying drugs in this study indicate that this animal model is sensitive to exogenously induced immunomodulation by xenobiotics. Rats treated with the immunosuppressive drugs, CY and DEX, generally had reduced immune functions, while animals treated with the immune stimulating compounds, AVD and NPT 15392, generally had enhanced immune responsiveness. These results suggest that other immune responsemodifying agents, such as environmental toxicants, could also be detected by these methods and thereby may represent a practical immunotoxicological screen. Immunomodulating agents which potentially could be detected include those which either augment immune responsiveness and thereby predispose to development of autoimmune-related dis-
394
EXON
ET AL.
TABLE 4
Treatment’
Antibody productionb (Meanabsorbance at 405 mm (GE))
Delayed hypersensitivity’ (mean mm swelling (GE))
Natural killer cytotoxicityd (% killing (*SE) ratio 50: 1)
Avridine (AVD) and NPT 5 1392 (NPT) Control
1.41 + 0.18
3.4OkO.16
26.4 + 1.6
1 mg AVD/kg 25 mg AVD/kg
1.63 *0.15 1.57 + 0.22
4.46 +- 0.49’ 4.21 f 0.21’
32.6 + 2.0 37.8 f 2.0’
0.1 mg NPT/kg 1.Omg NPT/kg
1.62kO.13 1.4450.16
4.92 + 0.41’ 3.95 + 0.39
26.5 2 2.0 33.9 k l.3e
Cyclophosphamide (CY) and Dexamethasone (DEX) Control
1.40 f 0.21
4.10 + 0.20
23.6 + 1.9
25 mg CY/kg 15 mg CY/kg
0.42 f 0.06’ 0.26 -c 0.10’
2.32 f 0.87’ 0.86 f 0.2 le
6.3 + 0.8e 5.2 + 1.4’
0.1 mg DEX/kg 1.Omg DEX/kg
1.40 + 0.26 1.31 +0.13
2.88 + 0.10’ 1.35 + 0.17’
13.0 f 1.3’ 7.9 f 1.7’
’ See Table 2 for treatment times. b By an indirect ELISA to KLH; 1:1000 serum dilution. ’ By a footpad-swelling assayto BSA. d By a 4-hr 5’Cr release assayto YAC- 1 target cells; ratio 50: 1 = NKC:target cells. ‘p G 0.05 by analysis of variance and least-squares mean comparison to respective control.
eases, or those which depress the immune system and render the host more susceptible to immunodeficiency-related disorders. Furthermore, since all of the immunoassays are performed in each animal, it is possible to compare the relative overall alteration of different types of immune responses in the same rat following exposure to a chemical or drug. This multiple-immunoassay model is an extremely economical method of assessing immune competence, since all immunoassays are performed concomitantly in a single animal. Consequently, a minimal number of animals and space is required, thereby reducing the overall cost of purchasing and maintaining experimental animals. Furthermore, certain types of experimental variation/error are minimized since all assays are performed at approximately the same time in the same animal. Also, multiple responses can be cor-
related in individual animals. A limitation of the multiple immunoassay approach is the availability of adequate numbers of personnel to perform assays and the numbers of immunocytes available from each animal. Cell numbers available for use in each assay could, however, be increased by pooling cells from inbred strains. The multiple-immunoassay model can be very versatile. Although the ELISA, DTH, NKC cytotoxicity, immunocytokine production, and pathotoxicologic techniques were utilized in this study, any suitable representative immunoassay could be substituted, depending on the investigator’s preference or the chemical tested. Also, additional assays could be easily incorporated into the model (e.g., bone marrow progenitor, helper/suppressor T-cell ratios, complement fixation, macrophage and T-cell cytotoxicity). This immunotoxicologic testing model is
MULTIASSAY
IMMUNOTOXICOLOGY
395
TABLE 5 THE
EFFECTSOFIMMUNERESPONSEMODIL~ERSONIM~OREGULATORYCYTOK~NEPRODUCTION
Treatment’
Interleukin 2 productionb (mean units/ml (+SE))
Interleukin 1 production’ (mean CPM/l.5 X lo6 cells @SE))
Prostaglandin E2 productiond (mean ng PGEr/ 10’ cells (+SE))
Avridine (AVD) and NPT 15392 (NPT) Control
73.7 * 6.5
11,950 f 2,798
54.0 + 4.0
1 mg AVD/kg 25 mg AVD/kg
93.4 f 8.0e 76.1 f 5.2
25,394 f 3,678’ 10,278 & 3,873
58.4 f 4.0 32.5 f 4.6’
0.1 mg NPT/kg 1.O mg NPT/kg
88.4 f 9.0 73.6 k 5.5
22,900 f 5,906 19,633 + 3,900
53.0 + 4.3 22.4 + 4.0’
Cyclophosphamide (CY) and Dexamethasone (DEX) Control
70.3 f 2.3
9,858 + 2,113
59.9 f 2.8
25 mg CY/kg 75 mg CY/kg
53.3 -t 1.6 36.3 -t 2.0’
13,440 + 2,040 22,821 t 1,904e
84.4 f 3.5’ 85.7 2 3.5’
0.1 mg DEX/kg 1.O mg DEX/kg
45.7 e 2.3 29.7 f 1.5’
8,874 + 1,815 2,665 + 503e
55.6 + 2.8 52.6 1?I2.8
’ See Table 2 for treatment times. b By Con A-induced splenocytes; CTLL cells were used as the IL2-dependent T-cell clone. ’ By LPS-stimulated resident plastic adherent peritoneal cells; IL1 levels determined by stimulation of PHA-induced mice thymocytes. d By LPS-stimulated plastic adherent peritoneal cells; PGE, levels determined by a double antibody radioimmunoaSSay. ep < 0.05 by analysis of variance and least-squares mean comparison to respective control.
relatively inclusive in that the functions of the prevalent populations of effecter/regulator immunocytes are assessed. The immunoprofile obtained from each rat includes assessment of major types of immune responses, immunocyte populations, and immunoregulatory pathways. For instance, the production of antibodies to a T cell-dependent antigen such as KLH requires the interaction of B cells, regulator T cells, and macrophages (Julius, 1982). Any effect on antibody production, as measured by the ELISA or any other suitable method, could result from altered reactivity of any one of these immunocyte populations. Macrophage function is further tested by their capacity to produce the immune response-regulating monokines, IL 1 and PGE2 . The function of T effector cells in-
volved in specific CM1 responses is assessed by their capacity to elicit a DTH response. Regulatory T-cell populations are further tested by their capacity to produce IL2, a potent immunoregulatory lymphokine which is required for optimal cytotoxicity functions of NKC and antigen-specific clonal expansion of T cells mediating the DTH response and cytotoxicity reactions. The NKC cytotoxicity assay assessesthe function of these first-line host defense immunocytes to kill tumor cells. Therefore, the assays composing the multiple-immunoassay model collectively test for the competence of B cells, effecter/regulator T cells, effecter/regulator macrophages, and NKCs. The functions tested relate to humoral immunity, specific acquired CM1 immunity, natural nonspecific CM1 (i.e., mac-
396
EXON
ET AL.
rophages and NKCs), and production of relevant immune assays for use in immunoassessment must potent endogenous immunoregulatory cy- toxicologic/pharmacologic tokines. The model does, however, lack an be determined by extensive testing and valiadequate assay for hypersensitivity reactions, dation, especially before any recommendawhich should be included in any tier 1 screen. tions can be made with regard to an appropriThe model also lacks a host resistance assay ate battery of immunoassays which may be although, due to the in vivo immunoselectivrequired by regulatory agencies in general tier ity of many of the pathogens used in these 1 or tier 2 toxicologic testing protocols. types of assays, their feasibility for inclusion REFERENCES in tier 1 testing is questionable and they may be more appropriately included in the tier 2 panel. BERKE, G. (1983). Cytotoxic T-lymphocytes. How do they function? Immunol. Rev. 12,5-4 1. This general model could be adapted to alDEAN, J. H., LUSTER, M. I., AND BOORMAN, G. A. most any species, depending on preference. (1982). Immunotoxicology. In Research Monographs The rat was chosen for use in this model since in Immunology. Immunopharmacology (P. Sirois and it is often the species of choice in toxicologic/ M. Rola-Pleszczynski, eds.), Vol. 4, pp. 349-398. pharmacologic research, and thus facilitates DEAN, J. H., LUSTER, M. I., BOORMAN, G. A., LAUER, a direct comparison of immunotoxic effects L. D., AND LUEBKE, R. W. (1980). The effect of adult exposure to diethylstilbestrol in the mouse: Alterations with other toxicologic parameters and could in tumor susceptibility and host resistance parameters. be readily incorporated into the standard 90J. Reticuloendothel. Sot. 28,57 l-583. day subchronic toxicity testing protocols cur- DEAN, J. H., PADARATHSINGH, M. L., JERRELLLS, T. R., rently in use. Furthermore, a substantial KEYS, L., AND NORTHING, J. W. (1979). Assessment amount of baseline toxicologic data has been of immunobiological effects induced by chemicals, drugs or food additives. II. Studies with cyclophosphacompiled for this species. Therefore, there is mid& Drug Chem. Toxicol. 2,133- 153. a need to be able to assess immunotoxicity J. H., AND KOLLER, L. D. (1983). Effects of chloin the rat. The immune system of the rat is EXON, rinated phenols on immunity in rats. Int. J. Immunorelatively well-characterized, and state-ofpharmacoi. 5, I 3 I- 136. the-art reagents and inbred strains are availEXON, J. H., AND KOLLER, L. D. (1984). Toxicity of 2chlorophenol, 2,4-dichlorophenol and 2,4,6-trichloroable for detailed study of the immunologic phenol. In Water Chlorination: Chemistry, Environfunction. Greater numbers of immunocyte mental Impact and Health E&cts (R. L. Jolley, ed.), subpopulations can be collected from the rat Vol. 5, pp. 307-330. Lewis Publishers, Chelsea. as compared, for instance, to the mouse. This EXON, J. H., HENNINGSEN, G. M., KOLLER, L. D., AND facilitates the multiple-assay approach, TALCOTT, P. A. ( 1986). The selectivity of isoprinosine, NPT 15392, avridine and cyclophosphamide on mulThe rat model described in this report for tiple immune responses in the rat. Int. J. Immunopharuse in immunotoxicity testing of drugs and macol. 8,53-62. chemicals is comprehensive, economical, EXON, J. H., HENNINGSEN, G. M., OSBORNE, C. A., AND versatile, and apparently sensitive to immuKOLLER, L. D. (1984a). Toxicologic, pathologic and nomodulation. However, it is not the intent immunotoxic effects of 2,4-dichlorophenol in rats. J. Toxicol. Environ. Health 14,723-730. of this report to recommend that this particular model system is the best for use in the de- EXON, J. H., KOLLER, L. D., AND KJZRKVLIET, N. I. (1979). Lead-cadmium interaction: Effects on viral-intection of immune-modulating xenobiotics. duced mortality and tissue residues in mice. Arch. EnThe species and specific immunoassays utiviron. Health 34,469-475. lized in the model are secondary, in many re- EXON, J. H., KOLLER, L. D., HENNINGSEN, G. M., AND OSBORNE, C. A. (1984b). Multiple immunoassay in a spects, to the main concept of assessing mutisingle animal: A practical approach to immunotoxicople concomitant immune responses in each logic testing. Fundam. Appl. Toxicol. 4,278-283. animal. Also, several of the assays may be EXON, J. H., PATTON, N. M., AND KOLLER, L. D. more appropriate for tier 2 testing (e.g., IL1 , (1975). Hexamitiasis in cadmium-exposed mice. Arch. IL2, PGE2). The optimal species and most Environ. Health 30,463-464.
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