Modification by Suppressor Cells and Serum

Transcription

1 Modifiction by Suppressor Cells nd Serum Fctors of the Cell-Medited Immune Response in Experimentl Pyelonephritis THOMS MILLER, LESLEY SCOTT, ELINE STEWRT, nd DEREK NORTH, Deprtment of Medicine, ucklnd Hospitl, ucklnd, New Zelnd B S T R C T mrked suppression of the thymusderived (T)-lymphocyte response to concnvlin hs been demonstrted in vitro during renl infection. Suppression of the T-lymphocyte response in vitro ws seen s erly s 2 h fter the induction of renl infection, but mximum suppression ws found h lter. popultion of suppressor cells in the splenic lymphocyte popultion, generted during the host's response to infection, contributed to the depressed lymphocyte response. Removl of suppressor cells rtestored the mitogenic responsiveness ofthe remining splenic lymphocytes. Conversely, in co-culture experiments, suppressor cell present in the splenic lymphocyte popultion ofpyelonephritic nimls ws shown to be cpble of suppressing the mitogenic responsiveness of norml splenic lymphocytes. Significntly reduced host vs. grft responses by the pyelonephritic nimls confirmed, in vivo, the depression of cellmedited immune mechnisms. n dditionl suppressive fctor ws found in the serum of pyelonephritic nimls which depressed in vitro the mitogenic responsiveness of splenic lymphocytes from norml nimls. Support for the suppressive role of this serum fctor ws found when splenic lymphocytes from pyelonephritic nimls were tested in vivo in the bsence of homolgous serum (grft vs. host). nder these conditions, the lymphocytes showed n enhnced rection compred with lymphocytes from norml nimls. The presence of suppressor cell popultion nd serum fctor, both cpble of depressing cell-medited mechnisms, my be mjor fctors contributing to the estblishment of infection in the kidney. INTRODCTION lthough the epidemiology nd nturl history of renl infection is well documented, convincing studies tht Received for publiction 16 My 1977 nd in revised form 3 November explin the peculir susceptibility of renl tissue to infection re lcking. Recently, number of investigtors hve studied the nture of the immune response to renl infection nd hve chrcterized the biology of the host response to infectious chllenge (1-8). s result, detils of the locl nd systemic immune response ginst the invding orgnism hve been described in detil, but the biologicl significnce nd reltionship of the ctivity of the immune system to the pthogenesis nd immunobiology of pyelonephritis is still uncler. Erly studies indicted tht the renl environment ws not fvorble milieu for the expression of some host defense mechnisms (9) nd recently it hs been reported tht thymus-derived (T)-lymphocyte function my be depressed in the renl environment (1, 11), which suggests n ssocition between the persistence of infection nd n inmpirment of cell-medited immune responses. Other experiments hve indicted tht the immune response to infection not only is ineffective in eliminting the invding orgnism, but ctully protects the orgnism from host defense mechnisms (12). In the present experiments we describe the modultion of the host's immune response to renl infection coinciding with the period when rpid bcteril growth is tking plce in the kidney. These studies hve estblished tht in vitro mrked suppression of T- lymphocyte response to mitogenic stimultion occurs during renl infection with Escherichi coli nd tht the functionl cpcity of the T-lymphocytes in vivo is lso significntly reduced. n immunosuppressive mteril ws found in the serum of the pyelonephritic nimls nd popultion of cells cpble of suppressing the splenic lymphocyte response to T- lymphocyte mitogen ws lso detected. Removl of the suppressive cells restored the mitogenic responsiveness of the lymphocytes from the pyelonephritic nimls to norml levels. 964 J. Clin. Invest. The mericn Society for Clinicl Investigtion, Inc., /78/ $1.

2 METHODS niml strins. Femle rts weighing g obtined from n inbred strin of D rts were used. Inbred rts of the HS nd S2 strins, which hve mjor incomptibility t the g-b locus, were used in the grft vs. host rection. Bcteril strin. The strin of E. coli.75 used in these experiments ws the sme s tht used in previous studies of experimentl pyelonephritis (5-8). Production of renl infection. Pyelonephritis ws induced by direct inocultion of E. coli into the surgiclly exposed kidney with glss microcpillry. Detils ofthis method hve been given previously (13). Bcteril content ofrenl tissue. Nutrient gr pour pltes of seril 1-fold dilutions of homogenized kidneys were mde to obtin the bcteril count per grm of wet renl tissue. In vitro nlysis of concnvlin -stimulted mitogenesis of T lymphocytes. Lymphoid cells were obtined from the spleens by mincing them with curved iris scissors nd the tissue frgments were tesed through 6-mesh stinless steel guze to produce single cell suspension. This ws wshed once nd then plced in cold Egle's miniml essentil medium (MEM)l (Grnd Islnd Biologicl Co., Grnd Islnd, N. Y.), buffered with Hepes to finl ph of 7.2 (Hepes-MEM). fter removing cell clumps (5 min t 1 g) the cell suspension ws counted nd sufficient cells were smpled to provide culture in triplicte; ech culture contined 5 x 16 lymphocytes. These lymphocytes were gin centrifuged nd then deposited cells resuspended in "insert" medium (see below). Roswell Prk Memoril Institute complete medium (RPMI- 164) ws used in ll the experiments nd contined 7 ml of 2.8% sodium bicrbonte per liter to mintin the culture t ph 7.2 in n tmosphere of 1% CO2 in ir. The medium lso contined 1,tg/ml of penicillin nd 1,ug/ml of streptomycin. Rt serum obtined from control nimls t the time of scrifice ws dded to the complete medium to give finl concentrtion of 5% rt serum nd this provided the "insert" medium in which the cells were cultured. 5 x 16 splenic lymphocytes in 1 ml of insert medium were suspended on dilysis membrne in Mrbrook culture vessel (14), contining 1 ml of RPMI-164 medium in the externl reservoir. 5 mg of concnvlin (Con ) ws dissolved in 5 ml of phosphte-buffered sline nd 1,ul ws dded to the contents of ech "insert" tube. nstimulted cultures contining splenocytes without Con were included s control with ech cell suspension ssyed. Cultures were held for 3 dys t 37 C in humid tmosphere of 1% C2 in ir. 16 h before hrvesting on the 4th dy, 1 /Ci of [3H]thymidine (2 Ci/mmol) ws dded to ech culture (The Rdiochemicl Centre, mershm, Englnd). The mount of [3H]thymidine incorported into cellulr DN ws determined fter terminting the culture by the ddition of distilled wter, nd filtrtion of ech culture through Whtmn GFC 2.5-cm glss fiber filter (Whtmn, Inc., Clifton, N. J.) held in mchined metl block. The contents of the insert were rinsed with further volume of distilled wter nd finlly with 1% nd 5% TC to precipitte mcromolecules retined in the fiberglss filter. Filters were trnsferred to glss vils for [3H]thymidine counting which ws crried out in Beckmn model B liquid scintilltion spectrometer (Beckmn Instruments, Inc., Fullerton, Clif.). The procedure gve consistent replicte vlues to within 1%. Con stimultion of 5 x 16 splenocytes from norml nimls resulted in [3H]thymidine incorportion of pproximtely 1, dpm/16 lymphocytes fter the ddition of 1,tCi of 'bbrevitions used in this pper: Con, concnvlin ; MEM, Egles's miniml essentil medium. [3H]thymidine. In unstimulted control cultures, less thn 1, dpm/16 lymphocytes were incorported. Grft vs. host nd host vs. grft response. The poplitel lymph node weight method of ssy for determining the grft vs host ctivity of rt lymphoid cells ws crried out s described by Ford et l. (15) with the Fl progeny of the g-b disprte rt strins S2 nd HS. 5 x 16 splenic' lymphocytes from control nd pyelonephritic prentl nimls of the S2 strin were injected subcutneously into the left foot pd of Fl progeny t 4-6 wk of ge. similr number of syngeneic cells were introduced into the right foot pd nd in third group, culture medium lone ws injected. 7 dys lter the recipient nimls were killed nd the poplitel lymph nodes were removed nd weighed fter the dherent connective tissue hd been removed. similr protocol ws used to determine the host vs grft response of norml nd pyelonephritic nimls. Pyelonephritis ws induced in prentl S2 nimls nd 5 x 16 splenic lymphocytes from the Fl progeny were introduced into the left foot pd of pyelonephritic nimls 2 dys fter chllenge. Syngeneic lymphocytes were injected into the right foot s control. 4 dys lter the prentl host nimls were killed nd the poplitel lymph nodes removed nd weighed (16). Frctiontion of splenic lymphocytes. Splenic lymphocyte suspensions were prepred in RPM by tesing tissue frgments through 6-mesh stinless steel guze. Clumps were removed by llowing the suspension to stnd on ice for 5 min. The splenocyte suspension ws then wshed once in RPM resuspended in 1 ml of the sme medium nd then centrifuged t 3 g for 2 min (17). Deposited cells were recovered s the "pelletble frction" nd the lymphocytes in the supernte were lbeled the "nonpelletble frction." Sttisticl nlysis. nlysis of the dt ws crried out with the Wilcoxon sum of rnks method. Procedurl controls. Norml nimls, subjected to shm chllenge with sline in plce of the bcteril inoculum, were used s the control group in these experiments. RESLTS Pyelonephritis. Injection of E. coli into the kidney with glss microcpillry results in consistent nd reproducible injection in the renl prenchym. The erliest gross pthologicl chnges re detected on the 4th dy fter infection s discrete, wedge-shped corticl lesions originting long the line of inocultion nd extending into the medull of the kidney. Histologicl evidence of ctive renl infection is detected 48 h fter chllenge. trnsient bcteremi occurs in the 3 min fter inocultion of bcteri into the kidney but subsequent blood cultures re sterile. smll number of bcteri re found in the spleen nd liver for 48 h fter the induction of infection, wheres the bcteril content of the pyelonephritic kidney remins high. The increse in bcteril numbers in the kidney during the first 48 h fter bcteril chllenge is shown in Fig. 1. In vitro response to Con of splenic lymphocytes from pyelonephritic nimls. In these experiments the bility of splenic lymphocytes to respond to Con stitnultion in culture ws used s n index of their potentil function s effector cells in the cell- Cell-Medited Immunity in Experimentl Pyelonephritis 965

3 C Go,D L- GC' hours fter chllenge FIGRE 1 Bcteril numbers in the kidney fter the direct inocultion of E. coli 975. Ech point is the men of the results obtined from six nimls±sem. medited immume response. In 55 consecutive cultures of splenocytes from norml nimls n verge of 119,726 dpm/16 lymphocytes ws found fter the ddition of 1,uCi of tritited thymidine to the culture. With ech experiment, ge-mtched shmoperted nimls were killed nd cultures prepred in prllel with the test nimls. The results (Fig. 2) re expressed s percentge of the response of lymphocytes from norml nimls. decrese in the response of T lymphocytes in the spleen ws found s erly s 2 h fter the initil chllenge, nd by 24 h drmtic decrese in the mitogenic responsiveness of splenic lymphocytes ws found which persisted for 48 h in ll of the pyelonephritic nimls. In 46% of the nimls the bility to respond to Con ws less thn 1% of norml nd in 7 of23 individul nimls the response ws less thn 5% of norml. 7 dys fter chllenge no difference between the response of norml nd pyelonephritic nimls could be demonstrted. Suppressor cell depression of the mitogenic response. ntigen dministrtion my result in decresed response of rt T lymphocytes to mitogen, medited through n ntigen-ctivted suppressor cell (18). In the present experiments serch ws mde for popultion of suppressor cells cpble of depressing the T-lymphocyte response. Splenic lymphocyte suspensions from norml nd pyelonephritic nimls were frctionted into pelletble nd nonpelletble frction. The pelletble frction consisted of men of 18% of the splenic lymphocytes in the pyelonephritic nimls (21 nimls killed 3 dys fter chllenge) nd 16% of the splenic lymphocytes from 15 shm-operted control nimls. Stndrd cultures in triplicte were estblished contining: () unfrctionted splenic lymphocytes from pyelonephritic nd control nimls, (b) nonpelletble splenic lymphocytes from the supemte of the centrifuged suspensions, nd (c) pelletble cells recovered fter low-speed centrifugtion. ll cultures were stimulted with Con nd their mitogenic response ws determined. s previ- - _ 6 14 Splenic lymphocytes ex control nimls... Splenic lymphocytes ex pyleo nimls...o I.2. C CL CD " S.S * S s t.- S ~~~~~~~~ ~ ~ h 8h 16h 24lh Intervl fter renl infection & L 2 dy 3 dy FIGuRE 2 Comprtive response to Con of splenic lymphocytes from norml nd pyelonephritic nimls. Ech point from both norml nd pyelonephritic nimls hs been clculted nd expressed s percentge of the men mitogenic response of ll 55 norml nimls which ws 119,726 dpm/16 lymphocytes. L *@, S 7 dy 966 T. Miller, L. Scott, E. Stewrt, nd D. North

4 ously demonstrted, the response to Con of splenic lymphocytes from pyelonephritic nimls, compred with shm-operted control nimls, ws mrkedly reduced. The mitogenic responsiveness of lymphocytes from the pyelonephritic nimls ws restored when cells, pelletble by centrifugtion t 3 g for 2 min, were removed from the suspension of splenocytes (Fig. 3). The response of the remining splenic lymphocytes (82%, lbeled "not pelletble" in the figure) to Con ws similr to the superntnt lymphocytes from norml nimls. The pelletble frction from shm-operted control nimls ws lso cpble of suppressing the response of norml splenic lymphocytes to Con but to much lesser degree. Co-culture demonstrtion of suppressor cell popultion. lthough restoring the immune responsiveness of splenic lymphocytes by removing the suppressor cells is strong presumptive evidence for the genertion of suppressor cell popultion, forml proof demnds tht the puttive suppressor cells, when exmined in co-culture, be ble to suppress the mitogenic responsiveness of norml splenic lymphocytes. This experiment ws crried out with splenic lymphocytes from norml nimls nd the pelletble frction from pyelonephritic nimls 48 h ; cl x cl [ 12[ S * 64 2 Iv. %P not not not pelletble pelletble not pelletble pelletoble frctionted f rctionted frct ionted f rctionted Splenic lymphocytes norml nimols Pyelonephritic nimls FIGRE 3 "Suppressor cell" depression of the mitogenic response. Suispensions of splenic lymphocytes from pyelonephritic nimls chllenged 72 h previously nd control nimls were prepred nd seprted by centrifugtion t 3 g into pelletble nd nonpelletble frction. The responses to Con of frctionted nd nonfrctionted splenic lymphocyte stuspensions were then determined. Ech point is the men of triplicte ecultuires. fter chllenge. Pelletble cells obtined from splenic lymphocytes from norml nimls were used s control. The results (Tble I) hve shown tht pelletble cells from pyelonephritic nimls, when compred to pelletble cells from norml nimls, cn gretly suppress the Con response of norml splenic lymphocytes. Similr suppression ws seen in experiments crried out 72 h fter chllenge. Response of T lymphocytes in vivo (host vs. grft response). dditionl experiments were crried out to ssess in vivo, in the pyelonephritic niml, the functionl cpcity of cell-medited immune mechnisms. Pyelonephritis ws induced in group of 1 dult nimls nd 48 h lter 5 x 16 lymphocytes bering n llogenic histocomptibility ntigen obtined from n Fl niml were injected into the foot pds of the pyelonephritic nd norml nimls. Poplitel lymph nodes were removed 4 dys lter to determine the extent of the host vs. grft response. The cell-medited immune responses ofthe pyelonephritic nimls to the llogeneic histocomptibility ntigens were significntly reduced compred to norml control nimls (P =.1, Fig. 4). Effect of serum from norml nd pyelonephritic nimls on the response of splenic lymphocytes to Con. Hving demonstrted the presence of suppressor cells both in vitro nd in vivo, the possibility tht serum of pyelonephritic nimls might contin suppressor ctivity ws next evluted. Duplicte tissue cultures were estblished with splenic lymphocytes from norml nimls with the ddition of seruim from norml nimls or pyelonephritic nimls 72 h fter chllenge. In this experiment two fctors ssocited with the effect of norml nd pyelonephritic serum on the responses of norml splenocytes were exmined. These were: () The effect of vrying concentrtions of norml serum nd sertum from pyelonephritic nimls on the response of splenic lymphocytes to Con. (b) The effect of' cuilturing splenic lymphocytes in vrying concentrtions of' norml nd pyelonephritic serum s bove but delying the ddition of Con to the splenic lymphocyte cultures for 24 h. There ws highly significnt depression of lymphocyte rectivity to Con when serum from pyelonephritic nimls ws dded to the culture medium. However, this ws demonstrble only fter n intervl of 24 h hd elpsed between the initition of the lymphocyte culture nd the ddition of the mitogen (Fig. 5). When the Con ws dded t the time the lymphocyte culture ws initited, no significnt depression of mitogenic responsiveness by the pyelonephritic serum ws f'ound (Fig. 5B). Functionl ctivity in vivo of isolted splenic lymphocytes from pyelonephritic nimls. The cpcity of isolted splenic lymphocytes from pyelonephritic nimls to initite grft vs. host responise in Cell-Medited Immunity in Experimentl Pyelonephritis 967

5 TBLE I Co-Culture Demonstrtion of Suppressor Cell ctivity in the Pelletble Frction of Splenic Lymphocytes from Pyelonephritic nimls Individul cultures Percent of suppression of norml response by Nornl Pyelonephritic Norml Co-cultures Pyelonephritic Norml splenic pelletble pelletble pelletble pelletble lymphocytes frction frction nticipted response Observed response frction frction () (B) (C) ( + B) ( + C) ( + B) ( + C) % 256, ,8 256,554* 267,2 49, , ,18 ± ,82 256, ,84 257,9t 279,6 4, , ±16,18 ±298 +5,84 ±5, , ,22 1,959 38, , ,789 1,597 27, ,18 ±51 ±7,215 +5,191 ±16,2 Conditions of co-culture: * 2.5 x 16 norml splenic lymphocytes +.6 x 16 pelletble cells. Mitogenic response Con dpm/16 lymphocytes±sem. t 2.5 x 16 norml splenic lymphocytes x 16 pelletble cells. 2.5 x 16 norml splenic lymphocytes x 16 pelletble cells. of lymphocytes to the bsence of suppressive serum fctors ws then investigted. Pyelonephritis ws induced in four groups of nine nimls nd the bility to initite grft vs. host response of T lymphocytes in splenic lymphocyte preprtions from pyelonephritic nimls ws determined. The pyelonephritic nimls were studied 24 us 5or 4[, 3C 1 c - = -O. 2 II.. E C1 ii 2 dy post-chllenge host vs. grft soo Control norml 'Pyelonephritic' Syngeneic ' Culture nimls nimls lymphocytes medium Source of splenic lymphocytes (grft) FIGRE 4 Host vs. grft nlysis. 5 x 16 splenic lymphocytes obtined from Fl hybrid nimls (S2 x HS) were injected into the footpds of pyelonephritic nd control nimls (S2) 48 h fter renl infection ws induced in the pyelonephritic nimls. Poplitel lymph node weights were determined 4 dys lter. h, 2, 3, nd 7 dys fter chllenge. Splenic lymphocyte preprtions from norml nimls were included s controls. Lymphocytes from pyelonephritic nimls 24 h fter chllenge hd the sme bility to induce grft vs. host response s splenic lymphocytes from norml nimls (P = >.1). Splenic lymphocytes studied in isoltion 48 h (Fig. 6) nd 72 h fter chllenge, however, showed n enhnced grft vs. host response compred with norml splenic lymphocytes (P = <.1). There were no differences in the grft vs. host response between lymphocytes tken 7 dys fter chllenge nd lymphocytes from norml nimls. Specific depression of lymphocyte function by renl infection. n inoculum of 3 x 15 vible E. coli ws used to induce unilterl renl infection in three nimls nd deep intrmusculr infection in the thigh of further group of three nimls. ll nimls, including control group, were scrificed 72 h fter chllenge nd the response ofthe splenic lymphocytes to Con ws determined. The mitogenic responsiveness of splenic lymphocytes from nimls with pyelonephritis ws considerbly reduced (Tble II), wheres muscle infection initited with similr chllenge did not hve ny mrked effect on lymphocyte responsiveness. DISCSSION feture of this study hs been the demonstrtion of popultion of suppressor cells ctivted or proliferting during the erly stges of renl infection which exerted regultory effect on normlly respon- 968 T. Miller, L. Scott, E. Stewrt, nd D. North

6 14 E 2 12I u -C c *>; 8 1 K,, ,E. 8 S OD i n6 c &~ 4 4, c 2 c- E 14 E _ 8 E s 2 E 6 c ~4 2 I I. I. * Serum -norml nimls Serum-pyelo. nimls I.. 5 percent 1 percent 2 percent Concentrtion of serum in tissue culture medium B. :. ** Serum - norml nimls Serum - pyelo. nimls I,... 5 percent 1 percent 2 percent Concentrtion of serum in tissue culture medium Immunosuppressive serum fctors elborted in FIGRE 5 pyelonephritis. Splenic lymphocytes from norml nimls were cultured in "insert" medium contining incresing concentrtions of serum (5-2%) obtined from norml nimls nd pyelonephritic nimls chllenged 72 h previously.., con dded 24 h fter the initition of the culture; B., Con dded t the commencement of the culture. Ech point from both norml nd pyelonephritic nimls hs been expressed s percentge of the men response of the norml nimls in ech group. The men mitogenic responses of the norml nimls in groups 5 nd SB were 84,28 nd 19,63 dpm/16 lymphocytes, respectively. 4-5r E 3 2 E 2 = 1.5 tit 4^ grft vs. host 2 dy postchllenge lesos (no, 9 Control norml I eonephritic I syngentic I culture nimls nimls lymphocytes medium Source of splenic lymphocytes (grft) FIGRE 6 Grft vs. host response. 5 x 16 splenic lymphocytes obtined from pyelonephritic nd control nimls (S2) 2 dys fter the estblishment of renl infection, were injected into the footpds of 8-wk-old Fl recipients (S2 x HS). Poplitel lymph node weights were determined 7 dys lter. sive lymphocytes, by suppressing the T-lymphocyte response to less thn 5% of its norml response. Suppression of the T-lymphocyte response to Con in vitro ws seen s erly s 2 h fter the induction of renl infection but mximl suppression ws found, h fter infection t time when rpid bcteril * repliction ws tking plce in the kidney. popultion * of suppressor cells in the splenic lymphocyte popul- tion ccounted for the depressed immune response of : the lymphocytes from the pyelonephritic nimls nd * removl of these cells restored mitogenic responsiveness to the remining splenic lymphocytes. In coculture experiments, ddition of suppressor cells to culture of splenic lymphocytes suppressed the norml response of the splenic lymphocytes to Con. Quntittive nlysis in vivo of cell-medited immune mechnisms ws lso nlysis in vivo of cell-medited immune mechnisms ws lso undertken using the TBLE II Specific Depression of the Mitogenic Response during Renl Infection Control group Muscle chllenge Renl chllenge 112,97* 16,369 9,126 (5,81) t (6,322) (3,526) * dpm/16 lymphocytes. Men of 12 cultures of splenic lymphocytes from three nimls. t SEM is shown in prentheses. Cell-Medited Immunity in Experimentl Pyelonephritis 969

7 host's response to chllenge of lymphocytes bering g-b disprte trnsplnttion ntigens (host vs. grft). Pyelonephritic nimls showed significntly depressed response to this strong ntigenic chllenge nd confirmed the results of the in vitro nlyses oft-lymphocyte function. n immuno-suppressive fctor ws lso found in the serum of pyelonephritic nimls nd this finding ws confirmed in experiments in which lymphocytes from pyelonephritic nimls, exmined in isoltion (grft vs. host rection), were shown to hve n enhnced ctivity. There is incresing evidence supporting our suggestion tht the depressed cell medited immune response disclosed in these experiments is of biologicl significnce in the pthogenesis of renl infection. () The depression of cell-medited immunity found in the pyelonephritic nimls coincided temporrily with the phse of rpid bcteril repliction observed when smll inoculum of E. coli ws introduced into the renl prenchym. (b) The diminished bility of the pyelonephritic host to mount response ginst strong chllenge, e.g., disprity t mjor histocomptibility locus, is good evidence for biologiclly significnt reduction in cell-medited immunity. (c) In recent experiments Rmshw et l. (19) were ble to show tht dministrtion of cyclophosphmide blocked the ctivity of suppressor cell popultion nd tht this led to the restortion of the cellmedited immune response. On the bsis of their experiments we hve treted pyelonephritic nimls with cyclophosphmide in the nticiption tht this would modify suppressor cell ctivity nd restore host immune function. This occurred nd highly significnt reduction of bcteril numbers in the kidney ws found when pyelonephritic nimls treted with cyclophosphmide were compred with untreted controls.2 (d) The fct tht the mitogenic responsiveness of splenic lymphocytes from pyelonephritic nimls could be restored fter the removl of the suppressor cell popultion nd normlly responsive lymphocytes filed to respond to mitogenic stimuli when co-cultured in the presence of suppressor cells. (e) Previously reported studies tht hve shown tht T lymphocytes forming the lymphocytic infiltrte in the pyelonephritic kidney nd obtined from the peri-renl node drining the pyelonephritic kidney show gretly reduced mitogenic responsiveness (1, 2). Finlly the fct tht the reduction in mitogenic responsiveness ws seen during renl infection, but not fter locl infection in muscle which further supports role for the present findings in the biology of renl infection. 2Miller, T. E., nd S. M. Phillips. The effect of cyclophosphmide on the immunobiology ofrenl infection. Mnuscript in preprtion. We re currently investigting the nture of the puttive suppressor cell which my resemble the regultory cell described by Folch nd Wksmn (21). In their initil report Folch nd Wksmn (22) described popultion of spleen cells cpble of inhibiting DN synthesis by mitogen-stimulted lymphocytes. Splenic lymphocyte suspensions from which glss-dherent cells were removed showed mrked elevtion in mitogenic responsiveness. Subsequently, Folch nd Wksmn (23) nd Bsh et l. (24) with T-lymphocyte-depleted nimls nd selected cytotoxic gents were ble to identify the suppressor cell s T lymphocyte with surfce properties defined by dhesiveness to glss. More recent experiments which hve demonstrted the ppernce of these suppressor cells s erly s 3-6 h fter ntigenic chllenge (18) re closely relted to our own results where depressed response of splenic lymphocytes to mitogenic chllenge could be demonstrted 2 h fter the induction of renl infection. The possibility of serum fctors which block or inhibit the cell-medited immune response ws lso investigted nd it ws demonstrted tht serum from nimls with cute pyelonephritis ws cpble of cusing significnt depression of the mitogenic response of T lymphocytes from norml nimls. Modifiction of lymphocyte function occurred only when serum from pyelonephritic nimls hd been in contct with lymphocytes in culture for 24 h before dding mitogen. Other studies hve shown the production of suppressor substnces in vriety of pthologicl conditions including children with recurrent infections (25), immunologiclly medited diseses such s grft vs. host rections (26), nd in tumor bering mice (27, 28). The nture of the suppresive mteril produced s result of these chllenges hs not been identified nd the present studies hve not differentited between the possibility tht the serum fctor is nonspecific inhibitor or cytotoxic mteril rther thn n immunoregultory molecule. Functionl testing of the lymphocytes from pyelonephritic nimls tht were nonresponsive to mitogens in vitro ws crried out in vivo with the grft vs. host rection. This showed tht the functionl ctivity of lymphocytes tested in vivo without homologous serum ws enhnced. The results of this experiment strongly support dt from the experiments crried out by Brooks et l. (29) which showed tht lymphoid cells tken from mice mde nonresponsive to H-2 incomptible skin llogrfts were still ctive in grft vs. host rections when the lymphocytes were removed from their nturl milieu. Our experiments re consistent with the hypothesis tht suppressor fctors in the serum of pyelonephritic nimls contribute to the depressed immune sttus of these nimls nd tht 97 T. Miller, L. Scott, E. Stewrt, nd D. North

8 this restrint is removed once the lymphocytes hve been wshed nd trnsferred s n isolted suspension to llogenic recipients. The concept tht cells with immune suppressor ctivity my be concerned in the biology of the immune suppressor ctivity my be concerned in the biology of the immune response to n infectious disese hs not been widely pprecited but one interesting clinicl study hs shown tht suppressor T-cell popultion my led to hyporectivity of potentilly responsive lymphocytes in ptients with fungl infections (3). In the present experiments we hve provided cler evidence for popultion of suppressor cells nd serum suppressive fctor, both of which re specificlly generted during renl infection nd which modulte the host's immune response. s the cell-medited immune response provides mjor link between the immune nd inflmmtory systems, suppressed response t such criticl time my be mjor fctor contributing to the susceptibility of the kidney to infection nd my explin the inbility of host defense mechnisms to cope with n infectiouis chllenge to the kidney. CKNOWLE DGM E NTS The uthors wish to cknowledge the ssistnce of Mr. Ry Hll in the breeding nd mintennce of the nimls used in these experiments. This study ws supported by the Medicl Reserch Council of New Zelnd. REFERENCES 1. Jodl,., S. hlstedt, B. Crlsson, L.. Hnson,. Lindberg, nd. Sohl Locl ntibodies in childhood urinry trct infection. preliminry study. Int. rch. llergy ppl. Immunol. 47: Smith, J., J. Holmgren, S. hlstedt, nd L.. 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9 in children with recurrent infections. Immunology. 3: McMster, R., nd J. G. Levy Immunosuppression of norml lymphoid cells by serum from mice undergoing chronic grft-vs-host disese. J. Immunol. 15: Whitney, R. B., nd J. G. Levy Effects of ser from tumour-bering mice on mitogen nd llogeneic cell stimultion of norml lymphoid cells. J. Ntl. Cncer. Inst. 54: Whitney, R. B., nd J. G. Levy Suppression of mitogen responses by serum from tumour-bering mice. Eur. J. Cncer. 1: Brooks, C. G., L. Brent, P. J. Kilshw, R. R. C. New, nd M. Pinto Specific unresponsiveness to skin llogrfts in mice. IV. Immunologicl rectivity of mice treted with liver extrcts, Bordetell pertussis, nd ntilymphocyte serum. Trnsplnttion (Bltimore). 19: Stobo, J., S. Pul, R. E. Vn Scoy, nd P. E. Hermns Suppressor thymus-derived lymphocytes in fungl infection. J. Clin. Invest. 57: T. Miller, L. Scott, E. Stewrt, nd D. North