Document RpoNG15G3JkGo1KxzBBb5R9RB

INVESTIGATIONS ON THE EFFECTS OF 2,3,7,8-TETRACHLORODIBENZO-/>-DIOXIN (TCDD) ON PARAMETERS OF VARIOUS IMMUNE FUNCTIONS Robert E. Faith' and Michael I. Luster Environmental Biology and Chemistry Branch National Institute o f Environmental Health Sciences Research Triangle Park. North Carolina 27709 * Introduction As interest in the biologic effects of environmental pollutants grows it becomes increasingly evident that a number of these chemicals can cause alterations in the immune function o r an exposed subject. Of the chemicals of environmental concern that have been shown to induce immune alterations 2,3,7,8-teirachlorodibenzo/7-dioxin (TCDD) has been investigated most extensively. TCDD is a chlorinated hydrocarbon found to be a contaminant in biocides (trichlorophenol). herbicides (2.4,5-TJ, and products from chemical sensitizing processes in which polychlorinated phenols are used.1This compound has previously been shown to induce atrophy of thymus and peripheral lymph nodes of rats. mice, and guinea pigs.:"* in addition, TCDD has been shown to suppress thymic-dependent immune function*"6 and resistance to bacterial infection.7 The suppression of cellmediated immune functions includes suppression of mitogen responsiveness.'1"6 suppression of skin graft rejection5 and suppression of delayed hypersensitivity responses.6' Suppression of these T-ccll-deoendent immune functions appears to -..cur without helper cell function being affected." Effect of Developmental TCDD Exposure on Imm une F unction in F ischer/ W istar R ats Figure I. Effect of developmental TCDD expo weights. . female animals exposed to TCDD postna 0. 1 and U ; , male animals exposed to TCDD po* female animals exposed to TCDD pre- and postn: gestational day 18 and on days 0. 7 and 14; and a . m; dosing as above. Relative thymus and spleen weights Previous studies utilizing rats to investigate the effects of developmental TCDD exposure on immune functions have utilized Fischer rats.561 Recently it was reported that the Fischer rat is a poor immunologic responder.9 In order to investigate the effects of developmental TCDD exposure on a strain of rat with a good immunologic response, experiments performed previously in Fischer rats6 were repealed in Fischer/Wistar rats.9 To accomplish this, female Fischer rats bred to male Wistar rats were randomly distributed into three dosage groups. (1) The control group was dosed with corn oil (0.1 m l/100 g body weight) on gestational day 18. At parturition, the neonates were cross-fostered among these females; all litters were standardized at eight pups per litter. Nursing rats were also dosed with corn oil (0.1 m l/100 g body weight) on postnatal days 0, 7 and 14. (2) Females in the postnatal group were allowed to litter, and neonates were cross-fostered among these females; litters were standardized at ` Present Address: Biomedical Research Center. Oral Roberts University. Tulsa. Oklahoma 74171. ' Age in Da vs body wt. I rcl. ihv. wt rcl. \plcen M 35 ) bodv wt rcl. thv. wt rel. spleen wt. i:s body wi. rcl. ihv. wt. rel. spleen wt, 270 body wt. rcl. ihv. wt. rel spleen " i Significant Variations f p < 0.05 All N S .` a a All N.S.* All N.S.' All N .S.1 All N.S.* nn". 5M y -= , s ', ,\s / N S ."CiC.ilev r..insit!n;!!:vjni differences from vni I EFFECTS OF -/-DIOXIN (TCDD) TMUNE FUNCTIONS .1 1. Luster mis try Branch ! Health Sciences Carolina 27709 :nial pollutants grows it becomes cals can cause alterations in the :micals of environmental concern lions 2,3,7,8-tctrachlorodibenzosively. o be a contaminant in biocides iucts from chemical sensitizing j.1This compound has previously ral lymph nodes of rats, mice, and n to suppress thymic-dependent cction.' The suppression of celln of mitogen responsiveness/"* sion of delayed hypersensitivity Timune functions appears to occur 1 j | t- xposure on Immune tar R ats ; effects of developmental TCDD * rats/''* Recently it was reported odcr/ In order to investigate the n of rat with a good immunologic Fischer rjts f were repeated in male W istar rats w-cre randomly 'ol group W2S dosed with corn oil At parturition, the neonates were e standardized at eight pups per (0 1 m l / 100 g body weight) on mial group were allowed to litter, .ties: litters were standardized at Kubcrts L'mvcrsitv, Tulsa. Oklahoma c l*J?o NVaS Daft ol 9 Figure 1. Effect of developmental TCDD exposure on body weight and selected organ ights. . female animals exposed to TCDD postnatally via maternal dosing (5 jig/kg) on day 7 and 14: . male animals exposed to TCDD posin3tally via maternal dosing as above; A. male animals exposed to TCDD pre- and postnatally via maternal dosing <5 on siarional day 18 and on days 0. 7 and 14; and A . male animals exposed to TCDD via maternal ..ino above. Relative thymus and spleen weights represent organ*to-bodv-eight ratios. Acc in Days 18 bods I. rd lhy 1. rd. spleen wt 35 body wi. rcl. thy. w*t. rd. spleen w-i. 12 tvxU sit rcl. lh) wi rd spleen w[ :?o NJ\ wt rd thj wi rd spleen ! Significant Variations from Control p < 0-05 C All N.S.* C A AU N S * All N S.* All N S.* Ail N S * *N S indicates n.tns^Mirtic.int d erenccs irniii COtiH>l p < 0.01 J. . A. A . A. A . A. A . A. A . A. A m fc r il 566 Annals New York Academy of Sciences eight pups per litter. Nursing rats were dosed with 5 jig/kg TCDD (Dow Chemical Co., Midland, Michigan, lot 851 - 144* 11, more than 99% pure) on postnatal days 0. 7, and 14. (3) Females in the prenatal-postnatal group were dosed with 5 jig/kg TCDD on gestational day 18. At parturition the neonates were cross-fostered to untreated females and the litters were standardized at eight pups per litter. The nursing females received doses of TCDD (5 Mg/'kg) on postnatal days 0, 7. and 14. All rat pups were weaned at 25 days of age. These animals were utilized to investigate the effects of TCDD exposure on various parameters of immune function. F igure I illustrates the cirect of TCDD exposure on body weight, relative thymus weight, and relative spleen weight. Developmental TCDD exposure resulted in depressed body and relative thymus weights in males and females of both treatment groups at 18 and 35 days of age. By 128 days of age body weights and relative thymus weights were still slightly suppressed in the prenatally treated males whereas values were normal or above normal for the other treated animals. This is similar to results previously obtained in Fischer rats.6 At 35 days of age the relative spleen weights in both groups of TCDD-exposed animals were greater than those of nonexposed animals. This ratio reflects the change in body weight, as actual spleen weights did not vary significantly between the groups. in vitro mitogen stimulation of lymphocyte cultures was performed using spleen and thymus cells from TCDD-exposed and nonexposed animals as previously described.6 Results of mitogen stimulation were evaluated by calculating stimulation indices (counts per minute incorporated into stimulated cultures divided by counts per minute incorporated into nonslimulated cultures) and relating the indices of stimula tion of cultures from TCDD-exposed animals to the indices of stimulation or cultures from nonexposed animals. Figure 2 illustrates the effect of developmental TCDD exposure on mitogen responsiveness of splenic and ihymic lymphocytes. Prenatal and postnatal and postnatal only exposure to TCDD suppressed the response of splenic and thymic lymphocytes to stimulation by the mitogens phytohemagglulinin (PHA) and Concanavalin A (Con A). The response of splenic lymphocytes had returned to normal by 270 days of age. Thymic cultures were not .performed at 270 days of age because of extreme thymic involution at this time. There was a trend towards recovery of mitogen responsiveness in thymic lymphocytes over the time course studied. Delayed hypersensitivity reactions (DHR) were utilized as an in vivo assessment of cell-mediated immune function. The DHRs were performed according to the radiometric assay method of LelTord,10 The effect of developmental TCDD exposure On delayed hypersensitivity responsiveness is illustrated in F igu re 3. TCDD exposure resulted in a suppression of delayed hypersensitivity responses at 35 and 133 days of age. By 270 days of age the response of exposed animals had returned to normal. The final assay of immune function investigated in the Fischcr/W istar animals was antibody production. Animals were immunized with primary and secondary injections of bovine gamma globulin (BGG) in complete Freund's adjuvant at various aces. Serum was obtained from the immunized animals at approximately weekly intervals for 5 weeks and antibodies to BGG assayed lor by passive hemagglu Inatron reactions. F iglrf 4 illustrates the antibody responses obtained in these animals. Developmental TCDD exposure had no apparent effect on antibody responses. These results indicate that at least one T-cell function, that of helper cell function, is spared from TCDD suppression, as BGG is a T-depcndent antigen. A lterations in Lvmphocyte H oming Patterns as a R esult of Developmental TCDD Exposure Inbred Fischer rats were exposed to TCDD .ts described lor the p:cn.iiai-po>tnatal e x p lo re group described above. At -15 days of age these animals' were uiib/ed to Faith & Luster: Effects of TCDt Day Fic.lrf 2. Effect of developmental TCDD exp mitflier.' I eaer.J .is in Fn.LKL : I Age in Da vs 25 Spleen PHA Con A ' Thymus PHA Con A 35 Spleen PHA 1 Con A Thymus PHA Con A Significant Variatio .I Spleen PH \ Con A Thymus PHA Con A 12S Spleen PHA Con A Thymus PHA Con A 270 Spleen PHA Con A All N. All N. ani difference fr.iin ,  of Sciences i 5 ^c/kc TCDD <D-m ChemieJ , 99% purci on postnatal days 0. 7, t were dosed with 5 pg/kg TCDD . were cross-fostered to untreated jps per litter. The nursing females vs 0. 7. and J4. All rat pups were .[zed to investigate the effects of function. re on body weight, relative thymus tal TCDD exposure resulted in les and females of both treatment body weights and relative, thymus ally treated males whereas values animals. This is similar to results age the relative spleen yyeights m eater than those of nonexposed hi. as actual spleen weights did not lures was performed using spleen mexposed animals as previously aluated by calculating stimulation ;ted cultures divided by counts per nd relating ihe indices of stimular indices of stimulation of cultures c effect of developmental TCDD hymic lymphocytes. Prenatal and pressed the response of splenic and ^ phytohemaeelutinin (PHA) and mphocyics had returned lonurmal med at 270 days of age because of trend towards recovery of mitogen c course studied. : utilized as an in vivo assessment -ere performed according to the if developmental TCDD exposure tied in Figl RH 3. TCDD exposure y responses at 35 and 133 days of nimals had returned to normal, rd in the Fischcr/W istar animals cd with primary and secondary plete Treund s adjuvant at various nimals at approximately weekly d for by passive bemaggluiinairon .mses obtained in these animals, feet on antibody responses. These i of helper cell function, is spared I amicon, P a 3 7 |-`k \ S \S \ !>D Fv'USt RI - .i: ; he pren.il.iJ-poe,:',.!!.;. :VC'C .nim.iU w eic utilized L' Faith & Luster: ElVccts of TCDD on \ urious Immune [ unctions ?67 Fica k 2. Effect of developmental TCDD exposure on in vitro lymphocyte responsiveness to miloccn stimulation. Legend as in FiGt-RE 1. Ace in Dass Spleen PHA Con A Thvmus PHA Con A Significant Variations from Control P : 0,05 1_J p ^ 0.01 D. a. A u . - A . A. . A. A A. A Spleen .PHA Con A Thvmus PHA Con A 59 Spleen PHA Con A Thymus PHA Con A 128 Spleen PHA Con A Thymus PHA Con A :7u Spleen PHA Con A !" 1 .A All V S * All \ S * * \ S indicates ni'iistj'mttc.int dincren.:e fruiv. n>nuol . A. A 3 . A. A Q . * , A. A 22. . A. A . A, A . A . A . A . A. A .A. A -i $ jjg *,k' if hi a1; j iftc jiil!"A n ! i !. I i ' J: ; iI 1 i patterns of lym phocytes were p significantly increases the thym ic v injected into nonexposed recipient; decreased homing to spleen and recipients. A t the sam e lim e thymi* decreased homing to thym us in no W hile there is controversy ab* homing patterns o f lym phocytes TCD D -induced m odification of noi i Doys of Age Figure 3. Effect of developmental TCDD exposure on delayed hypersensitivity responsive ness. . animals exposed to TCDD postnatal!}' through maternal dosing (5 ^g/kg) on days 0. 7 and 14; and A . animals exposed to TCDD pre- and postnatally by maternal dosing (5 ng/kg} on gestational day 18 and on days 0, 7. and 14. AT 35 and 133 days of age both TCDD dosage groups responded at a level significantly lower than noncx posed animals (p < O.nil. At 370 d m ' of age there as r..> significant difference between responses of exposed and noncxposed animals. RasporiM of 270 Day 14- - o86- investigate the effects of T C D D exposure on lym phocyte homing patterns. Lympho cytes from spleen, thym us, bone m arrow and lymph node were isolated from TCDD exposed and nonexposed anim als, labeled with ^ C r .'1and injected in TCDD-exposed and nonexposed recepiem s. T C D D exposure allered th e hom ing o f exposed spleen cells to the thym us in nonexposed an im als. In a d d itio n , T C D D exposure altered the pattern of homing o f nonexposed thym us cells in exposed an im als and the p attern of homing of exposed thym us cells in nonexposed anim als. C onclusions The highly toxic ch lo rin ated hydrocarbon . T C D D ap p e a rs to be equally as im m unosuppressive n a stra in o f ra t th a t is a good im m unologic responder ( F isc h e r/W ista r) as it is in the F ischer r a t / "-* which is a poor im m unolgie responder.'1 The im m unosuppression induced by developm ental exposure to T C D D in the F isc h e r/W ista r rat is long tastin g , as has previously been shown to be tru e in the Fischer ra t.0 T he exposed an im als do recover from the induced im m unosuppression by 270 days of age. however. At present the m echanism of induction o f im m unosuppression by T C D D is unknown. The results of these experim ents indicate that w hatever the m echanism the effects are reversible. In an effort to und erstan d m ore fully the types of d e fe c t' in im m une function induced b> exposure to T C D D , studies on the effects of T C D D exposure .m homme FiGURE 4. Effect of developmental ' globulin. . nonexposed animals; anim doling (5g/kc) on days 0, 7, and 14; ar of maternal doling t^ug/kg) o ar.ificar.; dificiciiuia were observed r* 1CCS paiierns of lymphocytes were performed. t( was found that TCDD exposure igii'.icanth increases the thymic uptake of spleen cells from TCDD-cxposed animals injected into nonexposed recipients. Thymic cells from non exposed animals showed decreased homing to spleen and increased homing to thymus in TCDD exposed recipients. At the same time thymic lymphocytes from TCDD exposed donors showed decreased homing to thymus in non-exposed recipients. While there is controversy about the role of cell surface markers in control of homing patterns of lymphocytes,11 the data obtained here arc consistent with TCDD-induced modification of normal cell-surface markers, This modification could XI 2*0 260 290 hypersensitivity responsiveosing (5 **g/kg) on days 0, 7 naicrna! dosing (5 */g/kg) on <of age both TCDD dosage nals {p < 0.01). At 270 days af exposed and nonexposed oming patterns. Lympho-vere isolated from TCDD njcctcd in TCDD*exposed tine of exposed spleen cells xposure altered the pattern id the pattern of homing, of appears to be equally as d immunologic responder oor immunoJgic responder.'1 >po$urc io TCDD in the ;n shown to be true in the jeed immunosuppression bv osupprcss'mn b \ T C D D is h.uever the mechanism the iefeets in im mune function I CDD e\po"ure on homing Fir.i p.i 4 Effect of developmental TCDD exposure on antibody response to bovine gamma globulin , nonexposed animal*. animal* exposed 10 TCDD pnsmat ally by means of maternal dosing (Sug/kgl on days 0. 7. and (4. and annua!* exposed io TCDD pre- and postnatally by me,ns-of maternal dosing (.V p 'l e ) on goiaiionaJ day IS and on days (>. 7. and M No significant differences were ohsrned l'i",i'rn nonexposed and TCDD-cxposed animals 570 Annals New York Academy of Sciences Faith & Luster: Effects of TCDC SUMV. The effects of TCDD exposure on pat developmental period were investigated. Ex rats. Fetal and neonatal rats were expo. (5pg/Kg) on day 18 of gestation and on da -\not her group of neonatal rats were expos Ju\s 0. 7. and 14 of postnatal life only (gr. weights were found to be suppressed up to 1 days of age in group 2. Parameters of cell were investigated. TCDD suppressed cell-r nc hummoral immune function. TCDD cell-mediated immune function by 270 da; A group of inbred Fischer rats was ex above. At 45 days of age these animals wen was found that TCDD exposure alters horn animals when adoptively transferred to un from nonexposed animals did not home no recipients. Referi SnJ l_-*C 5C fC ctl Typ Tron)*rr*d FiGLRE 5. ElTeci of developmental TCDD exposure on lymphocyte homing patterns. The open bars represent cells from nonexposed animals injected into nonexposed animals, the shaded bars represent cells from nonexposed animals injected into TCDD-exposed animals, and the crosshatched bars represent cells from TCDD-exposed animals injected into nonexposed animals. Significant differences occurred in splenic uptake of thymus cells. Nonexposed cells homed to nonexposed spleen in greater numbers than to exposed spleen (p = 0.06) and in greater numbers than exposed cells homed to nonexposed spleen (p < 0.01 ). In addition, nonexposed cells homed to exposed spleen in greater numbers than exposed cells homed to nonexposed spleen (p = 0.054), TCDD-exposed bone marrow cells homed to nonexposed spleen in greater numbers than nonexposed bone marrow Cells migrated to nonexposed spleen (p < 0.01). TCDD-exposed spleen cells homed to nonexposed thymus in greater numbers than nonexposed spleen cells homed to nonexposed or exposed thymus (p < 0.01). TCDD-exposed thymus cells homed to nonexposed thymus in greater numbers :han nonexposed thymus cells homed to nonexposed thymus (.P 0,01 ). Nonexposed bone marrow cells home to nonexposed liver in greater numbers than exposed bone marrow ceils homed to nonexposed liver (p < 0.01 ). All other comparisons were nonsignificant. be accomplished by at least two mechanisms: (I) a TCDD-induced change in cellular metabolism resulting in an altered cell membrane; or (2) alteration of the cell membrane by insertion of TCDD into the membrane. In any case, TCDD exposure appears to alter homing markers in the spleen and thymus, as well as on the lymphocyte, as evidenced by alteration of homing patterns of normal thymic and splenic lymphocytes when injected into TCDD-exposed recipients * I. MOORF. J. A. 1973 Prespeclivc on chlorina Health Perspec:. 5: ! 2. G c rrx . B. N.. J.O . Nus. J. A. Moore. J. clfecis of 2.3.7.8-tctrachlorodibenzo-/j-d: Perspea. 5: 125-140. 3. H arris, M. w ,, j . a Moore, J. G. Vos &.! TCDD in laboratory animals. Environ. F 4. Vos. J. G.. J. A. Moore & J. G. Z ink ' /j-dioxin on the immune system of la' ; 5:149-162. 5. Vos. J. G. & J. A. Moore. 1974. Suppre: maternal treatment with 2,3,7,8-ietrachl * Immunol. 47: 777-794. : 6. Faith, R. E. & J. A. Moore. 1977. lmpari ; exposure of the developing immune : \ (TCDD). J. Toxicol. Environ. Health3:- 7. T higpev J. E.. R. E. Faith. E. E. Me , susceptibility to bacterial infection as a se- /3-dioxin. Infect. Immun. 12: 1319-1324 8. F ai th. R. E.. M. 1. Lister & J. A. Mo function and delayed hypersensitivity res 9. T aDa. N,, K. Itaki.-Rx &. M. A iZawa. 19 inbred rats. J. Imrm.nogcnel. 1: 265. i 10. Lffiord. J. J. 1974. The measurement ol Allcrgv Appl. Immunol. 47: 570-585. i II. Morse.' H. C.. Ill & Asofsky. R. 1974. * homing patterns andgraft-verus-host n j Immunol. 11: 19-29. j 12, S o n FSrvfiFR. M. 1976. Cell surface re- Cumm 5: Sciences Faith & Luster: tilled* ul' TCDD on Various Immune Functions 5"! f S ummary i i The effects of TCDD exposure on parameters of immune function during the developmental period were investigated. Exposures were performed in Fischer/ Wistar i* rals Fetal and neonatal rats were exposed to TCDD through maternal dosing ^ g /K g ) on day 18 of gestation and on days 0. 7, and 14 of postnatal life (group 1). Another group of neonatal rats were exposed to TCDD through maternal dosing on 'dai's 0, 7; and 14 of postnatal life only (group 2). Body weights and relative thymus ; weights were found to be suppressed up to 135 days of age in group 1 but only up to 35 I davs of age in group 2, Parameters of cell-mediated and humoral immune function ! were investigated. TCDD suppressed cell-mediated immune function without afiecti mg hummoral immune function. TCDD-exposed animals had recovered normal I ccll-mediated immune function by 270 days of age. A group of inbred Fischer rats was exposed to TCDD as described for group 1 = above. .At 45 days of age these animals were utilized in lymphocyte homing studies. It j was found that TCDD exposure alters homing patterns of lymphocytes from exposed | animals when adoptively transferred to untreated animals. In addition, lymphocytes from nonexposed animals did not home normally when injected into TCDD-exposed recipients. i R eferences 1. Moore. J. A. 1973 Prespeciive on chlorinated dibenzodioxins and dibenzofurans. Environ. Health Perspcct. 5: 1. 2. GcpTa. B. N.. J. G. Vos. J. A. MoORf, J. G. Z inkl & B. C. Bui.LOCK. 1973. Pathologic effects of 2.3.7.8-telrachlorodibcnzo-/i-dioxin in laboratory animals. Environ. Health mphocyie homing patterns. The ' nonexposed animals, the shaded "CDD-exposrd animals. and ihe njectcd into nonexposed animals, ells, Nonexposed cells homed to * 0.061 and in greater numbers addition, nunexposed cells homed led to nonexposed spleen (p *= d spleen in greater numbers than x < 0.01). TCDD-exposed spleen onexposed spleen ceils homed to ymus cells homed to nonexposed led 10 nonexposed thymus (p < in greater numbers than exposed I- All other comparisons were ! i j PrT'peei- 5: 125-140. 3. H xKki*. M. J. a Moori. J. G. Vo> i. B V G; r" 1973. General biological effects of TCDD in laboratory animals. Environ. Health Perspcct. 5: 101-109. 4. Vos. J. G.. J. A. Moore & J. G. Z i* kl. 1973. Effect of 2.3.7.8-ietrachlorodibenzo- /7-dioxin on the immune system of laboratory animals. Environ. Health Perspcct. 5:149-162. 5. Vo*. J. G. & J. A. Moore. 1974. Suppression of cellular immunity in rats and mice by maternal treatment with 2.3.7.8-tctrachlorodiben20-/>-dioxin. Ini, Arch. Allergy Appl. Immunol. 47: 777-794. 6. Faith, R. E. & J. A. MOORF.. 1977, lmparimcni of thymus-dependent immune function by exposure of the developing immune- system to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). J. Toxicol. Environ. Health 3:451-464. 7. Tmigriv J. E.. R. E. Faith. E. E McCo ^ nei.l & J. A. Moore. 1975. Increased susceptibility to bacterial infection as a sequela of exposure to 2.3.7.8-teir3chlorodibenzop-dioxin. Infect. Immun. 12: 1319-1324. 8. F aith. R. E.. M. I. Listi r & J. A. MooRI . 1978. Chemical separation of helper cell j function.and delayed hypersensitivity responses. Cell. Immunol. In press, j 9. T aDa. N., K. Itaklka & M. Aizawa. 1974, Genetic control of the antibody response in ! inbred rats. J. Immunogcnct. 1: 265. D-induccd change in cellular 10. Ll.riORO. J. J. 197-J, The measurement of tuberculin hypersensitivity in rats. Int. Arch. 'r \2) alteration uf the cell Allergy Appl Immunol 47: 570-585. n any ease. TCDD exposure 11. MokSJ. H. Ill A; Amiim. 1 . R. 197q. r, i/iv effects of 3niithymocyie scrum on the thymu*, as well a* on the terns of normal thymic and teopicms i | homing p.tiicms and graft-crus-bnxi reactivity of murine splenic lymphocytes. Cell, Immunol 11: 19-29 >2 Si m i m m i I r. M 197d C d! sitrf.tcc receptors and lymphocyte migration Immunol Comm 5 :7 7 5 -"**5 ' t ti til