Suppressor Cell Regulation Cell-mediated Immune Responses ......Suppressor Cell Regulation ofCell-mediated ImmuneResponses in Renal Infection IN VITRO MODULATIONOFSUPPRESSORCELLACTIVITY

Suppressor Cell Regulation of Cell-mediated

Immune Responses in Renal Infection

IN VITRO MODULATIONOF SUPPRESSORCELL ACTIVITY

THONIAS MILLER and ELAINE MARSHALL,Depa rtnent of lMedicinie, AucklandHospital, Aucklanid, News Zeala 11(d

A B S T R A C T Infection-induced anergy is a fre(luentcomplication of bacterial, viral, and parasitic infection.A marked suppression of the thymus-derived (T)lymphocyte response to concanavalini A has beendemonstrated in vitro during renal infection and themechaniismiis by which suppressioni occurs have beeninvestigated. In particular we have considered the pos-sibility that suppressioni might result from the inhibi-tory effect of prostaglandins, secreted by activatedmacrophages with immunoregulatory potential. Theexperiments have shown that the T-lymphocyte effec-tor status in experimentally-induced renal infection isdetermined by two suppressor cells, one infectioni-in-duced and the other naturally occurring. The inabilityto respond to mitogenic stimulation was reversible andrestoration of immnune responsiveness to spleniclymphocytes from infected animals could be achievedin two stepwise manipulations; differential centrifuga-tion removed the infection-induced suppressor cells,and the suppressor activity of the naturally occurringsuppressor cells could then be inhibited by indometha-cin. Thus the two suppressor cells were distinguish-able on the basis of their physical characteristics andtheir response to indomethacin. The dominant factordetermininig the immune responsiveness of spleniclymphocytes from the pyelonephritic animals was,however, the infection-induced suppressor cell. Thiscell has been characterized as a sedimentable cell(30 g) with suppressor activity demonistrable in co-culture experiments. Plastic-adherent cells from thesedimentable fraction of pyelonephritic animals'splenic cells were shown to have suppressor activitythat was not inhibited by indomethacin. The infec-tion-induced and naturally occurring suppressor cellscan be viewed as prototypes for the equivalent cellsin man and may be useful models for studying the

Received for publication 22 October 1979 and in revisedform 6 Mall 1980.

J. Clin. Invest. (© The American Society for Clinical Investiga

role of these cells as dleterminiiants in the pathogeniesisof infectious disease.

INTRODUCTION

The ability of an infectious disease to initiate ani im-mune responise in the infected host has been kniownfor decades, but it has been recogniized only receintlythat as well as being stimiiulated by infectioin, immiiuineeffector functioins imav also be depressed. As a result,the ability of the host to mount an effective immuneresponse against ain invading pathogen may be severelycomproimiised and there are an increasiing number ofinfectious diseases in man and experimeintal animilalswhere a depressed cell-mediated immune response hasbeen associated with ain adverse clinical course (1, 2).

The elucidation of cellular interaction controllingthe immune response to infection is one of the principalobjectives in the study of the immunobiology of infec-tious disease. Most experiments have been directedtoward characterizing subpopulations of T cells, butthere is increasing evidence that activated macro-phages are also important cellular componients con-trolling the host's immune response to infectioin (3, 4).The direct inhibitory effect of prostaglandins secretedby activated macrophages is one likely regulatorymechanismii. Goodwin et al. (5) have described a prosta-glandin-producing suppressor cell in human periph-eral blood that inhibits in vitro, lymphocyte activa-tion by T cell mitogen. Suppressor cell activity thatacts through the production of prostaglandin' has beendescribed in both experimental animals and man (6,7). This indicates that the naturally occurring periph-eral blood suppressor cells in man and splenic sup-pressor cells in experimental animals may be relatedin both function and mode of action.

Knowledge of the relationship between the naturallyoccurring suppressor cell in man and the suppressorcell induced during infectious disease may make a sig-

tion, Inc. 0021-973818011010621/08 $1.00 621Volume 66 October 1980 621 -628

nificanit contribution to our uniderstanidinig of the im-ml-unoliology of infectious disease. In the present ex-perimiienits we have shown that the T-lymphocyteeffector status of splenic lymlphocytes in experi-mentally-iniduced renal infection is determinied by twosuppressor cells, one naturally occurring and the otherinifection iniduced. The two suppressor cells are dis-tinguishable oin the basis of their physical characteris-tics anid the capacity of the prostaglandin synthetaseinhibitor, inidomethacin, to block the immunosuppres-sive activity. These cells, we believe, can be realis-ticallv viewed as prototypes of the niaturally ocecurrinigsuppressor cell anid the inifectioni-indcluced suppressorcell in man1.

METHODS

Aniimal str-ainl. DA x AS2 F, hy)rid f'emiale rats weigh-iing 200-260 g were used in these experimiienits.

Bacterial strain The strain of Escherichia coli 075 usedl inthese experimiienits was the saimie as that used in previousstur(lies of experimiiental pyelonephritis (8).

Producction of renal inifectioni. Pyelonephritis was induce(dbN (lirect inoculationi of E. coli into the surgically exposedlkidney. Details of this metho(d halve been given previ-oulsly (9).

Characterilzationi of spleic cells. Phagocytic cells wvereidenitifiedl by their ability to ingest latex particles (0.84 ,Im,Dow Corninig Corp., Midland, Mich.) (10). Mononouclear andpolymorphonuclear phagocytic cells were differenitiated bydirect examliinationi of cytocenitritugedl preparationis. Lymphoidcells dlemiionistrating peripheral fluorescenice using fluores-ceiin-conijutgatedl rabbit anti-rat i mmunogloblilin (WVellcomeReagents Ltd., Kent, England) were idlentified as B cells.Surf:ace staininlg was performed accor(inig to Pernis et al.(11) with minor modificationis. T cells were identified usingthe Pta.A.1 lymphocyte-specific surf:ace anitigeni (12, 13). Thepresenice of the antigeni Pta.A.1 onl the surface of the Tlymphocytes was determinied by anl indclirect i mmiuniofluores-cenice technii(lue involving incubation of spleniic cells withLevis aniti-Fischer F344 (anti-Pta.A. 1) serumll followed byHuoresceini-conijtigated rabbit aniti-riat immunoglobtilin. In thisproceclure both T and B lymphocytes (lemiionstrated peripheralfluorescenice and the number of T lymphocytes was obtainedbv subtracting the number of cells with surface immlluno-globulil.

In vitro (ali/alysis of the iniitogenlic responise to T-lytinpho-ci te specific mnlitogetns. Cultures of splenic lymphocyteswere established in tissue culture tubes (Falcon Labware,Div. of Becton, Dickinson & Co., Oxnard, Calif.; 3033). Fulldetails of the culture procedure have been presented else-where (14).

Fractiontationi of splentic lynmphoci tes. Splenic lymphoidcells were prepared and the suspension was washed once inRPMI-1640 (Gibco Laboratories, Grand Island BiologicalCo., Granid Island, N. Y.), resuspended in 10 ml of the samemedium and then centrifuged at 30g for 2 min. Depositedcells were recovered as the "sedimenitable fraction" and thelymphocytes in the supernate were labeled the "nonsedi-mentable fraction."

Pla stic adheretnce. The sedimenitable fraction from normalor pyelonephritic spleens was resuspended in RPMI-1640medium containing 10% fetal calf serum. 2 ml of mediumcontaining varying numbers of sedimentable lymphocytes,as indicated in the text for individual experiments, were then

addledI to plastic petri dishes (Falconi Labware, 1008) audincubated aIt 37°C for 30 min. Nona(lherenit cells were re-moved, pooledl, and( cultured in test tubes with 2.5 x 106niormiial nonisedimenitable lymphocytes. The petri (lishes weretheni washed twice with fresh mediumiii. 3 ml of comiipleteimiedliumiii conitaininlg 5 x 10'i normiial nonsedimentable lympho-cvtes were theni add(le( to each petri dish. Proce(dures de-scribed previously (14) were then use(d to determiinle the(legree of [:3H]thymidfine incorporationi.

Preparation of agentsIn(lomethlacin. A stock solution of inldomethacini, 500 mg/

100 ml in 95% alcohol (indomethacini NF, 1475, Merck,Sharp and Dohbme Ltd., New Zealand(l) was (lilute(d inl 95%ethaniol so that 6 ,ul added to cultures of spleniic lvmpho-cytes gave final conicenitrationis of indomiiethacini in cultuire of 1or 10 Ag/mlll.

Prostaglandini El. A stock solution of( prostaglalnin El(Sigmiia Chemical Co., St. Louis, Mo.) vas prepatred by dis-solving 35.45 mig of prostaglanidini E, in 1 ml of 95% ethaniol.Dilutionis were made in ethanol so that 5 ,lA, wheni added tocultures of' spleniic lymphocytes, gave a ranige of concentra-tioins of rostaglandlin in cuiltuire betweeni 10 and 90 A1M.

Dibtytt r/I cyclic AMP. 83.1 nmg of dibutyryl cyclic AMP(Sigmiia Chemical Co.) was dissolve(d in 0.25 ml of phos-phate-bufferedl salinie anid filtered through a 0.45-,um filter.5 Al of this soilution and of a 1 in 10 dilutitio were a(ldle(l tolymphocyte cultures giving fincal co(Icentratio(Is of 1 mMald0.1 mIM dibutvryl cyclic AMP.

RESULTS

Characterization of the infectioni-itnducedsuppressor cell

Morphologic evaluiation of the coin positiotn of spleencellfractionisfromii niornmal atnd pyteloiephritic anlilimals.Renal infection led to a 43% increase in total spleencell numbers 72 h after challenge, but the percentageof T and B cells in the spleen was not altered.Centrifugation at 30g depleted the nonsedimentablefraction of mononuclear phagocytic cells. These cellswere concentrated in the sedimentable, suppressorcell-containing fraction that consisted of 24 and 27% ofthe total spleen cells from normal and pyelonephriticanimals, respectively (Table I).

Cells pelleted by lowc-speed cetntrifuigation havesuppressor activity. Splenic lymphocyte suspensionsfrom normal and pyelonephritic animals were frac-tionated into a sedimentable and nonsedimentablefraction by cenitrifugation at 30 g for 2 min. Standardcultures in triplicate were established using: (a) non-sedimentable lymphocytes from normal animals, (b)sedimentable cells from normal and pyelonephriticanimals sacrificed 48 h after challenge, and (c) co-culture of the sedimentable fraction from normal andpyelonephritic animals with nonsedimentable lympho-cytes from normal animals. The results (Table II) haveshown that cells in the sedimentable fraction frompyelonephritic animals, when compared with sedi-

622 T. Miller and E. Marshall

TABLE IEffect of 30 g Cenitrifuigationl otn thle Celilar Compositioni of Fractions of Splenic Cell Suspensions

fromti Normi al anidl Pyjelonephrtitic Aniimals

Normal* Ps eloinephritic t

Spleeti T'otall TotalCell spleen cells spleen cells

tratctioni x 10" T B PMN§ MPC u1D x 106 T B PNIN§ MPC UID¶

Nonfractioncate(d 114 45 36 6 7 6 164 46 33 14 8 0Nonsedimentable 72 40 40 6 1 13 102 40 41 4 1 14Sedimentable 27 30 29 11 10 20 44 31 31 19 13 6

Mean, ninie niormiial rats.Mean, eight pyelonephritic rats.

§ Polymorphonuclear leukocytes.Mononuclear phagocytic cells.Unii(lentifie(d cells.

imenitable cells froimi normal aniimlals, cain greatlystuppress the Conicaniavalin (Con) A response of normalspleinic lymphocytes (81 vs. 8%). Similar suppressionwas seein in experiments carried out 72 h afterchallenige.

Sedinen table vp)pl)Cressor cells (lisplay adherenitcharacteristics. Sedimentable cells were obtaiinedfrom splenic cell suspensions prepared from pyelo-nephritic and normal animals. Varying numbers from1 x 1063 to 10 x 106 of sedimeintable cells were added to35-imm plastic petri dishes and allowed to adhere over a30-mmlii incubation period at 37°C. Nonadherent cellswere removed and co-cultured with nonsedimentablelyimiphocytes from normal aniimlals. These cells did notdepress the mitogenic responsiveness of normalsplenic lymphocytes; rather, enhanced responseswere found when cells from the normal sedimentablebut nonadherent fractioin were added to the culture

(Fig. 1). Marked suppression could be demonstratedwhen sedimiienitable adherent cells from pyelonephriticaniimlals were added to normiial lymphocytes. The effectwas (lose dependeint and could readilv be differenti-ated, on a (uanatitative basis, from the suppressiveactivity of naturally occurring suppressor cells in theequivalenit fractioin of adherent cells from normal ani-miials (Fig. 2).

Effect of iniclomiiethlacini on n1atu rally occ urrintg an1ditifectioni-inidced stuppressor cells. The suppressionof mitogeinic responses in vitro has been attributedto prostaglandin synthesis by cellular componeints. Inthese experiments we have determined whether sup-pressioni of T lymphocyte functioin, attributed tonaturally occurring and infection-iinduced suppressorcells, could be inhibited by the prostaglandin syn-thetase inhibitor, indomethacin. Addition of indo-methacin to uinfractionated splenic lymphocytes froml

TABLE IICo-cuiltuire Demtionistrationi of Suppressor Cell Activity in the Sedimientitable Fraction

of Splenic Lb1ymphocy tes frotmz Pylelontephritic Animiiials

Individual cultures* Stippressioni of n)ormiialresponises

Nonsedinmentable Sedimiienitablelymphocytes cells Sedimentable cells

Pvelone- I Pvelone-Normiial phritic Normiial Aniticipated response Observed responise phritic Normiial

(A) (B) (C) (A + B) (A + C) (A + B) (A + C)256,200 334 10,800 256,600 267,000 49,280 253,900 81 8+16,020 ±156 +3,820

* 2.5 x 106 spleen cells: dpm/106 lymphocytes±SE.M.2.5 x 106 normal nonsedimentable lymphocytes + 0.6 x 106 sedimentable cells. -Mitogenic response of lylphocytes

to Con A dlisintegrations per minute per 106 lymphocytes.

Stippressor Cell Modulation in Renal Itifection 6623

40E

30CL;20

E

10

0 0.08 0.15 0.30 0.60nonadherent sedimentable cells (x 106)

P(; f1'CcItff Ato lmdo wro-lIl\ t s(dil]elwltilbl(} ( v]ls mI

lnorma1, l mit)ge l ci resplnse S. 1() x 10" s(lidillwiltaille splellic(elIs froMl n110I alA (U) 01 pvel mwplbI ritie n ii mias (LI) weredllv(d(l to t(1dlwit to p)lstic- petr dishes. The iiomid(lheeiilt

cill frt-rtioll wi5s reov(ered and in creasill in e-rs (al ibsisscis\\v('i'i (stalblisjlldl iII to-ClIture with 2.5 x 10' iioniaml nioni-S(}dlllwl)t~lbh} l-ymphl!(wvUs. 1Vao-.l1 b'11- 1vp("w11etS t1w 1wa'of tlip)lieatt, itlvl'5s(S.

io)0inal rats inctlease(l tle 1:l1 Itilh iilym ildie uiptake lbyli I i t( 1re u-st jIila ite dlIvilhoctcs fr()om 8,900() dpimto :321 ,90() dlpm/(I l0 ymphOcv s (Fig. 3). I dlom0ethacilcii)

400

300

200

E

100

x 50E

~40

30

20

10

0 1x104 1.75x104 1.25x106 3.3x106 5.4 x106adherent sedimentable cells

Fic ';u _, 2 EfL Cet (i atd1rivit ((1 11() lilble el tIs ()11 n l()ilaliitogllii resp)miises. 1, 2.5, 5, a.5, ld ( ) x 10"' sedIimilenit-il)lt spl]ii ic' (eells firom01 ui0iirmiI (U) id pyelonemphritic anli-

iliil.s (17) \v(1v( atllowed ttoatdhere lto plastic p)etJri tlislhes. TheX11(1ildieiit cells wer-e diser-d(led leaxivlg varvilng 1iiuiliers of'

esisduial atdherent c'ells ()sci ssa). .5 x 10"; norimal nonsed(li-li.lltble splenie lymphloevtes were theii d(de(d to the petridli.shs to fhirl .1 eo-eciltire Eachki- r ri)preseiits the meinof quiiitup i o,o)l.iete alnalyse s.

350

300

a, 250

E 200

a)

1502

E

100

50

0

Nrmal

a,\1

%

I' c0 t

I

I.~~~~~~~~~~~~~~~~~~02505l 55

350 r |Pyetonephritic 1

300 [

,, 250a)

E 200

a)

150ExECL

100

50,6 --A

AN

-10.25 0.5 1.0 2.5 5.0

/ ml Con A

Fi(;'n I: 3 1 ff ( t f indlmniethaetin (1 /Ig/l on1 tliit(ito. ei ir-sp)olsii\i( slicss of iomifl irctioiiiatedl splelli it l\ 1iiuot fites 0looniii)riiii1 anl d p (mo phlrhitic animals. ath pmoint is the necnof anl allnaisiSu maried mi it ii tril)licate. of a pooled Sple ,ic

lx1))pl)ete popuil itiom f-mli three normal an mals. Fach (c11-ture coiitaiid 2i .5 i)x 10)spleie lmIiph)locyVtecs. 0 mid

sAIleit l'itr a tcd t fi(lic lyolil ll t1s 1) t loi0el)h aititd pvilo;

aiso intceras. tl the immnetill responsis ixelsS of sp)leiiitclymphocytes ff1om11 pyelonephritic animals, l)lt al-

tlhouighi the percen)tage iincrese iii 1HJtlhymi(inc uip-t.ake was tomipl)arable to that seen with normnal animals(263 and 351 c4( ,respectively), the mlitogenitc re-sponsiveness of imopibethacin-treated liii poCytesf'romii the pvelonephritic an inials (39,02() (1O)11p/10('lymphocytes) was still oily 129/ (f' the respmoiise ofthe compIl)arable po)ulation of lymilpocytes from- 1normalanimals (321,90() dpm/10' lymphi eOcts). The eXperi-ineits atlso showed that the effect of iidomlethaciniwas related to the concenitration of' (C'o A bnt niot in-versekl related ats hadi l)een found previously (1.5).

I)cprc-.s'.s'd (cll-ttlC(liated c )10111 ii(' 1'/)OliSCS'll1carc ducto alnl ipll(1rctiletliaci-in.s'cnl.siticc 'so pprc-.s.soc cell. Theresuilts (If the prexiotis experiments suggested that theactivity (If the ilofectioll-in(utcec(l souppressor tel asniot inhihited by ilidoniethlaclini. This was clnfirmedl illthe Pi-rsenit ex)erimilenit where it waIs fouind that intlo-inethac- iin was unable to block the immuilnostul)pres-sive activity o(f stirfnce-aclherenit snppressor cells. These(limllentahle firaction fromi pyelonephritic spleens wasallowed tol adhere to the h)ottom (If a 3.5-inil ti sstie

c-ultture (lish for 30 min.m lN adherent cells were thieniremoved by repeatedI washings wvith culture 13)etlitlml.Duplicate stispenisionis of spleniic lymphocytes fromniormllal animaictls were established in a series of 35-mm

624 1'. .\lille r-o(1a .E. M s/ha/ll

pi/

petri dishes, half of which conitained the adherentcells from the spleens of pyelonephritic animals. Tri-tiated thymidine incorporatioin by Con A-stimulatedlymphocytes in the absence of adherent suppressorcells (807,200 dpm/culture) was reduced to 112,000dpm/culture when lymphocytes were culturedconjointly with adherent cells from pyelonephritic ani-mals (Fig. 4). The additioni of indomethacin (1 ,ug/ml) atthe samiie time as the cultures were established didnot inhibit the suppressor activity of the adherent cellsand simiiilar results were obtained when 10 times theconicenitrationi of indomiiethacini was added to theculture.

I in ulnlCe responsiveness can be restored toSplen ic ly m)hocytes from pJyelon)ephritican imcals itn tWvo steps(a) Phljsical remioval of the itnfection1-inldtice'd s17p-

)res.sor cell. Spleniic lymphocytes responid to mitogeniiestimiiulationi in vitro if suppressor cells are removed.Previous studies have shown that fractioncationi ofsplenic lymphocytes from pyelonephritic animals 1y30 g centrifugationi enhaniced the immune responsive-ness of' the nonisedimiieintable fraction (16). Spleniclymphocytes f'rom normal and pyelonephritic animilcalswere centrifuged at 30 g for 2 mimi anid cultures of thenonsedimentable cells were established. Fractionia-tion by centrifutgationi at 30 g resulted in a small in-crease in mitogeniic responsiveniess of the nionisedi-mentable cells from normiial animiials (76,100-98,780dpm/106 cells) (Table III). In conitrast there was amilarked enihaiieeinent in the uptake of [3H]thymidineby the nonsedimentable cells from pyelonephriticaniimals (4,150-53,600 dpmn/106 lymphocytes).

(b) Pharmiiacologic inihibitiotn of suippressor cell ac-tivitt bt1 indotmethacin. The immilunie responsiveness

* splenic lymphocytes

- Splenic LymOes

E 5M Spienic lymphocytes,600 Adhered cells 1- 10'

o0 Indomethacin 1pg/ml

Lx. Splenrc lymphocytes4

Adhered cells 1x10'Kt Indomethacin lOpg/ml

P 300

Em 200

100

FIGURE 4 Effect of ini(idomethacin. (1 ani(d 10 gg/nml) on thesuppressor activity of aclherenit splenic cells fromii pyelo-niephritic animilals co-ctultture(l wvith nonsedimentable 1villpho-cv-tes fro011 inorial animilals.

of the nonsedimentable spleniic lymphocytes cani befurther enhanceed bL indomiiethacin treatmiienit. Dupli-cate suspensionis of nlonsedimentable, pooledl spleniiclymphocytes froi 01 ormiial anid pyelonephriti c animalswere culturecl in vitro. The additioni of iindomiethlacito half the ecultures of' nonsedimentable spleniielymphocytes fromii niormlal animilals resuilted in a miiarke(dinierease in their mitogeniie responisiveniess. Frac-tioniated splenic ilymphocytes f'romii pyelonephritic anii-miials, after stuppressor cells had beeni remiiovecl b1 30 gcenitriftugationi, also showed a miiarked inierease in imiito-geniic responisiveniess wheni inidomiiethacini was addled( tothe 1 lmphocyte ctultture. Tritiated thvmiiidiine uiptatkeinerease(d f'romi 81,570 to 217,200 dpm/10 lymphocytesanid exceedled the miiitogeniic responsiveness of al comi-parable spleinie lymphocyte populationi fromii nioralllcl

TABLE IIIEffrct of FJractioniationi bil 30 g Cen trifugation on the Alitogcn ic Re.spon.se.s

(of Spl nic Llinphocitte.s fr-om,, Normtial a11(1 Pilcloncplh ritic Aniimals

Noralllsl animiaslls Pyelomiephritic ialiimaulls

Inrealse with Increase withA.ninmals Nonifractionated8 Fractionated t tractiounationl§ Nonflractiomated Fractionated I fractioniationt

dpinIO" him11n/pocltes tIpminO0 hi/??II)/h(ci/tes c'

1 76,220 97,400 23 4,550 110,1(( 2:3202 91,630 82,580 0 5,700 15,600 1733 66,290 119,500 8( 1,750 71,840 40104 76,100 87,800 15 5,980 48,810 7155 57,000 76,590 34 2,300 39,450 16106 89,200 128,800 44 4,640 35,850 672

MCeain±SE 76,100) 5,400 98,780(8,58() 31±+12 4,150( 715 53,600) 13,54() 1,580±+578

* UnimaniipuilatedI whole spleeni suispenlsioII.4 Nonseclimentahle fractioni obtained fromii the whole spleen suspension followilg 30 g celntrifutgationi.§ Increase in Coni A responsiveness of lymphocytes following the removal of the Se(limentabl)le fractioni 1y 30 g centriflogattiol.

S up)pr-e.S.Sor- Cell Modulation in Rena(il Infection 6625

TABLE IVEffect of Indomethacin on the Nonsedimentable Splenic Lymphocytes from Normal

and Pyelonephritic Animals after Removal of the "30-g" Suppressor Cell

Normal animals Pyelonephritic animals

Con A per Without Increase Without Increasemilliliter indo- Indo- with indo- indo- Indo- with indo-of culture methacin methacin* methacin methacin methacin* methacin

,ul dpm/106 lymphocytes % dpmIlO6 lymphocytes %

5.0 86,270 178,400 106 81,570 217,200 1662.5 78,120 108,500 39 58,270 89,970 521.0 35,150 36,420 4 18,930 22,120 160.15 22,530 24,370 8 15,290 15,190 00.25 17,000 11,010 0 5,400 6,570 21

* 1 ,ug/ml of culture.

animals (Table IV). In Table V the results of the presentexperiments have been summarized and show that theimmune potential of splenic lymphocytes is deter-mined by two separate suppressor cells. A naturallyoccurring suppressor cell, inhibited by indomethacin,was demonstrated in splenic lymphocyte suspensionsfrom normal animals (87,900-321,900 dpm/106 lympho-cytes). When the infection-induced suppressor cellswere first removed by 30 g centrifugation, however, theimmune responsiveness of the nonsedimentablelymphocytes from control animals and the pyelo-nephritic group was comparable (86,270 and 81,570dpm/106 lymphocytes, respectively). In the absence ofthe infection-induced suppressor cell, lymphocytesfrom pyelonephritic animals, whose immune re-sponsiveness had been previously only partially en-hanced by indomethacin (39,020 dpm/106 lymphocytescompared with 321,900 for normal animals), respondedto mitogenic stimulation at a level that exceeded nor-mal splenic lymphocytes examined under the sameexperimental conditions (217,200 vs. 178,400 dpm/106lymphocytes).

DISCUSSION

The experiments have demonstrated that T lympho-cyte effector status in experimentally induced renalinfection is determined by two suppressor cells; oneinfection-induced and the other naturally occurring.The inability to respond to mitogenic stimulation wasreversible and restoration of immune responsivenessto splenic lymphocytes from infected animals wasachieved in two stepwise manipulations. Differentialcentrifugation removed the infection-induced sup-pressor cells and the suppressor activity of the naturallyoccurring suppressor cells could then be inhibited byindomethacin. Thus the two suppressor cells were dis-tinguishable on the basis of their physical characteris-tics and their response to indomethacin. The -infec-tion-induced suppressor cell was further characterizedas a sedimentable cell (30 g) with suppressor activitydemonstrable in co-culture experiments. Plastic-ad-herent cells from the sedimentable fraction of spleniccell suspensions from pyelonephritic animals sup-pressed the mitogenic responsiveness of normal

TABLE VEffect of the "30-g" Suppressor Cell on the Indomethacin Enhanceable Mitogenic Response

Nornal animals Pyelonephritic animals

Nonfractionated splenocytes* 30 g fractionated splenocytest Nonfractionated splenocytes* 30 g fractionated splenocytesI

Without Without Without Withoutindo- Indo- indo- Indo- indo- Indo- indo- Indo-

methacin methacin§ methacin methacin§ methacin methacin§ methacin methacin§

dpmIlO lymphocytes dpmIlO6 lymphocytes

87,9001 321,9001 86,27091 178,40011 8,6401 39,0201 81,5709 217,20091

* Unmanipulated whole spleen suspension.t Nonsedimentable fraction obtained from the whole spleen suspension following 30 g centrifugation.§ 1 ,ug/ml culture."i See Fig. 3.¶ See Table IV.

626 T. Miller and E. Marshall

lymphocytes but the immunosuppressive activity ofthe infection-induced suppressor cell was not inhibitedby indomethacin.

Suppression of cell-mediated immunity has beendemonstrated in infectious diseases both in man andexperimental animals (17, 18). In addition to the pres-ent experiments suppressed cell-mediated immunityhas also been demonstrated in tumor-bearing animals(19), experimentally induced malaria (20), schisto-somiasis (21), trypanosomiasis (22), and after the injec-tion of adjuvants (23). Thus infection-induced immunenonresponsiveness may be a much more commonresponse of the host to infection than has been recog-nized previously but the nature of the biologic mecha-nisms that induce the immunosuppressive state is notwell understood. One of the objectives of these ex-periments was to investigate the role of the prosta-glandins as immunoregulatory molecules controllingcell-mediated immune responses to infection. Prosta-glandins of the E series are produced by macrophagesin substantial amounts after activation and can inhibitlymphocyte functions (24, 25). In the present experi-ments the addition of indomethacin, a specific prosta-glandin synthetase inhibitor, to cultures of spleniclymphocytes from normal and pyelonephritic animalsresults in a considerable increase in the mitogenic re-sponse (351 and 263% for normal and pyelonephriticanimals, respectively). The response of pyelonephriticlymphocytes, however, was still only 12% of that of thecomparable population of lymphocytes from normalanimals.

Although the activity of the naturally occurring sup-pressor cell is inhibited by indomethacin and thusshares this and other characteristics with the naturallyoccurring suppressor cell found in man (5), the infec-tion-induced suppressor cell appears to be unique. Thelatter cell differs from the naturally occurring suppres-sor cell both in physical characteristics (i.e., removableby 30 g centrifugation) and biologically in that its sup-pressor activity is not inhibited by indomethacin.

A number of investigators have described the im-munosuppressive activity of surface-adherent cells,presumed to be mononuclear phagocytes, obtainedfrom the spleens of animals challenged with a varietyof stimuli (26-28). The mechanism of action of thesuppressor cell has yet to be defined but a reasonableassumption is that because activated macrophages syn-thesize increased amounts of prostaglandin, this agentcould lead to depressed cell-mediated responses. Thepresent experiments have shown that the naturally oc-curring suppressor cells can depress cell-mediated im-mune function through prostaglandin activity. How-ever, the mechanism by which the infection-inducedsuppressor cells act has yet to be defined.

One important conclusion is that although theunmanipulated splenic lymphocytes from pyelo-

nephritic animals are nonresponsive in culture, fullimmune responsiveness can be restored. The abilityof these cells to respond to mitogenic stimulation isdetermined by two suppressor cells. One, a naturallyoccurring suppressor cell, is inhibited by indometha-cin but only in the absence of the infection-inducedsuppressor cells. A recent observation that an indo-methacin-resistant splenic suppressor cell developedin mice moved from germ-free to conventional condi-tions (6) supports our concept of microbial infectionas a factor stimulating the generation of an indo-methacin-resistant suppressor cell that can be distin-guished from the naturally occurring suppressor cell.It seems likely that the cell described by Mattinglyet al. (6) and the infection-induced suppressor celldescribed in the present experiments may be derivedfrom the same cell lineage.

These experiments have shown that the infection-induced suppressor cell is the dominant factor deter-mining the immune responsiveness of splenic lympho-cytes from pyelonephritic animals, but unlike thenaturally occurring suppressor cell, the immunosup-pressive activity of the infection-induced suppressorcell is not inhibited by indomethacin. Our intention isto use both these cells as models to study the roleof suppressor cells in the pathogenesis of infectiousdisease in man.

ACKNOWLEDGMENTThe assistance of Mrs. Jan Nelson with the morphologicalanalyses carried out in this study is gratefully acknowledged.

The study was supported by the Medical Research Coun-cil of New Zealand.

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628 T. Miller antd E. Mlarshall