Mercury-induced Renal Autoimmunity: Changesin RT6+ T-Lymphocytes of Susceptible andResistant Rats

1 2Linda L. Kosuda, Dale L. Greiner, and Pierluigi E. Bigazzil1Department of Pathology, University of Connecticut Health Center, Farmington, CT06030 USA; 2Department of Medicine, University of Massachusetts, Worcester, MA01655 USA.... ... ........................- -

.'.'.'.'.'.'.'. '!: ., ..a... ... . , ~

trMi g~I sponses and disease (1). However, stud-

Husually quite difficult because the absorp-presor.ce1kI,.'.'eidoyeR'nt4dnt. tion of low doses of xenobiotics may

thaotghdisease retrospective study

e.e:o.............

typic flmhoye sboi *' Therefore, investigations using experi-thespeewn p-od-o mental animals have provided

N g XLsyte mb sestevidenc l in sarato immune-r...f....T

t io d rse a ms to wa nstr a-

reicv (els of ache irnd ed haurt-i ut ye

eS-B---~~~~~~~~~sual qut diwsfficuts whecause 1otherstabsoarpl-~~~~~~~~~~in oflo doseso xenoitc ma

oX o c cupera ats,a aon me ic Ri e eated

eQeoanlesse saxpos ure bt ca e the a utoimmune

tit re sponsr es tore nal dtheinratio n angentsm ; y - - i -imercury havaanietrrand.had.CO.as~.Three ired bestrin ofn rat eBperio

Inresmdnglto icolohe Farexamp e, thwad m i istra

atsy are sul sin epti le dtesoff cts rc thu utr in mr

yin rb t ra t and m ice icaRpl a

Tosure to HgCl2pra re sautere-s

gee;' d'e 'en "l' '..d *m nth ti

eo sa BNje la

plaBanai ( 0) rlsa vusel blo do stes eo

effn cb ts wh ereat e: for aie am pe thes

st nt4i:: Th me s

hl bc ffd

XI. bytreatm~~~~Wen njete with HgCiou ane imals foroms

branorusy glomerusoneHrit)uisngcharacer-iedt byoprteinuriadmandstheprdutionofbcta

aeutoa ntib todie to raepitopestiof , nhe eala

ti nc -1ey diss o

derchas ha vesbelf-li ours e me htiv

~~~~~~~imilart thats ofd m exa p lerim hen

talmallergic enfcphaomyegls in

rats:tautoimmuel reach0 theiry

pe akhtapproxia te 2uc lweek tho e

beginin ngth ofm treat mx ent, cthe

regress spontaneously in spite of continu-

ous administration of HgCl2. BN rats

that have recovered from this glomeru-......................lonephritis are subsequently resistant to

additional treatment with mercury.

Recent studies from our laboratoryhave shown that treating BN rats withHgCl2 causes a decrease in peripheralRT6+ T-lymphocytes, a change that coin-cides with the appearance of circulatingautoantibodies to renal antigens (17). Wehave proposed that autoimmune kidneydisease induced by mercury in geneticallysusceptible rats may be a consequence ofinteractions between this metal and a sub-set of regulatory (RT6+?) T-cells. In con-trast, Lewis (LEW) rats are resistant to theautoimmune effects of mercury and report-edly develop a subpopulation of suppressorT-lymphocytes (18,19). Therefore, it wasof interest to compare the effects of mer-cury on lymphocyte subpopulations fromBN, LEW, and (BN x LEW) F1 rats. Thehybrids between the mercury-susceptibleBN and the mercury-resistant LEW strainare susceptible to the autoimmune effectsof HgCl2, a trait inherited in a dominantfashion (4). We show here that LEW ratsinjected with HgCI2 do not have signifi-cant decreases in RT6+ T-lymphocytes ordetectable autoimmune responses. In-stead, both BN and (BN x LEW) F1hybrid rats experience autoimmune re-sponses to renal antigens as well as similarchanges in RT6+ T-cells after mercurytreatment.

MethodsWe obtained 107 female rats [39 LEW, 36(BN), and 32 (BN x LEW) Fl hybrids,hereafter referred to as F, hybrids] from acommercial source (Harlan Sprague Daw-ley, Indianapolis, IN). All animals werehoused in plastic cages with wood shav-ings, in an automated light cycle environ-ment (12:12 hr) and received standard ratdiet and water ad libitum. Treatment of allrats followed the standard National Insti-tutes of Health guidelines: light ether anes-thesia was used for all procedures, includ-ing bleeding from the retro-orbital plexusor exsanguination followed by transectionof the thoracic aorta at the termination ofthe experiments.

Previous studies have shown that treat-ment with mercury in its various chemicalforms, using different routes of administra-tion (subcutaneous injection, oral inges-tion, inhalation), results in autoimmuneresponses to a variety of antigens (7-10).Relatively low doses of mercury have beenfound to be effective in both rats and mice;for example, the immunotoxic effects of

Address correspondence to P.E. Bigazzi. This workwas supported by U.S. Public Health Service grantsES03230 (to P.E.B.) and DK36024 (to D.L.G.).We are grateful to Marcia Nahounou, AnitaWayne, and Kathleen Fitzgerald for excellent tech-nical help. An abstract of these results was present-ed at a poster session of the 1993 Society ofToxicology meeting in New Orleans, Louisiana.

Environmental Health Perspectives178

If-

HgCI2 occur in BN rats at doses (3-25,g/100 g body weight) that are muchlower than those causing the commonnephrotoxic effects (11,12, Bigazzi et al.,unpublished data). However, in the exper-iments reported here, we used higher levelsof HgCl2 (100 ,ug/100 g body weight) toreplicate the doses used in previous studiesof BN and LEW rats (17,18,20).

BN, LEW, and F1 rats were randomlydivided into experimental and controlgroups and treated with mercury or water,respectively, as previously described (17).We injected 80 experimental rats (31LEW, 25 BN, and 24 F1 hybrids) subcuta-neously on the abdomen with 0.1 ml of amercuric chloride solution (HgCl2, 1mg/ml) per 100 g body weight. We dis-solved HgCl2 in distilled water and record-ed the pH before use (range 3.9-4.2). Ratswere injected under light ether anesthesia.We injected rats three times a week for 2weeks, for a total administration of 600 ,ugHgCI2/ 100 g body weight/rat. Four LEWrats died under ether anesthesia and one F1hybrid injected with mercury died on day12 of mercury treatment: these animals arenot included in the Results section. Weanesthetized and injected 27 control rats (8LEW, 11 BN, and 8 F1 hybrid of same ageand weight as those injected with mercury)with distilled water at pH 4.2 (acidified byadding 0.01 N HCI) following the sameregimen.

ELISA for circulating autoantibodies torenal GBM and anti-laminin antibodieswas performed as previously described(17). In brief, we incubated sera frommercury-treated and control rats in ELISAplates that had been coated previously withrat GBM or mouse laminin (500 ng/well).After incubation and washing, horseradishperoxidase-conjugated rabbit antibodies torat IgG Uackson ImmunoResearch Labor-atories, Inc., West Grove, PA) were used todetect the binding of rat anti-GBM toGBM (or anti-laminin to laminin). Weread plates using a Titertek ELISA Readerat OD414.

We determined rat IgG bound to renalGBM and tubular basement membrane(TBM) by direct immunofluorescence aspreviously described (17). In brief, cryo-stat sections of rat kidney were stainedwith fluoroscein isothiocyanate (FITC)-conjugated rabbit antibodies to rat IgG(Sigma Chemical Company, St. Louis,MO) diluted 1:20 in phosphate-bufferedsaline. We read the sections using a LeitzDialux fluorescence microscope equippedwith epi-illumination.

As previously described (17), spleen,lymph node, and thymus cell suspensionswere obtained by gently pressing the tissuesthrough a stainless-steel screen (50 mesh).The six proximal cervical lymph nodes

were recovered for total cell counts andsubset analysis in all experiments. Wewashed the cells in two changes of coldmedium (RPMI 1640) and determined cellviability by trypan blue exclusion. Cellsuspensions were labeled with anti-RT6.2or anti-RT6.1 rat monoclonal antibodies(mAb) and developed for immunofluores-cence with an F(ab')2 fragment of anFITC-conjugated goat anti-rat IgG (heavyand light chain specific; Caltag Laborator-ies, San Francisco, CA). Controls includedthe FITC conjugate alone and "irrelevant"primary rat mAb developed with the samesecondary reagent: values obtained fromthese controls represent surface Ig-positive(B) lymphocytes and were subtracted fromthe counts of RT6+ cells. When cells werelabeled with mouse mAb directed againstother cell surface markers (CD4, CD5,CD8, etc., see below), we developed themwith an F(ab')2 fragment of an FITC-con-jugated goat anti-mouse IgG antibody thathad been absorbed by affinity chromatog-raphy on a rat IgG column to removecross-reacting antibodies. Controls rou-tinely included the FITC conjugate aloneand irrelevant mouse mAb. Cell suspen-sions were fixed before flow cytometryusing saline-buffered 10% formalin andthen analyzed using a fluorescence-activat-ed cell sorter IV (Becton Dickinson,Mountain View, CA), according to relativelow-angle light scatter and relative fluores-cence intensity as described previously(21,22). We excluded dead cells and cont-aminating red blood cells from analysis byelectronic gating. At least 50,000 nucleatedcells were analyzed for relative fluorescenceintensity.

Hybridoma cell lines secreting mAb tothe RT6.1 (clone DS4.23) and RT6.2(clone 6A5) alloantigens are maintained inour laboratory (21,22). Clone DS4.23 wasdeveloped in the laboratory of Frank Fitchin collaboration with Lubaroff and Greiner(23). The 6A5 cell line was a gift of D.M.Lubaroff of the University of Iowa and wasoriginally developed and characterized byB. Carpenter of Harvard University (24,25). The RT6 alloantigenic system in therat is not linked to the MHC and consistsof two alloantigens, RT6.1 (expressed inrats of the Lewis and PVG strains) andRT6.2 (expressed in rats of the BrownNorway and Wistar Furth strains) (17)'The RT6 antigen is expressed only by T-lymphocytes and approximately 45% ofCD4+ and 70% of CD8+ peripheral T-cellsubsets. No RT6+ thymocytes, bone mar-row lymphocytes, or B-lymphocytes havebeen detected, nor has RT6 been identifiedon other tissues, including brain tissue.RT6+ T-cell subsets are severely decreasedin diabetes-prone BB rats (i.e., animals thatspontaneously develop insulin-dependent

diabetes mellitus, both clinically and bio-chemically analogous to human diabetesmellitus) (26,27). Different lines of BBrats have been developed, one called "dia-betes-prone" (DP) and one called "dia-betes-resistant" (DR). In vivo depletion ofRT6+ cells induces diabetes in 50% of DRBB rats (28). Lymphocyte transfers fromDR to DP rats prevent diabetes mellitus,and this protection is associated with thepersistence of donor-origin RT6+ T-cells(29). In addition, concanavalin A-activat-ed spleen cells from RT6-depleted PVG orYoshida rats can transfer insulitis and/orthyroiditis to histocompatible athymicrecipients (30).

We obtained MAb to additional ratlymphocyte markers from Seralab, Ac-curate Chemical & Scientific Corporation(Westbury, NY) and Harlan Bioproductsfor Science, Inc. (Indianapolis, IN): MRCOX-19 (anti-CD5, pan-T-cells), R7.3(anti-TCRap), MRC OX-8 (anti-CD8 T-cell subset and natural killer cells), W3/25(anti-CD4 T-cell subset and monocytes),MRC OX-39 (anti-IL-2 receptor), MRCOX-4 (anti-common class II MHC).

We obtained the total number of RT6+lymphocytes from their percentage of thetotal cell numbers in spleen or lymphnodes. Then we calculated the total num-ber of RT6- cells by subtracting the totalnumber of RT6+ lymphocytes from total T(CD5+) cell numbers. Finally, we ob-tained the RT6- to RT6+ ratio by dividingthe total number of RT6.1- or RT6.2- T-lymphocytes by the total number ofRT6. 1+ or RT6.2+ lymphocytes.

All ELISAs were performed in dupli-cate, and the data obtained are expressed asmeans ± SEM. Flow cytometric analysiswas performed on single samples from eachrat, and the data from all experimental (orcontrol) groups are expressed as means ±SEM. We statistically evaluated resultsobtained by ELISA and flow cytometry byone-way analysis of variance (ANOVA),followed by post-hoc means tests (Fisher'sProtected Least Significance Difference,Scheffe's S, Games-Howell, and Dunnett'smultiple comparison procedures). Twocomputer programs (StatView II andSuperANOVA, Abacus Concepts, Inc.,Berkeley, CA) were used for this purpose.

ResultsAutoimmune Responses to RenalAntigensWe first examined the effects of mercury intwo matching groups of LEW and BNrats, treated with mercury ("experimental")or injected with water ("control") and sac-rificed on day 16 of treatment, i.e., thetime when autoimmune responses to renalantigens are known to be at their peak in

Volume 101, Number 2, June 1993 179

. * *,

Table 1. Autoimmune responses to laminin and renal immunopathology in rats treated with mercury

% Ratswith serum % Rats

Serum antibodies antibodies with renalNo. of to laminina to laminina Ig depositsb

Strain rats Treatment Day 0 Day 16 (Day 16) (Day 16)LEW 7 HgCI2 0.093±0.006 0.106±0.005 0 0

4 H20 0.093 ± 0.009 0.104 ± 0.007 0 0

BN 7 HgCI2 0.118±0.006 0.289±0.035* 86 1004 H20 0.102±0.018 0.136±0.010 0 0

(BN x LEW) F1 24 HgCI2 0.065±0.003 0.182±0.018** 83 1008 H20 0.075 ± 0.007 0.077 ± 0.007 0 0

aDetermined by ELISA (data are expressed as mean OD414 ± SEM).Determined by direct immunofluorescence.

*Statistically significant compared to H20-treated controls (p = 0.0106). ** Statistically significant whencompared to H20-treated controls (p = 0.0055).

BN rats (14,1/). None of the experimentalLEW rats developed antibodies to laminin(Table 1) or to renal GBM (data notshown). On the other hand, approximately86% of experimental BN rats producedantibodies to laminin (Table 1).Autoantibodies to rat GBM were also de-tected by ELISA in the sera of the same ani-mals (data not shown). None of the con-trol rats (LEW or BN) produced auto-anti-bodies to GBM or laminin. To ensure thatthe choice of the timing of observation (day16) had no negative influence on ourresults, we performed a kinetic study inanother group of LEW rats and sacrificedthem at regular intervals (days 12, 16, 23,and 30) during their exposure to mercury.Again, none of these animals producedautoantibodies to GBM or laminin. Forexample, there were no statistical differencesin the anti-GBM ELISA values ob-tainedwith sera from control (0.211 ± 0.021) orexperimental rats (day 12 = 0.271 ± 0.114;day 16 = 0.235 ± 0.040; day 23 = 0.288 ±0.104; day 30 = 0.238 + 0.052). Instead,sera from experimental BN rats containedautoantibodies to rat GBM (0.606 ± 0.032).These findings confirm that rats of the LEWstrain are resistant to mercury-inducedautoimmunity, as previously reported byother investigators (4).

We then investigated the autoimmuneconsequences of exposure to mercury in Flhybrid rats. Antibodies to laminin werepresent in the sera of 83% of experimentalanimals (Table 1). Autoantibodies againstrat GBM were found in the same sera (datanot shown). None of the control rats pro-duced autoantibodies to GBM or laminin.These serological observations were con-firmed by renal immunopathology data.Direct immunofluorescence of kidney sec-tions revealed rat immunoglobulins boundin a linear fashion to renal GBM and TBMin 100% of experimental BN and F1 hy-brids, whereas all experimental LEW ratsgave negative results (Table 1). All controlanimals were also negative. Our observa-

tions confirm previous reports that ratsfrom the BN strain and their F1 hybridswith resistant strains are susceptible to mer-cury-induced autoimmunity (4). Therefore,these animals provide an excellent model todetermine possible changes in regulatory T-lymphocytes of the RT6+ subpopulation.

Phenotypic Analysis of LymphocyteSubpopulations in LEW RatsLymphocytes from spleens and cervicallymph nodes of experimental and controlLEW rats, sacrificed at various intervalsduring treatment (days 12, 16, 23, and 30),were counted and examined by flow cytom-etry for a variety of surface markers, withthe following results.

As shown in Table 2 and Figure 1,experimental LEW rats had a moderate butsignificant increase in numbers of spleenand lymph node cells. Higher spleen cellnumbers were noted on days 12, 16, and23 of treatment (93% in-crease, p =0.0518, 86%, p = 0.0065 and 108%, p =0.0289, respectively). Lymph nodes alsocontained higher numbers of cells on days16 (232% increase, p = 0.0004) and 23(46%, p = 0.0846).

There were no significant differencesin percentage of RT6+ cells from thespleens of control and experimental LEWrats on days 12, 16, and 23 (Table 2).Values for day 30 showed a moderateincrease (26%, p = 0.0233). There werealso no percentage changes in lymphnodes for days 12 and 30, but a slightdecrease was observed on days 16 (12%, p= 0.0565) and 23 (16%, p = 0.0438). Thetotal number of RT6. 1+ spleen lympho-cytes showed a moderate increase on eachday of treatment (day 12 = 92%, p =0.0083; day 16 = 83%, p = 0.0045; day 23= 112%, p = 0.0018; day 30 = 92%, p =0.0083), whereas lymph node cells had asignificant change only on day 16 (185%increase, p = 0.0001). The ratio of RT6-to RT6+ cells did not usually vary in ex-perimental animals versus controls, exceptfor a modest increase (37%, p = 0.0186)observed in lymph node cells only on day23. As shown in Figure 2, which com-pares values obtained from animals of dif-ferent strains, there was no significantchange in the RT6 ratio of LEW rats sac-rificed on day 16.

The percentage of T-cells did not varysignificantly in experimental versus controlrats. Table 2 provides data obtained withmAb OX-19 (anti-CD5), but similarresults were also obtained with mAb R7.3(anti-TCRoa). On the other hand, totalnumbers of T-cells had increases compara-ble to those observed in total cell numbers(data not shown). In the spleen, T-cellnumbers were higher on days 12 (99%, p =0.0442), 16 (65%, p = 0.0425), and 23(107%, p = 0.0363). In lymph nodes theyincreased only on days 16 (224%, p =0.004) and 23 (48%, p = 0.0763). Percen-tages of CD8+ and CD4+ lymphocytes var-ied little either in spleen or lymph nodes.Total numbers of CD8+ T-cells did notchange significantly in the spleen but were258% higher in lymph nodes (p = 0.0009)

Table 2. Lymphocyte subpopulations in control and experimental LEW rats

Treatment(day of No. of Total no.

sacrifice) rats Cells (x 106) %RT6.1 %T-cells %CD8 %CD4 %B-cellsH20 7 Spleen 127 ± 42 26.7 ± 2.1 59.9 ± 2.0 28.8 ± 3.7 50.3 ± 1.5 41.8 ± 3.0

LN 52 ± 7 41.1 ±1.7 75.5 ±1.0 28.2 ± 2.1 59.9 ± 0.9 33.5 ± 1.4HgCI2 5 Spleen 246 ± 24 26.9 ±1.7 64.4 ± 1.3 27.9 ±1.3 52.2 ± 0.9 36.6 ± 0.3

(day 12) LN 66 ± 10 42.4 ± 1.2 77.3 ±1.1 30.0 ± 1.5 62.6 ± 0.9 28.8 ±1.6HgCI2 11 Spleen 236 ± 10 26.6 ± 1.7 54.8 ±1.6 27.5 ± 1.6 46.6 ± 0.9 38.3 ±1.4

(day 16) LN 173 ± 21 36.6 ± 1.7* 73.6 ±1.2 30.0 ± 1.4 53.0 ±1.5 39.2 ±1.1HgCI2 5 Spleen 265 ± 25 26.4 ± 2.0 61.7 ± 0.7 28.8 ± 3.8 52.3 ± 0.9 35.1 ± 1.6

(day 23) LN 76 ±11 35.5 ± 1.8** 76.1 ± 0.6 35.3 ±1.1 57.1 ± 0.8 38.9 ± 1.7HgCI2 5 Spleen 195 ±17 33.7 ± 1.2***61.3 ± 1.1 26.4 ± 1.4 51.7 ± 1.6 34.7 ± 1.2

(day 30) LN 69 ± 12 41.9 ±1.8 74.7 ± 2.5 30.1 ±1.2 56.2 ± 2.0 36.8 ± 2.5LN, lymph nodes. Flow cytometric analysis of spleen and cervical node lymphocytes; data expressed asmeans ± SEM.*Slight statistically significant decrease compared to H20-treated controls (p = 0.0565). **Slight statisti-cally significant decrease compared to H20-treated controls (p = 0.0438). ***Moderate statistically signif-icant increase compared to H20-treated controls (p = 0.0233).

Environmental Health Perspectives180

-Z. .9 - - a-. -El-

I

LEW BN Fl

Rat strains

Figure 1. Subcutaneous injection of rats with HgCI2 results in various degrees of splemnphadenopathy. Data obtained after 16 days of mercury treatment show a moderate incinumbers in the spleen (p = 0.0065) and lymph nodes (p = 0.0004) of LEW rats. Similarly,treatment with HgCI2 there was a considerable increase of total cell numbers in both0.0033; lymph nodes, p= 0.0001) and (BN X LEW) F1 hybrid rats (spleen, p= 0.01; lymph nc

BN

I

L

I Fl

II

z ji z -i

+ U)+

'- + +

v CIA

aU 3: In zjul L m

-I

T

z ~i z -i

I ~ i IL

v- + % +

T- N|

_-IL _- IL _-

IL IL

Figure 2. Treatment with HgCI2 for 16 days causes an increase in the ratios of RT6- to RTin both lymph nodes (LN) and spleen (SPL) of BN and (BN X LEW) F1 hybrids. Similarlyexperience only minor variations of those ratios.

on day 16 and 84% (p = 0.0189) on day23. Numbers of CD4+ T-cells in bothspleen and lymph nodes showed increasesparalleling those of total cell numbers.Total numbers of B-cells in-creased inboth spleen and lymph nodes, again paral-leling the higher total cell numbers.Finally, numbers of lymphocytes positivefor MHC class II antigens were increasedon days 12, 16 and 23 (data not shown).

In summary, mercury

LEW rats resulted in mod4cant increases in the numbe

RT6+ T-lymphocytes did not change in asignificant fashion in the spleen andchanged only on day 23 in lymph nodes.

Phenotypic Analysis of LymphocyteSubpopulations in BN RatsWe have previously reported that RT6+ T-

* Ln. cells lymphocytes from spleen and cervicallymph nodes of mercury-treated BN ratsshow a relative decrease, particularly evi-

Spi. cells dent in the ratio between RT6- and RT6+cells (17). In the present series of experi-ments we examined the phenotypes of cellsfrom a new group of experimental andcontrol BN rats, sacrificed on day 16 (i.e.,at the time when autoimmune responses torenal antigens reach their peak in animalsinjected with HgCI2), with the followingresults.

omegaly and lyi- As shown in Table 3, the number ofrease of total cell spleen cells in experimental BN rats was

after 16 days of considerably higher than in controls, a dif-h BN (spleen, p= ference that was statistically significant)des,p=0.0001). (218%, p = 0.0033). A more striking

increase in total cell numbers (860%, p =0.0001) occurred in cervical lymph nodes(Table 3), confirming previous observa-tions of hyperplasia in peripheral lymphoidtissues from BN rats exposed to mercury(17,31-33).

After 2 weeks of mercury treatment,the percentage of RT6+ cells in lymphnodes was significantly decreased as com-pared to controls (100%, p = 0.0009),whereas spleen cells had a less markedchange (55%, p = 0.0062). The total

Water number of RT6.2+ lymphocytes of experi-mental BN rats was moderately increased

* Mercury in the spleen (78%, p = 0.0578), but washigher in lymph nodes (307%, p =0.0033). On the other hand, the totalnumber of RT6.2- cells was greatly in-creased and proportionally more than thatof RT6.2+ cells (332%, p = 0.0084 in thespleen; 572%, p = 0.0602 in lymphnodes). As a consequence, the ratio ofRT6- to RT6+ positive cells was higherboth in lymph nodes and spleens of experi-mental BN rats (Fig. 2).

In the spleen, there was no significantchange in the percentage of T-cells inexperimental versus control rats (p =

r6+T-lymphocytes 0.2643), but the total numbers of T-cellstreated LEW rats increased by 173% after mercury treat-

ment (p = 0.0045). Similarly, the percent-age of spleen CD8+ lymphocytes did not

treatment of show major changes (p = 0.3048), but theirest but signifi- total number was increased (316%, p =r of spleen and 0.0313). In contrast, CD4+ cells were pre-

lymph node cells, and as a consequence

most subpopulations of lymphocytes were

numerically increased. There were onlyslight and sporadic changes in the percent-age of RT6+ T-cells, but their numberswere higher in experimental than in con-

trol animals. The ratio between RT6- and

sent in a lower percentage in experimentalBN (38% change, p = 0.0004) but showedslightly higher numbers (1 19% change, p =

0.0114). The percentage of B-cells was

not changed (p = 0.337), but their numberincreased by 230% (p = 0.002). Lympho-cytes from cervical lymph nodes gave simi-

Volume 101, Number 2, June 1993

a-0

S '

= 4w

8 Ia I

o-

1400-

1200-

1000-

800-

600-

400-

200-

0-

LEW

IT

t2.5-

a 2

ID 1.5-

(0

°1-

a

o 05-om 2

1 O

I

181

. m~~~ **rqr_ -

Table 3. Lymphocyte subpopulations in mercury-treated BN and (BN x LEW) F1 hybrids

Total no.Strain Treatment Cells (x 106) %RT6.1 %RT6.2 %T-cells %CD8 %CD4 %B-cellsBN HgCI2 Spleen 535 ± 68 0 14.4 ± 1.3* 32.6 ±1.5 15.5 ±1.6 28.5 ±1.1 47.2 ±1.4

LN 484 ± 50 0 22.0 ± 2.8** 34.0 ± 2.8 11.7 ± 2.0 32.4 ±1.5 49.8 ± 3.4H20 Spleen 168±23 0 22.3±2.4 35.9±2.4 12.4±2.1 39.3±2.9 44.8±1.6

LN 50 ±13 0 44.0 ± 5.9 64.0 ±1.4 6.3 ± 0.4 54.4 ±1.5 30.9 ±1.6(BNxLEW)F1 HgCI2 Spleen 397±33 13.1 ±0.5*** 17.2±0.6*** 38.2±0.7 19.6±0.6 31.0±1.7 54.5±1.1

LN 580 ± 42 22.1 ± 1.4*** 29.9 ± 1.6*** 45.2 ± 1.4 17.5 ± 1.0 37.2 ± 1.3 51.0 ± 1.7H20 Spleen 223±30 27.5±2.1 37.7±2.0 47.4±1.7 14.6±0.8 40.1 ±1.7 40.3±1.1

LN 41±7 42.4± 1.0 60.3±1.0 70.2±2.3 10.7 ±0.5 62.3±1.7 20.1 ±1.5LN, lymph nodes. A total of 25 BN (18 experimental and 7 controls) and 32 F1 hybrids (24 experimental and 8 controls) were sacrificed on day 16 from the start ofthe experiments. Flow cytometric data are expressed as means ± SEM.Percentages of RT6.1+ and RT6.2+ T-lymphocytes significantly decreased in HgCI2-treated rats as compared to H20-treated controls: *p = 0.0062; **p = 0.0009;***p= 0.0001.

lar patterns, with moderate decreases in thepercentage of T-cells and CD4+ cells (89%and 68%, p = 0.0001 for both), and signif-icant increases in their numbers (374%and 442%, p = 0.0001 for both). On theother hand, CD8+ cells presented no majorpercentage change (p = 0.1083) but hadhigher numbers (1625%, p = 0.0167).Finally, the percentage of B-cells in lymphnodes increased by 61% (p = 0.0028), andtheir number increased by 1417% (p =0.0003) in experimental BN rats.

In summary, mercury-treated BN ratsshowed marked hyperplasia of peripherallymphoid organs, significant decreases inthe percentage of RT6+ cells, and propor-tionally higher numbers of RT6- cells. Asa result, the balance between these twosubpopulations of lymphocytes was alteredin favor of RT6- cells.

Phenotypic Analysis of LymphocyteSubpopulations in F1 Hybrid RatsBecause F1 hybrids between susceptibleBN and resistant LEW rats are also suscep-tible to mercury-induced autoimmunity,we determined the phenotype of peripherallymphocytes in experimental or control Frats, with the following results.

As shown in Table 3 and Figure 1, thetotal number of spleen cells in experimen-tal F1 rats was increased by 78% as com-pared to controls (p = 0.01). A more strik-ing increase in total cell numbers (1315%,p = 0.0001) occurred in cervical lymphnodes (Table 3).

The percentage of cells expressing theRT6 phenotypes was significantly de-creased in both spleens and lymph nodesof experimental animals as compared tocontrols (Table 3). In the spleen, percent-ages of RT6.1+ and RT6.2+ were lower by110% and 1 9% (p = 0.0001 for both).Similar reductions were observed in lymphnodes (92% and 102% change, p =0.0001 for both). The total number ofRT6.1+ and RT6.2+ spleen lymphocytesdid not show significant changes, but RT6-cells were increased (137%, p = 0.0016 for

RT6.1- and 296%, p = 0.0001 for RT6.2-).Therefore, the ratios of RT6- to RT6+spleen cells were increased by 197% forRT6.1 and 385% for RT6.2 (p = 0.0001for both; Fig. 2). In cervical lymph nodesof experimental rats, the total number ofRT6.1+ and RT6.2+ lymphocytes was sig-nificantly increased (623% and 587%, p =0.0001 for both). However, RT6- cellswere present in even greater numbers(1114% change for RT6.1- and 2372% forRT6.2-, p = 0.0001 for both), resulting inaltered ratios of RT6- to RT6+ cells (Fig.2). An increase of 89% was observed forRT6.1-/RT6.1+ (p = 0.0802) and 258%for RT6.2-/RT6.2+ (p = 0.0064).

In the spleens of experimental F, ani-mals, we observed a 24% decrease in thepercentage of T-cells (p = 0.0001) and a41% increase in T-cell numbers (p =0.0766) as compared to controls. Bothpercentage and total numbers of spleenCD8+ lymphocytes were increased (35%, p= 0.0001 and 140%, p = 0.0021). In con-trast, CD4+ cells were present in a lowerpercentage in the spleen of experimental Flrats (29% change, p = 0.0063), but theirnumbers were not significantly higher (p =0.1156). Both percentage and number ofspleen B-cells were increased (36%, p =0.0001 and 142%, p = 0.0038). Lympho-cytes from cervical lymph nodes gave simi-lar patterns, with decreases in percentage ofT- and CD4+ cells (55% and 68% change,p = 0.0001 in both cases) and strikingincreases in their numbers (816% and732%, p = 0.0001 for both). On the otherhand, lymph node CD8+ cells presentedincreases both in percentage and numbers(64%, p = 0.0004 and 2393%, p =0.0001). Finally, there was a definiteincrease of B-lymphocytes in the lymphnodes of experimental F1 rats: their per-centages were 154% (p = 0.0001) higher,and their numbers rose by 3665% (p =0.0001).

In summary, treatment of F1 hybridswith mercury resulted in hyperplasia ofperipheral lymphoid tissues similar to that

observed in BN rats under the same cir-cumstances. In addition, exposure to mer-cury was followed by significant decreasesof RT6+ and increases of RT6- cells. Theresulting changes in the ratio betweenthese lymphocyte subpopulations revealedthat mercury had altered their balance.Thus, F1 hybrids exhibited the samebehavior of their mercury-susceptibleparental strain (BN), both in their autoim-mune responses to renal antigens andchanges in lymphocyte subpopulations.

DiscussionThe existence of T-suppressor cells is oneof the most controversial topics of im-munology. However, Todd and Steinman(34) state that "suppressor cells are back infashion, since they refuse to go away andcrop up repeatedly in autoimmune diseasesas a plausible explanation for peripheraltolerance." Previous studies relied exclu-sively on a few markers (e.g., CD4 andCD8) as representative of functional(helper/inducer versus suppressor/ cytotox-ic, respectively) activities, which mayexplain both controversial results and lackof correlations between T-cell subsets,severity of autoimmune disease, and/orkinetics of autoimmune responses. Therecent demonstration of T-cell heterogene-ity, including various functional pheno-types of CD4+ cells (35-38), has led tomore refined dissections of the complexregulatory network. In rats, peripheral T-cells differ in their expression of variousmembrane determinants, i.e., CD4, CD8,QCA-1, Thy-1, CD45RC, and RT6.Thus, combining these markers allows theidentification of a large number of pheno-typically different subsets of peripheral T-lymphocytes (39). Previous investigationsin our laboratory have demonstrated thatBN rats repeatedly injected with mercuricchloride experience a relative decrease ofperipheral RT6.2+ T-lymphocytes and analtered ratio of RT6- to RT6+ T-cells (1/).These changes coincide with the appear-ance of circulating autoantibodies to renal

Environmental Health Perspectives182

A - - - * -e -Z

antigens (e.g., laminin). In the presentstudy we extended our phenotypic analysisto rats of another strain (LEW) and exam-ined additional numbers of BN rats as wellas F1 hybrids between BN and LEW.

We have confirmed that LEW and BNrats differ in their responses to the admin-istration of HgCI2. It was known thatLEW rats do not develop renal autoimmu-nity after mercury treatment (4). How-ever, there was no information on the be-havior of RT6+ T-cells after exposure ofLEW rats to mercury, and it is of interestthat their percentage did not significantlydecrease. Numbers of RT6+ cells wereactually higher in both spleens and lymphnodes of experimental rats, with increasesthat reached statistical significance. In thespleen, the ratio between RT6- and RT6+T-lymphocytes showed a modest decreaseonly on day 30, whereas in the lymphnodes it was increased only on day 23, sug-gesting that treatment with mercury didnot have consistent effects on this lympho-cyte subpopulation. The lack of changesin this natural balance may explain theLEW resistance to the autoimmune effectsof this metal. On the other hand, we con-firmed our initial findings that BN ratsinjected with HgCl2 experience autoim-mune responses to renal antigens as well asa decrease in the percentage of RT6+ cells.In spite of strikingly higher total numbersof spleen and lymph node cells, the num-bers of RT6+ cells did not proportionallyincrease, resulting in an altered balancebetween RT6- and RT6+ T-lymphocytes(Fig. 2). This mercury-induced effect isquite similar to our previous results fromthree different groups of BN rats, whichshowed a definite decrease in the numberof RT6+ spleen cells in one group and rela-tive decreases in the other two (e.g., a 6%increase versus controls, in spite of a 52%increase in total cell numbers) (17). Dif-ferences in total numbers of RT6 cellsbetween various groups of BN rats seem todepend on the magnitude of mercury-induced splenomegaly and lymphadenopa-thy, which vary considerably with age,weight, and microbial status of the individ-ual rats. However, percentage values ofRT6+ cells and ratios between negative andpositive cells are quite reproducible andprovide a reliable indication of the im-munotoxic effects of mercury on this sub-population ofBN rat lymphocytes.

We observed that F1 hybrid rats (i.e.,hybrids between susceptible and resistantrats), behaved like their BN parent strainwhen exposed to mercury: they had bothrenal autoimmunity and significantly lowerpercentages of RT6+ T-lymphocytes. Asfar as autoimmunity tO kidney antigens isconcerned, there was no detectable differ-ence between BN and F1 rats (Table 1).

All animals had linear immunoglobulin de-posits in their kidneys: previous work hasdemonstrated that such deposits containantibodies to laminin and other renal anti-gens (14,15). Antibodies to laminin andrat GBM were also present in the sera ofmost experimental rats. Flow cytometricanalysis showed that both RT6.1 andRT6.2 surface markers are expressed onlymphocytes of F1 rats. Control animalshad a higher percentage of RT6.2+ cellsthan BN rats and a percentage of RT6.1+cells similar to that of LEW (Tables 2 and3). Previous studies by single-label im-munofluorescence staining of lymph nodecells obtained from F1 hybrids betweenWistar Furth (RT6.2+) and diabetes-proneor diabetes-resistant BB (RT6.1+) rats alsorevealed the presence of RT6.1+ andRT6.2+ lymphocytes in both hybrids (40).Dual-label flow cytometric analysis of theselymphocytes for RT6.1 and RT6.2 allo-antigens has shown that the two moleculesare co-expressed on most of the cells. Inaddition, those F1 hybrids had a higherpercentage of RT6.2+ than RT6.1+ cells,which is confirmed by our findings inhybrids between two additional rat strains.The reasons for such a difference areunknown but are probably related to theepitope(s) recognized by the monoclonalantibodies against RT6 alloantigens ratherthan lack of expression of RT6.1 on someRT6.2+ cells. In any case, experimental Frats had a lower percentage of both RT6. 1and RT6.2+ cells in their spleens andlymph nodes. Again, total numbers ofthese cells were decreased in the spleen.On the other hand, as previously observedin BN rats, there was a relative decrease ofRT6+ cells in cervical lymph nodes, i.e.,their number did not show the strikingincrease observed in total cell numbers. Asa consequence, these changes resulted in analtered balance within the RT6 T- lym-phocyte population, as demonstrated bythe ratios between positive and negativecells (Fig. 2).

Exposure to mercury had other im-munotoxic effects on peripheral lymphoidtissues (see Fig. 1). Total cell numbersincreased in the spleen and lymph nodes ofall experimental rats; however, the increasewas rather modest in LEW rats as com-pared to that observed in BN rats.Changes in the spleens of Fl hybrids wereintermediate between those of the parentalstrains, whereas numbers in lymph nodeswere much higher. Other investigatorshave previously reported that spleens andlymph nodes were not much enlarged inLEW rats injected with HgCI2 and therewere no major changes in totaf cell num-bers (18,33,41). However, the modestincreases we observed may be easilyexplained by differences in age and weight

of our LEW rats or the use of a differentsubstrain, possibly more susceptible tomercury effects. The marked proliferationof BN spleen and lymph node cells aftertreatment with HgCl2 has been previouslynoted by various investigators (17,18,32,33,41). On the other hand, the lym-phadenopathy and splenomegaly of F1 hy-brids have not received much attention.

In addition to numerical increases ofspleen and lymph node cells, we alsonoticed changes in most lymphocyte sub-populations. For example, flow cytometryof lymph nodes from experimental LEWrats revealed a percentage decrease of B-cells on day 12 and an increase on days 16and 23. Parallel percentage changes of Ia'cells were also observed on the same days.The percentage of T-cells in the samelymph nodes never varied significantly andthat of CD8+ cells was increased only onday 23. In contrast, CD4+ cells differedon each day of treatment, with a percent-age increase on day 12 and decreases there-after. Values for the spleen were moreconstant, with the only significant percent-age changes noted on day 30, when therewas a decrease of B- and Ia' cells. On theother hand, total cell numbers of almost allsubsets were higher on days 12, 16, and 23of treatment, proportional to the moderateincrease in total cell numbers. Data forexperimental BN rats were obtained onlyon day 16, i.e., at a time when their auto-immune responses to renal antigens are attheir peak (17). B-cell percentages andnumbers were both higher than in con-trols, whereas T-cell percentages de-creasedand numbers increased. Percentages andnumbers of CD8+ T-lymphocytes wereincreased. In contrast, percentages ofCD4+ T-cells decreased and their numbersincreased. Almost identical changes wereobserved in spleen and lymph nodes ob-tained from F1 hybrids on day 16 of treat-ment with HgCl2.

Of particular interest is our findingthat both percentage and total numbers ofCD8+ T-lymphocytes did not show consis-tent and significant increases in the spleensof experimental LEW rats. In the lymphnodes, CD8+ cells increased in percentageonly on day 23 and in total numbers onlyon days 16 and 23, proportional to thehigher total cell numbers. In contrast,experimental BN and Fl rats had increasedpercentages as well as higher numbers ofthese lymphocytes. These data show thatBN and F1 rats, at the peak of autoim-mune responses induced by the adminis-tration of mercury, have increased levels(both as a percentage and numerically) ofCD8+ T-lymphocytes, whereas LEW rats,resistant to the autoimmune effects of thismetal, usually show only numerical in-creases. On this basis, it is difficult to

Volume 101, Number 2, June 1993 183

A - *~~~*..-

attribute an immunosuppressive role toCD8+ T-lymphocytes either in LEW orBN rats. In earlier studies, Pelletier et al.(18,20) observed significant increases inboth percentage and/or numbers of CD8+T-cells in LEW rats on days 14, 28, and 35of mercury treatment. The same groupreported that the administration of HgCI2to LEW rats for 2 weeks before immuniza-tion with certain autoantigens had im-munosuppressive effects on the experimen-tal induction of autoimmunity (e.g.,Heymann's nephritis and EAE) (19,42).As far as BN rats are concerned, sequentialstudies of peripheral blood lymphocytepopulations during HgCI2 treatment haveshown a transient decrease in the percent-age of CD8+ T-cells before the appearanceof serum anti-GBM antibodies, followedby elevated levels of these cells duringdown-regulation of the response (43).Aten et al. (32) also noted that CD8 levelswere higher in BN rats on day 21 than onday 0. These observations led to the sug-gestion that CD8+ cells, which at that timewere still defined as "suppressor/cytotoxic"T-lymphocytes, might have an immuno-regulatory role in both LEW and BN rats.Differences in techniques of phenotypicanalysis and the use of various substrains ofrats may explain the discrepancies betweenthe data available in the literature. In anycase, considerable doubts have later beenraised about the existence of T-suppressorcells in general and the immunosuppressiveproperties of CD8+ T-lymphocytes in par-ticular. Our findings suggest that increasesof CD8+ T-cells may actually have nomajor role in the lack of autoimmuneresponses or the down-regulation of renalautoimmunity observed in LEW and BNrats, respectively, after mercury treatment.

Finally, our data underline the impor-tance of a complete and detailed phenotyp-ic analysis of lymphoid cells in studies ofimmunotoxicity and xenobiotic-inducedautoimmunity. Traditional markers suchas CD4 and CD8 may not be sufficient tocharacterize different subpopulations of T-cells, in view of the heterogeneity demon-strated by functional studies (38). The useof additional surface markers (e.g., theRT6 alloantigens) together with cytokineprofiles of T-cell clones may be necessaryfor a better understanding of immunoregu-latory processes. T-cells of the RT6 pheno-type are known to be severely decreased inspontaneously hyperglycemic diabetes-prone BB/Wor rats, and their deficiencymay be an important predisposing factor insusceptibility to insulin-dependent diabetesmellitus. It has also been suggested thatRT6+ T-cells have a regulatory role in self-tolerance (44). The parallel changes ofRT6+ T-lymphocytes observed in both BNand F1 hybrids as well as the opposite

behavior detected in LEW rats also suggesta regulatory role of RT6 lymphocytes.The obvious question then arises: Are thereequivalents for the RT6 marker in hu-mans? To our knowledge, none have beenreported to date. However, two recentstudies have shown an abnormally highproportion of CD4+ T-cells of theCD45RA+ phenotype in patients withinsulin-dependent diabetes mellitus ormixed connective tissue disease (45,46).These results still need to be confirmed,and their functional significance remainsto be determined (47). However, theysuggest that phenotypic and functionalchanges first observed in rat models ofautoimmunity may have similar, if notidentical, counterparts in human disease.

In conclusion, on the basis of theresults presented in this paper we proposethe hypothesis that there are both endoge-nous and exogenous components of mer-cury-induced autoimmunity. The firstcomponent, genetically determined, con-stitutes the "terrain" of the individualsexposed to the xenobiotic and comprisesTCR, MHC, and an immunoregulatorynetwork based on a delicate balance be-tween cells with suppressor (e.g., RT6+ T-lymphocytes) and helper activities. Theexogenous component is represented by anenvironmental factor, e.g., mercury, capa-ble of altering the balance within theimmunoregulatory network. All these ele-ments must be present and interact forautoimmunity to occur. The rat modelsdescribed in this paper lend themselves toexperimental manipulations, currentlyongoing in our laboratory, to dissect therole of these various components in auto-immunity induced by mercury.

REFERENCES

1. Bigazzi PE. Autoimmunity induced by chemi-cals. J Toxicol Clin Toxicol 26:125-156(1988).

2. Bigazzi PE. Lessons from animal models: thescope of mercury-induced autoimmunity. ClinImmunol Immunopathol 65:81-84(1992).

3. Goldman M, Druet P, Gleichmann E. TH2cells in systemic autoimmunity: insights fromallogeneic diseases and chemically-inducedautoimmunity. Immunol Today 12:223-227(1991).

4. Druet E, Sapin C, Gunther E, Feingold N,Druet P. Mercuric chloride-induced anti-glomerular basement membrane antibodies inthe rat. Genetic control. Eur J Immunol7:348-351(1977).

5. Henry GA, Jarnot BM, Steinhoff MM, BigazziPE. Mercury-induced renal autoimmunity inthe MAXX rat. Clin Immunol Immunopathol49:187-203(1988).

6. Aten J, Veninga A, Bruijn JA, Prins FA, deHeer E, Weening JJ. Antigenic specificities ofglomerular-bound autoantibodies in membra-nous glomerulopathy induced by mercuricchloride. Clin Immunol Immunopathol 63:89-102(1992).

7. Druet P, Teychenne P, Mandet C, Bascou C,Druet E. Immune-type glomerulonephritisinduced in the Brown-Norway rat with mer-cury-containing pharmaceutical products.Nephron 28:145-148(1981).

8. Bernaudin JF, Druet E, Druet P, Masse R.Inhalation or ingestion of organic or inorganicmercurials produces auto-immune disease inrats. Clin Immunol Immunopathol 20:129-135(1981).

9. Andres P. IgA-IgG disease in the intestine ofBrown-Norway rats ingesting mercuric chlo-ride. Clin Immunol Immunopathology 30:488-494(1984).

10. Knoflach P, Albini B, Weiser MM. Auto-immune disease induced by oral administrationof mercuric chloride in Brown-Norway rats.Toxicol Pathol 14:188-193 (1986).

11. Bellon B, Capron M, Druet E, Verroust P,Vial M-C, Sapin C, Girard JF, Foidart JM,Mahieu P, Druet P. Mercuric chloride inducedautoimmune disease in Brown-Norway rats:sequential search for anti-basement membraneantibodies and circulating immune complexes.Eur J Clin Invest 12:127-133(1982).

12. Michaelson JH, McCoy JPJ, Hirszel P, BigazziPE. Mercury-induced autoimmune glomeru-lonephritis in inbred rats. I. Kinetics andspecies specificity of autoimmune responses.Surv Synth Pathol Res 4:401-411(1985).

13. Sapin C, Druet E, Druet P. Induction of anti-glomerular basement membrane antibodies inthe Brown-Norway rat by mercuric chloride.Clin Exp Immunol 28:173-179(1977).

14. Fukatsu A, Brentjens JR, Killen PD, KleinmanHK, Martin GR, Andres GA. Studies on theformation of glomerular immune deposits inBrown Norway rats injected with mercuricchloride. Clin Immunol Immunopathol 45:35-47(1987).

15. Bigazzi PE, Michaelson JH, Potter NT. Epi-bodies in autoimmunity: antisera againstautoantibodies to the renal glomerular base-ment membrane react with idiotypes as well aswith autoantigens. Autoimmunity 5:3-16(1989).

16. Guery JC, Druet E, Glotz D, Hirsch F,Mandet C, De Heer E, Druet P. Specificityand cross-reactive idiotypes of anti-glomerularbasement membrane autoantibodies in HgCl2-induced autoimmune glomerulonephritis. EurJ Immunol 20:93-100(1990).

17. Kosuda LL, Wayne A, Nahounou M, GreinerDL, Bigazzi PE. Reduction of the RT6.2+ sub-set of T-lymphocytes in BN rats with mercury-induced renal autoimmunity. Cell Immunol135:154-167(1991).

18. Pelletier L, Pasquier R, Rossert J, Druet P.HgCl2 induces nonspecific immunosuppres-sion in Lewis rats. Eur J Immunol 17:49-54(1987).

19. Pelletier L, Rossert J, Pasquier R, Villarroya H,Belair M-F, Vial M-C, Oriol R, Druet P.Effect of HgCl2 on experimental allergicencephalomyelitis in Lewis rats. HgCl2-induced down-modulation of the disease. Eur JImmunol 18:243-247(1988).

20. Pelletier L, Rossert J, Pasquier R, Vial MC,Druet P. Role of CD8+ T-cells in mercury-induced autoimmunity or immunosuppressionin the rat. Scand J Immunol 31:65-74(1990).

21. Greiner DL, Handler ES, Nakano K, MordesJP, Rossini AA. Absence of the RT-6 T cellsubset in diabetes-prone BB/W rats. J Im-munol 136:148-151(1986).

22. Angelillo M, Greiner DL, Mordes JP, HandlerES, Nakamura N, Mckeever U, Rossini A.Absence of RT6+ T-cells in diabetes-prone bio-

184 Environmental Health Perspectives

I WA- M. ME - MEE.

breeding/Worcester rats is due to genetic andcell developmental defects. J Immunol 141:4146-4151(1988).

23. Ely JM, Greiner DL, Lubaroff DM, Fitch FW.Characterization of monoclonal antibodies thatdefine rat T cell alloantigens. J Immunol130:2798-2803(1983).

24. Milford EL, Paradysc JM, Carpenter CB. Amonoclonal alloantibody that detects HLA-B7-associated polymorphism. Transplant Proc15:1974-1975(1983).

25. Solomon DR, Cohen DJ, Carpenter CB,Milford EL. Regulation of the immune re-sponse to alloantigens: suppressor and helpercells generated in the primary MLR of the rat. JImmunol 131:1065- (1983).

26. Georgiou HM, Lagarde A-C, Bellgrau D. Tcell dysfunction in the diabetes-prone BB rat.A role for thymic migrants that are not T cellprecursors. J Exp Med 167:132-148(1988).

27. Guberski DL, Butler L, Kastern W, Like AA.Genetic studies in inbred BB/Wor rats. Anal-ysis of progeny produced by crossing lym-phopenic diabetes-prone rats with nonlym-phopenic diabetic rats. Diabetes 38:887-893(1989).

28. Greiner DL, Mordes JP, Handler ES, AngelilloM, Nakamura N, Rossini AA. Depletion ofRT6.1+ T-lymphocytes induces diabetes inresistant biobreeding/Worcester (BB/W) rats. JExp Med 166:461-475(1987).

29. Burstein D, Mordes JP, Greiner D, Stein D,Nakamura N, Handler E, Rossini A. Preven-tion of diabetes in the BB/Wor rat by a singletransfusion of spleen cells: parameters thataffect the degree of protection. Diabetes38:24-30(1989).

30. McKeever U, Mordes JP, Greiner DL, Appel

MC, Rozing J, Handler ES, Rossini AA.Adoptive transfer of autoimmune diabetes andthyroiditis to athymic rats. Proc Natl Acad SciUSA 87:7618-7622(1990).

31. Pelletier L, Pasquier R, Guettier C, Vial MC,Mandet C, Nochy D, Bazin H, Druet P.HgCl, induces T and B-cells to proliferate anddifferentiate in BN rats. Clin Exp Immunol71:336-342(1988).

32. Aten J, Bosman CB, Rozing J, Stijnen T,Hoedemaeker PJ, Weening JJ. Mercuric chlo-ride-induced autoimmunity in the BrownNorway rat. Am J Pathol 133:127-138(1988).

33. Levine S, Saltzman A. The topography of mer-curial lymphadenopathy in Brown Norwayrats. Lymphology 21:161-168(1988).

34. Todd JA, Steinman L. Genetic dissection oftolerance. Curr Opinion Immunol 4:699-702(1992).

35. Bottomly K. A functional dichotomy in CD4+T-lymphocytes. Immunol Today 9:268-274(1988).

36. Powrie F, Mason D. Phenotypic and function-al heterogeneity of CD4+ T-cells. ImmunolToday 9:274-277(1988).

37. DeKruyff RH, Ju S-T, Hunt AJ, MosmannTR, Umetsu DT. Induction of antigen-specificantibody responses in primed and unprimed B-cells. Functional heterogeneity among Th I andTh2 T cell clones. J Immunol 142:2575-2582(1989).

38. Mosmann TR, Coffman RL. TH 1 and TH2cells: different patterns of lymphokine secretionlead to different functional properties. AnnuRev Immunol 7:145-173(1989).

39. Kampinga J, Groen H, Kiatter F, MeedendorpB, Aspinall R, Roser R, Nieuwenhuis P. Post-thymic T cell development in rats: an update.

Biochem Soc Trans 20:191-197(1992).40. Crisa' L, Greiner DL, Mordes JP, MacDonald

RG, Handler ES, Czech MP, Rossini AA.Biochemical studies of RT6 alloantigens inBB/Wor and normal rats. Evidence for intactunexpressed RT6a struLctural gene in diabetes-prone BB rats. Diabetes 39:1279-1288(1990).

41. Hirsch F, Couderc J, Sapin C, Fournie G,Druet P. Polyclonal effect of HgCl2 in the rat,its possible role in an experimental autoim-mune disease. Eur J Immunol 12:620-625(1982).

42. Pelletier L, Galceran M, Pasquier R, Ronco P,Verroust P, Bariety J, Druet P. Down modula-tion of Heymann's nephritis by mercuric chlo-ride. Kidney Int 32:227-232(1987).

43. Bowman C, Green C, Borysiewicz L,Lockwood CM. Circulating T-cell populationsduring mercuric chloride-induced nephritis inthe Brown Norway rat. Immunology 61:515-520(1987).

44. Mordes JP, Handler ES, Greiner DL, GottliebP, McKeever U, Tafuri A, Thomas VA, RossiniAA. Immunomodulation of autoreactivity:studies in RT6.1-depleted diabetes resistantBB/Wor rats. In: Frontiers in diabetes research.Lessons from animal diabetes (Shafrir E, ed).London:Smith Gordon, 1991; 106-113.

45. Faustman D, Eisenbarth G, DaleyJ, BreitmeyerJ. Abnormal T-lymphocyte subsets in type Idiabetes. Diabetes 38:1462-1468 (1989).

46. Becker H, Langrock A, Federlin K. Imbalanceof CD4+ lymphocyte subsets in patients withmixed connective tissue disease. Clin ExpImmunol 88:91-95(1992).

47. Mason D, Fowell D. T-cell subsets in autoim-munity. Curr Opinion Immunol 4:728-732(1992).

A LONG-STANDING TRADITION CONTINUES.........>.. ....

Environmental Health Q^

THE MOST RECENT Iz~r~issue of the Environmental Health Perspectives Supplements,Vol. 101, Supplement 1, is the complete collection of Technical Reportabstracts through 1992 as taken from the published reports of theNational Cancer Institute Bioassasy Program (1971-1978) and its {><successor, the National Toxicology Program. Because the Technical ,iReport series now represents over 400 volumes, it was felt thereCwas a need to compile a summary of all chronic tumor studies intoCa single reference volume.

To order your copy, contact:_GOVERNMENT PRINTING OFFICE, WASHINGTON, DC 20402Cor call 202-512-2406.C

Volume 101, Number 2, June 1993 185