Document O312pNO343NV1aZz05z9vjJNj

Enhanced Suppressor Cell Activity as a Mechanism of Immunosuppression by 2,3,7,8-Tetrachlorodibenzo-p-dioxin1 (41275) DAVID A. CLARK, JACK GAULDIE, MYRON R. SZEWCZUK, and GEORGE SWEENEY Departments o f Medicine and Pathology. Host Resistance Program. McSiaster University. 1200 \fnht Street HVj7 . Hamilton. Ontario L 8 S JZS. Canada Abstract. TCDD (2.3.7,8-tetrachlorodibenzo-p-dioxin). a toxic halogenaied aromatic hy drocarbon. acts in the body as a cumulative poison. The chronic immunotoxic effects of TCDD were studied in C57B1/6 male mice. Total doses of 100 pg/kg or greater produced cellular depletion in thymus, spleen, and lymph nodes, and the animals appeared sick. No cellular depletion was seen following 0.4 pg'kg. and only thymus was affected by 4 and 40 Pg'kg. The antibody response to SRBC and TNP-fir//i'W/o abortus was impaired following 40 pg'kg TCDD. the delayed hypersensitivity response to oxazalone was impaired by 4 ig/kg and the generation of alloantigen-specific cytotoxic T cells (CTL) was sensitive to as little as 0.004 tg/kg TCDD. In vitro analysis of the mechanism of suppression using limiting dilution techniques showed that TCDD did not deplete the precursors of CTL but generated cells capable of suppressing CTL generation in vitro. Halogenaied aromatic hydrocarbons (HAHs)2 such as polychlorinated biphenyls (PCBs)-. polychlorinated naphthalenes, polybrominated biphenyls (PBBs), poly chlorinated dibenzofurans, pentachlorphenols (PCP), and polychlorinated dibenzo-pdioxins, pose a serious man-made environ mental threat to health (1 -9 ). They are fat soluble, poorly metabolized, and tend to accumulate in the environment and in the body where they act as persistent poi sons (1). Polychlorinated dibenzo-pJdioxins such as TCDD (2,3,7,8-tetrachlorodibenzo(p)-dioxin) formed industrially by pyrolysis of chlorphenols, a particularly toxic subgroup of the halogenated aromatic hydrocarbons, pro vide a good model system for studying HAH toxicity. Allhough species vary in the types of 1Presemeli in pan ai the 23rd Annual Meeting of the Canadian Federation of Biological Societies. June II. 1980, and published in abstract form Voi 23, ab stract 295. *Abbreviations used: HAHs. halogenated aromatic hydrocarbons: PBBs. polybrominated biphenyls: Tf DO. 2.3." 8-terrachlorodibenzo-p-dioxin:'-SRBC. 'beep red Mood cells. TNP-BA. trinitrophens laied <"' - *7- ' .......... . . 'r e m ili: : ' P I A . pvnphei.il Isrnph .t .iv ' , br.tchi.il. ' C !'L . c> u o o \ :c l b m p h o c .jt c t VI p p r e d ir '* '! o i' C M . P I T ; pl.tque- :.'* 'iCve'l * '.t/.: ' V J v! li*1vt h '. tene 2 r h e n . lesions developing after TCDD exposure (8, 10, 11), a consistent observation in all animal species which have been studied is atrophy of the thymus. TCDD-treated ro dents show impairment of delayed hyper sensitivity responses, reduced ability to mount a graft-versus-host reaction and re duced ability to reject allografts, and show an increased susceptibility to certain infec tious agents (8, 10. 12- 15). Thus, one of the potentially important aspects of toxicity of TCDD and related HAHs is the effect on the immune system. In order to better un derstand the mechanism of TCDD immunotoxicity. we have analyzed the effects of different doses of TCDD on several types of immune function of C57B1/6J male mice. Particular attention has been directed to the chronic effects of TCDD since chronicity typifies human exposure. We find that the generation of cytotoxic T lymphocytes (CTL) following alloantigen challenge is particularly susceptible to impairment by low doses of TCDD, and this impairment is caused by a disturbance in the cells which regulate the CTL response. Measurement and characterization of this disturbance may provide a NCiiMtixe test for presence oi biologically significant amounts i'f TCDD m the body, Materia Is and Methods V/, C'~!il h 2vii 81 (Xi2v t M usui .otH) ritrn* t*\ the S.sicMy IWI \f*crjfnrntAl Bilious jnJ Mejivinr male mice age 6 - 8 week i from the Jackson Labo bour, Maine, and hous Teklad chow (6% fat) am vided ad lib. DBA/2J mi obtained and were used f< P-815-X2 (H-2rt) mastocy rial ascities transfer. C31 j p, and C57B1/6J x DBA/2 I produced in our own co | mice were purchased frorr. i erinary College, Guelph, < TCDD treatments. TCI 144-2 purity >99% was ob i Chemical, Sarnia, Ontari [ hundred and fifty micro solved in p-dioxane and di ^ Mice were injected ip weel corn oil containing speci f TCDD and studied 1 week I dose. Cell preparations. The and control mice were ki * dislocation. The sp leen \ peripheral lymph nodes (a: and inguinal) were asepti and a single-cell suspensic \ by pressing minced tissue mesh stain less-steel scr medium. Culture mediur. q-MEM supplemented wit! bovine serum (Grand Island 1 Grand Island, N .Y .), 100 ui i and 100 p.g streptomycin/j Hepes buffer. 2-Mercapi added at a concentration of : cell culture experiments. I An:i-Thy 1.2 treatment. ^ serum was purchased fr> Laboratories, Hornby, OnU The proportion of thy 1.2-1 peripheral lymph node (PL: sions was determined in tv suspension initially preparec phosphate-buffered saline w 4 tion V bovine serum album , by incubation with N a =5,C | giand Nuclear) and washed. 'T'm] were in.ubated wii :i 1-70 dilution at 4` for 45 Pended in ,i 1 1Z dilution of 1 v^'iink uu " t cd.iri.mc I Alter vt) min of incubation at X)-:y9ri'isn IMMUNOSUPPRESSION BY 2.3.7.8-TETRACHLORODIBENZOv-DlOXIN 291 .nosuppression by i /CZUK. AND taster University. Ja led aromatic hyoioxic eiTects of reatcr produced peared sick. No ;ied by A and 40 paired following s impaired by 4 is sensitive to as 3n using limiting 'L but generated r TCDD exposure (8, observation in all have been studied is s. TCDD-treated roit of delayed hyper- reduced ability to tost reaction and reallografts, and show ility to certain infec15). Thus, one of the spects of toxicity of *Hs is the effect on i order to better un;ism of TCDD imanalyzed the effects DD on several types C57B1/6J male mice, been directed to the 3D since chronicity ~e. We find that the .ic T lym phocytes ntigen challenge is to impairment by d this impairment is e in the cells which mse. Measurement f this disturbance test for presence of mounts of TCDD in ods. .U/< r. C57BI/6 male mice age 6 - 8 weeks were purchased of MCr release was calculated and percent from the Jackson Laboratory, Bar Har age lysis by anti-thy 1.2 plus complement bour, Maine, and housed six per cage. calculated by subtraction of the comple Teklad chow (6% fat) and water were pro ment control. Since TCDD treatment might vided ad lib. DBA.2J mice were similarly have altered the sensitivity of the cells to obtained and were used for maintenance of complement, a second method was used. P-815-X2 (H-2d) mastocytoma cells by se PLN were incubated in a 1/4 dilution of rial ascities transfer. C3H/HeJ x DBA/2J anti-thy 1.2 serum for 45 min at 4, washed, Fj and C57B1/6J.X DBA/2J F, hybrids were and then incubated in a 40-/xg/ml solution of produced in our own colony. RNC nude fluorescein-labeled protein A (Pharmacia). mice were purchased from the Ontario Vet After washing, the proportion o f cells erinary College, Guelph, Ontario. showing membrane immunofluorescent TCDD treatments. TCDD Lot No. 851- staining was determ ined by counting 144-2 purity >99% was obtained from Dow 200 - 400 cells under indirect ultraviolet il Chemical, Sarnia, Ontario, Canada. Two lumination (Leitz). hundred and fifty micrograms was dis Antibody response. The antibody re solved in p-dioxane and diluted in com oil. sponse to two different antigens was mea Mice were injected ip weekly with 100 lI o f sured. Mice were injected intraperitoneally corn oil containing specified amounts of with either 5 x 107 washed sheep red blood TCDD and studied 1 week after the fourth cells (SRBC) or, 0.1 ml of an 8% suspen dose. sion of TNP-conjugated Brucella abortus Cell preparations. The TCDD-treated (TNP-BA). Spleens were removed from and control mice were killed by cervical SRBC- or TNP-BA-challenged mice 3 - 7 dislocation. The sp leen , thym us, and days later and tested for antibody-forming peripheral lymph nodes (axillary, brachial, cells against SRBC or TNP-SRBC, respec and inguinal) were aseptically removed, tively, using a plaque assay as modified for > and a single-cell suspension was obtained slides by Dresser and Greaves (16). Freshly by pressing minced tissue through a 60- frozen guinea pig serum absorbed with 50%- mesh sta in less-steel screen into cold packed SRBC was thawed and added at a medium. Culture medium con sisted of final dilution of 1/30 as a source of comple ar-MEM supplemented with 10% v/v fetal ment to detect "direct** plaque-forming bovine serum (Grand Island Biological Co., cells. To detect IgG and IgA PFC, direct i Grand Island, N .Y .), 100 units penicillin G IgM PFC were first blocked by adding a and 100 pg streptomycin/ml and 20 mM 1/200 dilution of goat anti-mouse p during Hepes buffer. 2-M ercaptoethanol was the initial incubation of target and spleen added at a concentration of 5 x I0-s M for cells. Indirect IgG PFC were then devel ceil culture experiments. oped by adding rabbit anti-mouse IgG at a A/m'-77iy 1.2 treatment. AKR anti-C3H predetermined optimal 1/200 dilution, and < serum was purchased from Cedarlane IgA PFC were developed using a 1/100 di Laboratories, Hornby, Ontario (Lot 5101). lution of rabbit anti-mouse IgA. The IgG- The proportion of thy 1.2-bearing cells in developing serum was prepared by im peripheral lymph node (PLN) cel! suspen munizing rabbits with purified mouse IgG sions was determined in two ways. PLN (Miles. Lot 51). and showed only a single suspension initially prepared in Dulbecco's line against m ouse serum and IgG on phosphate-buffered saline with 0.2% Frac- Ochierlony plates. The IgA-developing lion V bovine serum albumin was labeled serum was prepared in rabbits using pure by incubation with NaA'CrO., (New En mouse myeloma MOPC 315 (IgA A2) and gland Nuclear) and washed. Labeled cells was depleted of anti-light-chain activity UF/ml were incubated with antiserum at by passage through a murine IgG-Seph- 'd 3 20 dilution a l"4c for 45 min and resus arose-immunoadsorbent column. Pure mouse pended in a 1/12 dilution of Low-Tox rabbit IgA or IgG added to the PFC assay inhib 1 complement (Cedarlane Laboratories). After 90 min of incubation at 37e, the degree ited only IgA or IgG PFC, respectively, when the developing sera were used. 292 IMMUNOSUPPRESSION BY 2,3.7,8-TETRACHLORODI BENZO-p-DIOXI N IMMUNOSUPP Delayed hypersensitivity response. In the bation period at 37 in a 4-hr slCr-release . first method used, TCDD-trcated and con assay using 2 x 104 P-815 target cells. ; trol mice were sensitized by injecting 1 -2 In some experiments, thymocytes from x ID1 SRBC iv and 5 days later the mice treated or control mice were added to the I were tested by injecting 2 x 108 SRBC in 20 cultures. Second, the frequency of precur p\ into one of the hind footpads (17). Saline sors of CTL (CTLp) was measured using (20 d) was injected into the other footpad the limiting dilution microculture method of 1 and the degree of swelling was measured Teh et ai. (20). Briefly, small numbers of after 24 hr. The net amount of swelling ob C57BI/6J spleen or PLN cells were cul served following SRBC challenge was cor tured in V-bottom microwells (Linbro 96- i rected for-swelling produced by medium MVC-TC) in 0.2 ml culture medium with 2 1 alone. Ill a second method, TCDD treated x 10s irradiated Fj spleen and 2 x 10s RNC * or control mice were sensitized by paint nude spleen cells. Here, the nude spleen j ing both ears with 3% oxazalone (4-eth- cells provide " help" which allows the de- * oxymethylene-2-phenyl, BDH Chemicals, velopment of CTL from single precursor i Poole, England) dissolved in 95% ethanol. cells (20). After 6 days incubation at 37`, i Nine to sixteen days later, sensitized and 2000 51Cr-labeled P-815 target cells were | unsensitized control animals were chal added to each well and the amount of lysis lenged with Wc oxazalone in olive oil (18). was determined after 5 hr. Those wells Ear thickness was measured prior to chal showing release exceeding the 95% confi- lenge and at 24 hr using a constant-pressure dence interval of spontaneous release from micrometer. negative control wells containing F, stim- 1 Generation and assay o f cytotoxic T ulators and nude spleen alone were scored cells. CTL were generated against the H-2d positive. Under limiting dilution condi alloantigen in vivo and in vitro. The in vivo tions. the number of lymphoid cells yield- 4 response to TCDD-treated mice was de ing 37% negative wells defines the cell termined following injection of 2 x 107 number containing; on the average, 1 CTLp P-815 tumor cells (H-2d) that had been according to the Poisson distribution: P(0) i treated with 5000 rad 60Co to prevent prolif = e , where P(0) represents the fraction of eration. Spleens were removed from the re negative wells and u the mean frequency of cipients 3 - 6 days later and the CTL activ CTLp per well. The value of u was calcu ity was assayed in a 6-hr MCr-release assay lated using a computer program derived against 51Cr-labeled P-815 targets as de from Porter et at. (21). The value of u al- \ scribed in detail elsewhere (19). The titra lowed an estimate o f the number of CTLp ' tion curve (percentage specific 51Cr release present in the tube MLC cultures and thus, vs concentration of sensitized spleen cells) a determination of the yield of CTL per was analyzed by computer for the parame CTLp (Nat x 103 per l CTLp). ter Mat which is proportional to the number R esults. Effect o f different doses of j of CTL lytic units in the cell suspension. TCDD on cellularity o f lymphoid organs. ^ 693 Nat x 103 units is equivalent to 1 lytic Figure 1 shows the effects of different doses * unit (the number of cells required to pro of TCDD on the cellularity of different or- j duce 50% lysis in the absence of interfering gans of the mouse immune system. The re- | bystanders (19)). ported LD5) in mice for TCDD is approxi* 1 CTL were generated in vitro in two dif mately 100 /xg/kg (8). although the time of ferent ways. First, mixed lymphocyte cul death is quite variable and often delayed. ture (MLC) was performed in 17 x 100-mm When a total dose exceeding 40 /ig/'kg was ** polystyrene tubes (Falcon Plastics, No. administered, cellularity of the spleen. j 2057) in 3 ml culture medium. Briefly. 1 -3 thymus, and peripheral lymph nodes was : x 10': spleen or PLN cells from the markedly reduced and the animals ap C57BI-M mice were mixed with 10s C3D2F, peared ill. In contrast, doses of 0 .4 -4 0 ug oi !if>D2F, >pleen cells that had been inac- did not reduce cell numbers in the spleen .'aieu with 1500 rad. CTL content of the and peripheral lymph nodes and the .iniinnb culture was determined after a 5-Jays incu- appeared healthy. N e\ ertheless. cellular F ig. 1. Effect of TCDD on c mouse lymphoid organs. Data are SEM for spleen (A), thymus (B), ai nodes (C) studies in four to six i ments. 4 jig/kg represe'nts a dose parts perbillion. In three experimei after the last dose of TCDD, the 1.2-bearing cells was determ i complement-dependent lysis (two immunofluorescent staining (one t scribed under Materials and Meihc control mice contained 70.4-74' cells whereas PLN from mice give was 4 3 - 4 6 ^ . from mice givei 43-609r. and from mice given 40-55rr. The dotted line in C sh number of thy 1.2-positive cells in depletion was already app. l-tg/kg dose when the thym ined. Since these data sug| thymus was particularly se effects of TCDD compared phoid organs, we tested \v uected the peripheral T-cell determining the proporrio celK in PLN mj-.| 1 c.UvU .>nd control . P-DIOXIN IMMUNOSUPPRESSION BV 2.3.7.8-TETRACH LORODI BENZO-^-DIOXIN 293 F-815 target cells, fc s, thymocytes from I ;e were added to the [ frequency of precur- i was measured using f icroculiure method of iy, small numbers of ^LN cells were culcrowells (Ljnbro 96jlture medium with 2 een and 2 x 10s RNC ere, the nude spleen which allows the deom single precursor * ys incubation at 37, ` 15 target cells were d the amount of lysis r 5 hr. Those wells eding the 95% confitaneous release from t containing F, stimen alone were scored ting dilution condiiymphoid cells yieldells defines the cell the average, 1 CTLp 'On distribution: F(0) 'esents the fraction of le mean frequency of alue of u was calcu:er program derived ). The value of a al:he number of CTLp X cultures and thus, e yield of CTL per 1 CTLp). different doses of f lymphoid organs. cts of different doses iarity of different orlune system. The reDr TCDD is approxialthough the lime of : and often delayed, ceding 40 ug'kg was rity o f the spleen, il lymph nodes was id the animals apdoses of 0.4 - 40 m? m herN in the sp leen odes and ihe animals vcrihelcss. cellular experiments showed that the percentage thy 1.2-posilive cells decreased following TCDD treatment at all doses studied (see Fig. 1). The dotted line in Fig. 1C shows that the estim ated number o f thy 1.2- positive cells in mouse lymph nodes also decreased after TCDD treatment. A de crease in the absolute number of thy 1.2- positive cells was also seen in spleen in one experiment where this organ was studied. Effect o f TCDD on immune responses. Figure 2A shows a representative experi- ment illustrating the effect of TCDD on the humoral immune response to SRBC. The number of background PFC in the spleens of unimmunized mice was consistently increased by TCDD treatment and the greatest increase was observed at 4 tg/kg dose (Fig. 2, Day 0). Following immuniza tion with SRBC, PFC number increased in control (corn-oil-injected) mice reaching a peak on Day 5. TCDD at a dose of 40 /ig/kg of TCDD produced a marked suppression of the PFC response. This suppression was reproducible and all three isotypes (IgG, IgM, IgA) were affected (data not shown). The result shown in Fig. 2A could have re Fia. I. Effect of TCDD on cellular content of mouse lymphoid organs. Data are given as mean = 1 SK'! for spleen (A;. ihvnutN 1B1. and peripheral lymph nodes (Cl siudies in four to six independent experi ments. 4 represents a dose of approximately 4 pans per billion. In three experiments done 1--6 weeks after the last dose of TCDD. the proportion of thv 1.2-bearine cells was determ ined in PLN by complement-dependent lysis (two experiments! or by sulted from an effect of TCDD on the Tdependent component of the anti-SRBC re sponse (22). Therefore, the response to a relatively T -independent antigen (23)., TNP-B. abortus was tested. Similar to the result obtained using SRBC as antigens (Fig. 2A), Fig. 2B shows that the 0.4 and 4 P-g/kg doses of TCDD had little effect on the immunofluorcsceni staining (one experiment) as de anti-TNP-BA response and the 40 Mg/kg scribed under Materials and Methods. The PLN from .dose suppressed the response. We con control mice contained 70 .4 -7 4 ^ ihy 1.2-posilive cells whereas PLN from mice given 0.4 ^g;kg TCDD as 43-46^7. from mice given 4 ig kg TCDD and from mice given 40 kg TCDD. 4H-55r. The dotted line in C shows the estimated number of thy 1.2-positive cells in lymph nodes. cluded that the dramatic suppression of the antibody response to SRBC by 40 /g/kg TCDD could not be explained by elimina tion of T helper cells. The effect of various doses of TCDD on the delayed hypersensitivity response is shown in Table I. Footpad swelling elicited depletion was already apparent at the 4 by SRBC was only slightly reduced by the Mg'kg dose when the thymus was exam 40 /ig-kg dose of TCDD but this reduction ined. Since these data suggested that the was not statistically significant. The ear thymus was particularly sensitive to the swelling response to oxazalone appeared to cifecis of TCDD compared to other lym be more sensitive. It can be seen that the 4 phoid 'organs, we tested whether TCDD and 40 Mg'kg doses impaired the ear swell fleeted the peripheral T-cell population, by ing response whereas little effect occurred determining ihe proportion of thy 1.2- at the 0.4 |ig-kg dose. posnive cells in PLN suspensions from The effect of TCDD on the generation of TCDD- treated and control animals. Three CTL following alloantigcn challenge//! vivo 294 IMMUNOSUPPRESSION BY 2.3.7.8-TETRACHLORODIBENZO^j-DIOXIN IMMUNOSUP1 is shown in Fig. 3. Even the 0.4 /i.g/kg dose of TCDD impaired the ability to generate a maximal response on Days 6 and 7. We concluded that the CTL response was par ticularly sensitive to functional impairment by TCDD. We therefore decIi.ded to direct our subsequent investigations toward elucidation of the mechanismjof impairment of CTL generation by TCDD[. In vitro analysis of effect of TCDD on TABLE I. E ffect of TCDD T reatment on Delated H ypersensitivity Reactions Treatment given Increment in footpad swelling 24 hr after oxazaJonc challenge" (x 10"s mAf. mean = SEMI Increment in ear thickness 24 hr after oxazalone challenge* (x 10-1 mM. mean =! SEMl Immune recipient Control recipient Control (com oil) 0.4 ig TCDD 4 UK TCDD in ug TCDD 23.7 = 5.2 (10) 24.5 = 5 .t (10) 34.5 = 4,1 (10) ` )4.7 = 4.y [ 10) 10.9 * 1.9 (8) 16.8 = 4.1 (8) 6.8 r 1.4 t8T 7.4 - 0.7 (8)' 0.j|7 = 0.87 -0.83 = 1.35 -0.^3 = 1.35 0 r I 29 :r; e w e rv ^crtMin'.evi b;> in tra v e n o u s n je e tio n W 1 *-2 IO"' S R B C j i i J t o t e d 5 0. 2 - lir NKBC min the left hind footpad A -mutar v.dimu- of valine v u \ tmeeted mio the fo; :he non-pv-eilie elfeet" of ihe fhuj mieetioti Parcnthc-v- uivlov.- the number . I'li- ->,i- .*u'-iti/ed lo t'\.i.\tlone in ethanol and ear -w filing m -en-iii.vd and n :- . r *v . ; t t 'f v.t. .... . od MeiKli.antiv lonct than conilo! h> -ludeni - : and M.mn Whmiu\ / Is n .-P- il it' h ime hi hi rid loot;' le.t- U-UK'lV DAYS AFTER IMMU Fig. 3. Effect of TCDD treaim of CTL in tYo following alloantigi mice ( ) or mice given 0.4 (s). 4 were immunized with irradiate tumor ceils as described under M; CTL activity was measured in sp from croups of three to four mi* caieJ. CTL generation. The supp: TCDD treatment on the ge: was analyzed in vitro using tl that have been employed the mechanism of immunpregnant mice (24). Experi II shows that TCDD treatm ability o f spleen and P TCDD-treated mice to against the H-2'1alloantiger cultures. The frequency < ever, was normal or sligh TCDD-treated animals as limiting dilution microcultu vation indicated that TCDI direct toxic effect on CTLj with this conclusion was crease in CTLp frequent spleens of animals given a 4TCDD that produced gros; Lon in thiv organ. It can als* ,rom the experim ent th: v'MOtoxic activity per unit t-'Mj.iiJy 'mbnofmat when froTn T C D D H e a t e d . .m m . a I-vfe; i.tm. ,d 2 Pilous th.u < alleete.! b> dove's of I 'Of- DIOXIN 9) or mice treated u ith a nation with SRBC (At or ,,r of PFC was measured jinber - SLM ib shown. fore decided to direct ves ligations toward :hanism of impairment ' TCDD. f effect o f TCDD on VITV RtACTJOvs in ear thickness azalonc challenge'' mean - SEM) Control recipient 0.17 = 0.87 -OS? c 1.35 -0.8? z 1.35 0 z 1.29 t 5 da> > l a i d H inieonon the 11eht hniil liH'ip.n! io it'd ui m easuic merits, ind nonicnMtircd .mimah 0,05 IMMUNOSUPPRESSION 8V :.J.7.8-TETRACHL0R0DIBENZ0r D10XIN 0.004 fjLgikg. Furthermore, mixing cells from TCDD-treated mice with those of normal mice appeared to suppress the CTL response of the untreated cells. Demonstration o f suppressor cells in the thymus of TCDD-treated mice. The above observations suggest that TCDD may en hance suppressor cell activity. We exam ined this possibility by adding thymocytes from TCDD-treated or control mice to MLC cultures containing normal PLN cells and quantitated the number of CTL gener ated /Vi vitro. The effect of different num bers of thymocytes on the generation of CTL from normal PLN is shown in Fig. 4. Dramatic suppression of CTL generation was observed when 4 - 8 x 106 thymocytes OAYS AFTER IMMUNIZATION from mice given 0.4 pgfkg and 0.04 pgfkg Fig. 3. Effect of TCDD treatment on the generation of CTL in fii'o following alloanligen challenge. Control mice ( ) or mice given 0.4 (A). 4 (O), or 40 () kg were immunized with irradiated allogeneic P-815 tumor cells as described under Materials and Methods. CTL activity was measured in spleen cell suspensions from groups of three to four mice at the times indi cated. TCDD were tested. Less suppression was seen when thymocytes from mice given 0.004 /xg/kg were used. Direct addition of TCDD up to doses of 0.025 pg to the cul tures produced no suppression (data not shown). If the entire dose of TCDD given to a mouse was concentrated in the thymus (containing on the average 8 x I0T) cells thenj 4 x 106 thymocytes from a mouse CTL generation. The suppressive effect cf given 0.4 figikg would add 0.00050 p g TCDD treatment on the generation of CTL TCDD to the culture. These data suggest was anaJyzed in vitro using the same methods that the suppression of CTL peneraiinn jp that have been employed to characterize ^TCDD-treated mice represented suppressor the mechanism of immunosuppression in c?H activity and was not a direct toxic ef pregnant mice (24). Experiment 1 in Table fect of ~JCD Lb II shows that TCDD treatment impaired the ^ Discussion. The data in this paper show ability of spleen and PLN cells from that the type of immunotoxicity produced TCDD-treated m ice to generate CTL by chronic exposure to TCDD depends on against the H-2rt alloanligen in vitro in tube the dose. In agreement with the results of cultures. The frequency of CTLp. how Faith et ui. (25). we found reduced delayed ever. was normal or slightly increased in hypersensitivity reactions as assessed by TCDD-treated animals as determined by ear swelling at a 4 zg/kg dose of TCDD that limiting dilution microcullures. This obser had little effect on the antibody response. vation indicated that TCDD did not have a The most interesting finding, however, was direct toxic effect on CTLp. In agreement the exquisite sensitivity of the CTL re-^ with this conclusion was the marked in snon^e to TCDD. CTL generation was im crease in CTLp frequency seen in the paired in viva at the 0.4 tg kg TCDD dose spleens of animals given a 400 Aic;kg dose of which produced no th>mic atrophy or de rC.DD that produced gross cellular deple tectable impairment of antibody or delayed tion in ihis orghn. It can also be appreciaied hypersensitivity responses. Impairment of hum the experim ent that the yield of CTL generation could be demonstrated in c\W'io \ie activity per unit precup-O! wa- i i:r, > with TCDD d o,W' low as 0.004 usually subnormal when Iwnphoid cell- kg when washed spleen or lymph node from TCDD-treated an im als were studied cells from TCDD-treated animals were Experiment 2 shows that CTL generation stimulated in tube cultures with alloanligen. was affected bv doses of TCDD as low as Some insight into the cellular basis for Q. -J --C c c r^, C--Nr* O'T r-i "3S* n *s&v"! o 5 rnI^ftl iwr-t- f(onN*J ri*i~o rUy, >Ino--T,.N"m----m>-"-o---ln--^-f\OO' If?e--t56rNyN---s(^~S.5N--'J1 wI yC$ z --;--J --,,gr_s 3--'*TT~----rr----Si*r3----* "--5Zj "uu5,_-x_ I1: u2>. e--- f *r>q-c-i vrVOJ.Irr--C*J. =^5- vm,3r*- --S--8--c--o =; f'' "1 i. .. 1| * * - *> tC--ttn sxT--j-i 'mri-J'h-r--vv~,C<Or-**< 'Crr--i.sS--e'C----2 uy o2 vX zE3 Pfc ----^ <N(N M N (N -s3/rZb---oC 3U 3 so /, --3 *sC3 03 ^--s -s- -E>3> u c ^ y*, Uj -= ^ . -- j i "sG_ - 2- 2~Sfi "CU5J o y -3= sa-e=c 00 'Z u UE*. MEl t 2*10" 101 NUMBER OF THYM ADDED PER CUL Fie. 4. Thymocytes from TCDl press CTL generation in vitro. Thi numbers of thymocytes from contr mice given 0.004 (V). 0.04 kg (Ol. i on the generation of CTL from 1 x shown. Five to six thymus dont group. } this impairment was obtaine the frequency of CTLp in 1 microcultures. It Was foun quency of CTLp was not re ment with TCDD. Thus, the generation in vivo and in tub< represented a defect in th of CTLp into CTL. Thi i suggested that TCDD tre; those cell types which regul lion of CTL in tube MLC i example, TCDD could eitl I amount of " help" available | ceils and accessory cells, o i pressor T-cell activity that c under in vitro conditions allc cell-cell interaction (24). >n thy 1.2-positive cells i TCDD-treated mice (Fig. 1 ^`ith a reduction in T helj lions. On the other hand, si } lions have suggested that ^ j mu\ he generated. Figure ^ t-'**e:,,:Mn ol L 1' !: m \ n ' ' Unr.-u A 'i h that "ee.Jed v ltv n\ ,i*e f heipe " " : ' h i ) :u\t L`j ri> limes but seemed to s 1 i he lost Ivmph mule nr spleen cells from Ihc ircalcd mice were culimctl with n radialcU L, Minuilaior colls m ivu^e iuno cim uks .mu im. i . .v, MC ;tciivj(y Was measured after 5 days incubation. The number ul milial CTL.p per culture was calculated by multiplying cell number by the Cl Lp 01 OX I S I M M U N O S U P P R E S S IO N BY 2.3.7.K-TL l`RA C'H LORO OIBENZO-/-DIOX IN - 600 response continued. Secondly, mixing lymph node cells from TCDD-treated o 500 animals with those from normal mice pro duced suppression (experiment 2, Table II). 400 - Thirdly, thymocytes obtained from mice treated with low doses of TCDD (0.004-0.4 fj.g per kg) suppressed the generation of CTL/n vitro. This suppression could not be explained by the amount of TCDD that 200 * would be present in the thymocytes. Addi tional experiments have shown that the presence of thymic suppressor activity par allels the impairment of CTL generation in 2*10* 4*10' 8*10* PLN and that both alterations ultimately NUMBER OF THYMOCYTES ADDED PER CULTURE F ju . 4. Thymocytes from TCDD-treaied mice s u p p re ss CTL generation in vitro. The efTcct of different numbers of thymocytes from control mice ( ) or from disappear as the dose of TCDD is progres sively reduced to 0.00004 ig/kg. ((31), manuscript in preparation). Taken together, these data indicate that TCDD mav imnair mice given 0.004 (V), 0.04 kg (O). or 0.4 (A) fig per kg The generation of CTJ- rhrnughrhe genera on the generation of CTL from 2 x 10* normal PLN is t io n oi suppressor T cells. shown. Five to six thymus donors were used p e r 7 ne mecnanism by which TCDD alters group. the thymocyte population to produce sup pression is unclear. It is unlikely that the effects of low dose TCDD on thymus can be this impairment was obtained by measuring attributed to malnutrition or to an increase in the frequency of CTLp in limiting dilution cortisone levels in the body. Malnutrition microcultures. It was found that the fre tends to deplete cells in the murine spleen and quency of CTLp was not reduced by treat thymus and to increase the cellular and hu ment with TCDD. Thus, the impaired CTL moral immune response (28). Our mice showed generation in vivo and in tube MLC cultures normal antibody responses and DTH but represented a defect in the development impaired CTL generation at TCDD doses of CTLp into CTL. This observation that caused neither lymphoid organ atrophy suggested that TCDD treatment affects (Fig. 1) nor significant weight loss (data not those cell types which regulate the genera shown). Serum cortisol levels in TCDD- tion of CTL in tube MLC cultures. For treated animals have been found to be nor example, TCDD could either reduce the mal by Vos et a!. (10). TCDD is known to amount of "help" available from T helper interact with a cytoplasmic binding protein celK and accessory cells, or increase sup or "receptor" and this "receptor" is pres pressor T-cell activity that can be detected ent in the thymus as well as liver (29, 30). underm vitro conditions allowing sufficient Indeed, in the rat. the highest concentration cell-cell interaction (24). The reduction of " receptor" is found in thymus (30). in thy 1.2-positive cells in the PLN of Thus, it is possible that TCDD mav act_di- TCDD-treated mice (Fig. 1) is consistent rectlv on thymus to alter T-cell differentia w'ith a reduction in T helper subpopula-" tion or perhaps the life span of certain T- tions. On the other hand, several observa cell subpopulations thereby promoting the tions have suggested that suppressor cells ' generation of suppressor T cells. Alter n m he generated. Figure 3 shows that the n a te ly . the alteration in thymus could be generation of CTL in viv,> io allogeneic m e d ia t e d indirectly through effects of tumor cells that lack la antigen*' T C D D on h e p a tic e n z y m e a c tiv ity (5, 311. needed to activate T be 1pci cell*' Co. 2~' it ca n he se e n fro m T a b le 1 th at th e re w as normal in TCDD-tre.ited animals at a slig h t 'b u t not s ta tistic a lly sig n ific a n t! in etirK times hut seemed to " shut off" as the c r e a s e in o \ a 7 a lo n c - in d n c e d e a r sw ellin g RWIWOBL'IM 298 IM M U N O S U P P R E S S IO N B Y 2 .3 .7 .8 -T E T R A C H L O R O D IB E N Z O -p -D IO X JN JM M UNOSUPPRE after 0.4 ig/kg TCDD and a similar effect on footpad swelling after 4 fig/kg TCDD. The stimulatory effect of 0.4 and 4 fig/kg TCDD on the background level of PFC has already been noted. We also found that these doses of TCDD increased the number of spleen colony-forming cells in bone mar row by 2 5 -3 9 ^ (data not shown) and thus it is possible that the 0.4 and 4 /xg/kg doses of TCDD. have a small nonspecific stimu latory effect on a variety of different cell types in addition to suppressor cells that influence the immune system. Our data showing that low doses of TCDD in mice impairs the generation of CTL may have some relevance to the ef fects of exposure to other HAHs. We have recently found that PCBs also impair CTL generation in mice ((31), manuscript prepa ration). Bekesi et al. (32) have reported that Michigan residents exposed to PBBs in their diet show a reduced number of T cells in blood and impaired mitogen and MLC responses in vitro. The effects of TCDD on the murine immune system may therefore have some generality. The biologic significance o f the immunotoxicity detected in vitro following exposure to low doses of TCDD and HAHs is not yet known. Our data show that a suf ficiently high dose of TCDD produces im pairment of the antibody response to SRBC in mice. Cattle heavily exposed to PBBs develop mastitis and furunculosis due to bacterial infections (33), and TCDD-treated mice are reported to be more susceptible to Salmonella infection (14). However, we and others (25) find that lower doses of 1CDD imnair primarily the cellular arm of the immune response, and as shown in this paper, the generation of CTL appears to be particularly sensitive. One would predict from these observations that low doses of TCDD and related HAHs should increase susceptibility to only certain types of infec tious agents. For example, CTL appear to play an important role in the defense .ig;tin\i certain i>pes of virus infection (34, 35) Susceptibility to duck hepatitis virus 15' .;:k! vu-kCep; i'nii it > tit herpe'A i; Infec tion in mice i 3m incre.^ed by PCB expo sure l^xpc rime ills arc currently in proere^ in our laboratory to determine if the increased thymic suppressor cell activity produced by very low TCDD doses in mice confers an increased susceptibility to lethal viral infections. We thank Ann Robson. Angela Mulvihill, Dalgeet Banwat. Fred Krestynsk, Dr. Mark McDermott, anj Dr. David Basford for expert technical assistance and Dr. W. E. Rawls for helpful comments during prepa ration of the manuscript. We thank Roberta Axler for typing the manuscript and Rita Campbell for preparing the figures. Grant support from the Ontario Ministries of Environment and Labour is acknowledged. David A. Clark and Myron R. Szewczuk hold scholarships from the Medical Research Council of Canada. 1. Kimbrough. R. 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