Evidence for delayed-type hypersensitivity mechanisms in glomerular crescent formation

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1 Kidney Interntionl, Vol. 46 (1994), PP Evidence for delyed-type hypersensitivity mechnisms in glomerulr crescent formtion XIA Ru HUANG, STEPHEN R. HOLDSWORTH, nd PETER G. TIPPING Centre for Inflmmtory Diseses, Monsh University, Deprtment of Medicine, Monsh Medicl Centre, Clyton, Victori, Austrli Evidence for delyed-type hypersensitivity mechnisms in glomerulr crescent formtion. The role of CD4-positive T cells in glomerulr crescent formtion ws exmined in WKY rts. Glomerulonephritis (ON) ws induced by subnephntogenic intrvenous dose of sheep nti-rt GBM ntibody in rts previously sensitized to sheep globulin. This resulted in severe prolifertive nd crescentic ON, with mrked proteinuri [143 4 mg/24 hr (men SD), norml mg/24 hr] nd crescent formtion involving 59 8% of glomeruli t dy 1 (norml %). Humorl immunity to sheep globulin ws evident systemiclly by high titers of circulting nti-sheep globulin nd loclly by liner deposition of rt immunoglobulin in glomeruli nd cell medited immunity by cutneous delyed-type hypersensitivity (DTH) to intrderml injection of sheep globulin. Glomerulr ccumultion of CD5 positive T cells cells per glomerulr cross section (c/gcs), norml.18.1 c/gcs], CD4 positive T cells, ( c/gcs, norml.14.8 c/gcs), nd mcrophges ( c/gcs, norml.5.5 c/gcs), together with the ppernce of multinucleted gint cells ( c/gcs, norml c/gcs) suggested DTH-like rection in glomeruli. Sensitized rts given nti-gbm globulin were treted with monoclonl nti-cd5 or nti-cd4 ntibodies in protocol which prevented cutneous DTH to sheep globulin without ltering the humorl immune response. Both tretments significntly reduced gbmerulr ccumultion of CD5 nd CD4 positive T cells t dy 1. Crescent formtion ws significntly reduced (CD5 treted, 13 4% of glomeruli ffected; P <.1; CD4 treted 13 3% of glomeruli ffected, P <.1) compred to rts treted with n isotype-mtched irrelevnt monoclonl ntibody. Glomerulr mcrophge ccumultion, multinucleted gint cell formtion nd proteinuri were lso significntly reduced by both tretments. These studies demonstrte functionl role for CD4 positive T cells s effector cells within glomeruli, seprte from their role in humorl immunity, in the development of crescentic ON. The locl prticiption of CD4 positive T cells, mcrophges nd multinucleted gint cells in crescent formtion, nd the ttenution of these fetures by functionl T helper cell depletion suggest tht locl DTH-like mechnisms my contribute to glomerulr crescent formtion. Glomerulr crescent formtion is feture of the most severe forms of humn glomerulonephritis (ON), nd is ssocited with poor prognosis for renl function. Crescents show similr immunopthologicl fetures to delyed type hypersensitivity (DTH) T helper cell-dependent response. These fetures Received for publiction August 9, 1993 nd in revised form Februry 9, 1994 Accepted for publiction Februry 14, by the Interntionl Society of Nephrology include locl ccumultion of T cells nd mcrophges, prolifertion of djcent epithelil cells, multinucleted gint cell (MGC) formtion nd fibrin deposition. In humn crescentic GN, glomerulr ccumultion of both CD4 nd CD8 positive T cells is observed [1 3] often in the bsence of deposition of humorl immune meditors [4]. Olomerulr mcrophge ccumultion is prominent [5, 6] together with prolifertion of intrinsic glomerulr cells, fibrin deposition nd occsionl MGC. These pthologicl fetures suggest tht immunologicl mechnisms kin to DTH (involving n importnt locl role for CD4 positive T cells) my contribute to crescent formtion. There is now substntil evidence tht T cells hve the potentil to ct s locl effectors of cute glomerulr injury in GN. Bhn et l demonstrted tht trnsfer of sensitized T cells into rts with plnted glomerulr ntigen (heterologous nti- GBM globulin) leds to ccumultion of proliferting cells in glomeruli [7]. Bolton et l demonstrted histologicl evidence of glomerulr injury in chickens fter trnsfer of T cells sensitized to GBM [8]. Oite et l demonstrted tht rts sensitized to T cell hpten developed prolifertive GN nd trnsient proteinuri fter the ntigen ws infused into the renl rtery. This injury occurred in the bsence of glomerulr ntibody deposition [9]. The contribution of CD4 positive T helper cells to more severe nd delyed mnifesttion of gbomerulr injury hs not been studied. T helper cells ply n importnt role in both humorl nd cellulr immune responses to ntigens. Nude (T cell deficient) mice fil to develop n immune response or glomerulr injury following immuniztion with humn glomerulr ntigens [1]. Therefore, in order to determine the locl effector role of T helper cells in the development of glomerulr injury in ON, protocols must be employed in which immune sensitiztion nd humorl immunity to the disese initition ntigen re not ltered. In the current studies, the contribution of CD4 positive T cells ws studied in crescentic model of nti-gbm ntibody initited GN. T cell depletion ws induced in the effector phse of this disese in rts with n estblished immune response to the disese inititing ntigen, to ssess the locl contribution of CD4 positive T cells to glomerulr crescent formtion. Thus, the development of crescents ws ssessed in rts which hd high circulting titers of utologous ntibody, but were unble to develop cutneous DTH to the disese inititing ntigen. 69

2 7 Hung et!: Mechnisms of glomerulr crescent formtion Methods Animls Mle Wistr Kyoto (WKY) rts, 1 to 15 g in weight, were obtined from Monsh University Centrl Animl Services. Induction of nti-gbm GN Rts were pre-sensitized by injection of totl of 4 mg of sheep globulin in.75 ml of Freunds Complete Adjuvnt (FCA, Commonwelth Serum Lbortories, Prkville, Victori, Austrli) t two subcutneous sites. Five dys lter, GN ws initited by intrvenous dministrtion of sheep nti-rt GBM globulin t dose of 2 pg/g body weight. This globulin ws prepred by bsorption with rt red blood cells nd mmonium sulphte precipittion of serum from sheep immunized ginst prticulte frction of rt GBM s previously described [11]. At the dministered dose, this ntibody bound 3 pg of globulin per grm wet weight of kidney (determined by trce lbeling studies) nd did not produce proteinuri in non-sensitized rts. Assessment of proteinuri Rts were housed individully in cges to collect urine over the finl 24 hours of ech experiment. Urinry protein concentrtions were determined by the Brdford method [121, dpted to microtiter plte ssy. Twenty microliters of diluted urine were dded to 15 d of Coomssie regent (3 ml of 1 g!m1 Pge blue 9; BDH Chemicls Ltd, Poole, UK) in 5% ethnol! 8.1% orthophosphoric cid). After 1 minutes, the bsorbnce t 595 nm wvelength ws red on microtiter plte reder (MR7, Dyntech Lbortories, Chntilly, Virgini, USA) nd the protein concentrtions clculted by reference to bovine serum lbumin (BSA; Sigm Chemicl Co., St. Louis, Missouri, USA) stndrds. All smples nd stndrd were ssyed in duplicte. The 24-hour urinry protein excretion ws clculted from the 24-hour urine volume nd the urinry protein concentrtion. Mesurement of serum cretinine nd cretinine clernce Serum nd urine cretinine concentrtions were mesured by the lkline picryc cid method using n utonlyzer (Cobs Bio, Roche Dignostics, Bse!, Switzerlnd). Cretinine clernce ws clculted from the serum nd urine cretinine nd the urine volume. Assessment of glomerulr crescents nd MGC Kidney tissue ws fixed in Bouins fixtive nd 2 jim tissue sections were stined with periodic cid Schiff (PAS) regent to ssess light microscopic ppernces. Glomerulr crescent formtion ws ssessed in blinded protocol by counting glomeruli with crescents in minimum of 2 glomeruli per niml. The presence of fibrinous mteril nd/or cells in Bowmn's spce ws considered s crescent. Crescents rnged in extent from pproximtely one qurter to greter thn three qurters of the glomerulr circumference. Only glomeruli cut in equtoril cross section were scored. The percentge of glomeruli demonstrting crescent formtion ws clculted individully for ech niml. MGC were lso ssessed histologiclly in blinded protocol. Lrge multinucleted cells with > 2 nuclei were counted in minimum of 2 equtorilly sectioned glomeruli per niml, nd the results expressed s cells per glomerulr cross section (c/gcs). Im,nunohistologicl ssessment of glomerulr cell ccumultion Spleen nd kidney tissue from ech niml ws fixed in periodte lysine prformldehyde for four hours, wshed in 7% sucrose solution then frozen in liquid nitrogen cooled isopentne nd 4 jim sections cut on cryostt. Tissue sections were stined using three lyer immunoperoxidse technique to demonstrte T cells, T cell subsets nd mcrophges nd nturl killer (NK cells) in glomeruli. The primry ntibodies were: X19 (nti-cd5) s pn T cell mrker [13]; R73 [nti-!/3t cell receptor (TCR); Serotec, Oxford, UK] [14] W3!25 (nti-cd4) s T helper cell mrker [15]; OX8 (nti-cd8) s T cytotoxic/suppressor mrker [15], ED-! s mcrophge mrker [16] (Serotec); nd the monoclonl mouse nti-rt NK cell mrker ws used to detect NK cells [17] (gift of Dr. J. Hiserodt, Pittsburgh, Pennsylvni, USA). The second ntibody lyer ws rbbit nti-mouse IgG globulin (Dko, Glostrup, Denmrk) t concentrtion of 1 in 1. This ws followed by soluble complexes of horserdish peroxidse nd mouse monoclonl nti-horserdish peroxidse immunoglobulin (Dko) t concentrtion of 1:1. Sections were then incubted with dimino benzdine (Sigm), nd counterstined with Hrris hemtoxylin. Sections of spleen provided positive control for ech niml, nd protein purified mouse globulin ws substituted for specific monoclonl ntibody to provide negtive control. Glomerulr cell numbers were ssessed using blinded protocol. A minimum of 2 equtorilly sectioned glomeruli per niml were ssessed per niml nd the results were expressed s cells per glomerulr cross section (c/gcs). The coefficient of vrition for men glomerulr T cell counts on seprtely processed sections from n individul rt ssessed by the sme observer ws 8.5% for X19, 6.1% for R73, 5.% for W3/25 nd 2.5% for X8. Mesurement of circulting T cells nd subsets Rts were bled under ether nesthesi from the retro-orbitl venous plexus to obtin 5 to 8 jil of blood into EDTA nticogulnt. Red blood cells were lysed in.1% formic cid nd the leukocyte popultions nlyzed by fluorescence-ctivted cell sorting (FACS, EPICS 752, Coulter Electronics, Hileh, Florid, USA) fter stining with monoclonl nti-rt T cell ntibodies X19, R73, W3/25 nd X8, followed by Ft,'2 sheep nti-mouse Ig FITC (Silenus Lbortories, Melbourne, Austrli). Totl leukocytes were detected using OX-I ( common leukocyte ntigen mrker) nd n irrelevnt isotypemtched monoclonl ntibody ws used s negtive control. The percentge of circulting NK cells ws compred in norml WKY nd Sprgue-Dwley rts (N = 6 in ech group), using FACS nlysis with the NK cell mrker (3.2.3). The percentge lymphocytes stining with the NK cell mrker ws (men SD) in Sprgue-Dwley nd in WKY rts. Mesurement of circulting rt nti-sheep globulin ntibody Titers of rt nti-sheep globulin ntibody were determined by ELISA on serum collected t the end of ech experiment. Round-bottom polyvinylchloride microtiter pltes (Dyntech)

3 were coted with 1. g/ml norml sheep globulin in crbonte! bicrbonte buffer ph 9.5 by incubtion overnight t 4 C, nd then blocked with 2% BSA. The pltes were wshed twice in phosphte buffered sline (PBS)/. 1% Tween 2 prior to incubtion with seril dilutions of rt serum. After further three wshes in PBS!Tween, pltes were incubted with horserdish peroxidse conjugted rbbit nti-rt IgG (Sigm) t dilution of 1 in 4. The pltes were finlly wshed six times with PBSITween nd incubted with.1 M 2,2'-zino-di-3-ethylbenzthizoline suiphonte (ABTS, Boehringer Mnnheim, Sydney, Austrli) in.2% H22. The substrte rection ws stopped by dding n equl volume of.1 m citric cid!.1% sodium zide nd the bsorbnce t 45 nm ws red on microtiter plte reder (Dyntech). Serum from ech rt ws tested t doubling dilutions, strting from dilution of 1 in 1, nd serum from six non-immunized rts ws tested to provide norml controls. Mesurement of serum complement Circulting C3 ws mesured by ELISA, in round bottom microtiter pltes coted with got nti-rt C3 IgG (Nordic Lbortories, Tilburg, The Netherlnds) t concentrtion of 1 g!ml. The detecting ntibody ws rbbit nti-rt C3 serum (Bethyl Lbortories, Montgomery, Texs, USA) t dilution of I in 1. Both these ntibodies produce single precipitin rc ginst C3 in rt serum by immunoelectrophoresis. The indictor system consisted of horserdish peroxidse-conjugted swine nti-rbbit IgG (Dkopltts) t 1 in 2, followed by.1 M ABTS s described bove. Serum (1 jd) from norml, treted nd control rts ws ssyed in duplicte t dilutions from 1 in 1 to 1 in 1, nd produced prllel curves of bsorbnce (45 nm) versus serum dilution. The bsorbnce vlues t dilution of 1 in 1 were used to compre circulting C3 levels between groups. Assessment of cutneous delyed type hypersensitivity (DTH) to sheep globulin Rts were shved over n re of the bck nd the skinfold thickness ws mesured using skinfold clipers, with n ccurcy of.1 mm. Rts were then injected intrdermlly with 1 jig of sheep globulin in 1 pa of PBS, 24 hours prior to the end of ech experiment. As control, ech rt lso received similr intrderml injection of horse globulin. At the end of the experiment, skinfold thickness ws mesured t the site of chllenge nd skin lesions excised for histologicl exmintion in representtive nimls. Histologiclly, lesions t sites injected with sheep globulin showed typicl fetures of DTH rections with tissue edem, some fibrinoid mteril, nd dense mononucler cell infiltrte consisting minly of CD5 nd CD4 bering lymphocytes nd ED-i positive mcrophges. Some CD8 positive lymphocytes were observed. Their time course of development ws chrcteristic of DTH. Neutrophils were evident in the lesions; however, hemorrhgic necrosis typicl of n Arthus rection ws not observed. Skin swelling incresed progressively over the 24 hours from injection of ntigen, nd the increse in skinfold thickness (in mm) ws used s n index of the ntigen specific cutneous DTH response. Hung et l: Mechnisms of glomerulr crescent formtion 71 Assessment of glomerulr ntibody deposition Kidney tissue ws frozen in liquid nitrogen cooled isopentne for immunofluorescence microscopy. Cryostt cut tissue sections, 4 jim thickness, were stined with seril dilutions of FITC conjugted rbbit nti-rt IgG (Silenus) strting t 1 in 1, to ssess the glomerulr deposition of rt IgG. Glomerulr deposition of utologous rt ntibody ws lso ssessed quntittively using n 1251 lbeled nti-rt immunoglobulin ntibody binding ssy. A norml rt ws given sheep nti-rt GBM ntibody (2 jig/g body wt, i.v.) nd killed 24 hours lter. Glomeruli were isolted by grded sieving nd incubted in liquots of 2 glomeruli in 1 pa of PBS with vrying mounts of 125j lbeled rt nti-sheep immunoglobulin ntibody from 5 to 2 jig, for four hours. The glomeruli were then wshed five times in PBS t 4 C nd the glomerulr bound 125J ctivity counted in gmm-counter (LKB Wllc, Turku, Finlnd). The specific mount of rt ntibody bound t ech incubtion concentrtion ws then clculted from the known specific ctivity of the nti-rt immunoglobulin ntibody. Incubtions with the sme btch of glomeruli were performed in prllel under identicl conditions using unlbeled rt ntisheep immunoglobulin ntibody. These were wshed in the sme mnner to produce glomeruli with known mounts of unlbeled, bound rt ntibody. These glomeruli were then used s stndrds in binding ssy using 125J lbeled rbbit nti-rt immunoglobulin ntibody (Nordic Lbortories) to determine the mount of ntibody bound in glomeruli isolted from rts on dy 1 of the experimentl protocol. Glomeruli from experimentl rts were isolted in the sme mnner nd in prllel with "stndrd" glomeruli were incubted with 125j lbeled rbbit nti-rt immunoglobulin ntibody nd wshed extensively s described bove. The glomerulr bound 1251 ctivity ws counted nd curve of bound I25J rbbit ntibody versus glomerulr bound rt ntibody ws plotted, using the counts from the "stndrd" glomeruli. This curve showed liner reltionship for the rnge of glomerulr bound rt ntibody from.56 to 9.55 jsg!2 x i3 glomeruli (liner correltion coefficient, r2 =.924). The mount of rt ntibody in glomeruli from ech experimentl rt ws then clculted from the bound 1251 counts by reference to the stndrd curve. T helper cell depletion protocols T cell depletion ws induced by single intrvenous injection of 5 mg of protein G purified mouse monoclonl ntibody. Two monoclonl ntibodies were used: X19 (nti-cd5) [13] which depletes both T helper nd suppressor cells, nd W3!25 (nti- CD4) which inhibits CD4 positive T helper cell ctivtion in vitro [18] nd prevents immunologicl injury requiring T helper cells in vivo [19 22]. An irrelevnt isotype mtched (IgG1) mouse monoclonl ntibody (with specificity for humn milk ft globulin) ws used s control. Antibodies were dministered 24 hours fter injection of nti-gbm globulin. The extent of T cell depletion ws ssessed by FACS nlysis of blood on dys 5 nd 1 of the experimentl protocol nd lso by stining spleen sections from ech rt obtined on dy 1. As CD4 my be expressed on resident peritonel mcrophges [23] nd some tissue mcrophges [24], the effect of nti-cd4 ntibody on glomerulr mcrophges ws ssessed in model of nti-gbm GN induced in nive rts. High titer

4 72 Hung et!: Mechnisms of glomerulr crescent formtion utologous rt nti-sheep globulin (3 mg totl in 3 divided doses) ws dministered over 16 hours to WKY rts (N = 6) pretreted with subnephritogenic dose of sheep nti-rt GBM globulin six hours erlier. Significnt proteinuri developed over the subsequent 24 hours [ mg/24 h (men SD), control mg124 hr, P <.1]. Histologicl exmintion showed glomerulr mcrophge ccumultion (ED I positive cells c/gcs) in the bsence of T cells (X19 positive cells c/gcs). Rts given non-immune rt globulin insted of rt nti-sheep globulin did not develop proteinuri (1.8.3 mg124 hr, or glomerulr mcrophge ccumultion (.2.7 c/gcs). Tretment of rts with monoclonl ntibody W3/25 (5 mg, i.v.; N = 6), 24 hours prior to initition nti-gbm GN, did not reduce the subsequent proteinuri ( mg/24 hr) or the glomerulr ccumultion of mcrophges (ED I positive cells c/gcs), indicting tht this ntibody does not inhibit glomerulr mcrophge ccumultion or development of glomerulr injury in the bsence of T cell prticiption. Experimentl design Anti-GBM GN ws induced s previously described. Depletion protocols were commenced 24 hours fter dministrtion of nti-gbm globulin nd immune responses, proteinuri nd histologicl ppernces were ssessed 1 dys fter nti-gbm globulin. The following groups of rts were studied: (1.) norml rts (N 6); (2.) untreted rts with nti-gbm GN (N = 6); (3.) rts with nti-gbm GN, treted with n isotype-mtched irrelevnt monoclonl ntibody (N = 8); (4.) rts with nti-gbm GN, treted with nti-cd5 ntibody (N 8); nd (5.) rts with nti-gbm GN, treted with nti-cd4 ntibody (N 7). Results re expressed s the men stndrd devition of the men (SD). Sttisticl significnce ws nlysed by the Mnn Whitney U test. Results Crescentic nti-gbm GN Untreted rts developed severe prolifertive, crescentic GN, with prominent mononucler cell infiltrtion on dy 1 (Fig. 1A). This ws ssocited with significnt proteinuri [143 4 mg124 hr (men SD, rnge 78 to 196); norml, mg124 hr (rnge.3 to 1.94)], rise in serum cretinine (167 5 tm/liter; norml, 38 1 /LM/liter; P <.1) nd fll in cretinine clernce (.4.5 mi/mm; norml.64.3 mi/mm, P <.1). Crescent formtion ws observed in 59 8% of glomeruli (norml %) together with the ppernce of MGC ( c/gcs, norml ). Infiltrtion of both mcrophges nd T cells contributed to glomerulr hypercellulrity. Glomerulr mcrophge numbers were mrkedly incresed ( c/gcs) compred to norml (.5.5 c/gcs, P <.5; Fig. 1B). A significnt T cell infiltrte ws lso pprent (CD5 positive T cells, c/gcs, norml.18.1 c/gcs, P <.5; CD4 positive T cells c/gcs, norml.14.8 c/gcs, P <.5; CD8 positive T cells, c/gcs, norml c/gcs, P <.5). Occsionl NK cells were observed in nephritic glomeruli (.5.65 c/gcs) which were not observed in norml glomeruli ( c/gcs) (Fig. 1 C-F). Effects of ntibody tretment on circulting nd splenic lymphocytes Following tretment with OX-19, rts with nti-gbm GN on dy 1 demonstrted mrkedly reduced stining of CD5, TCR, CD4 nd CD8 positive cells in splenic tissue compred to norml rt spleen or splenic tissue from rts treted with isotype control ntibody. Stining for mcrophges with ED-i ws unltered. Rts with nti-gbm GN, treted with W3/25, demonstrted only minor reductions in CD5, TCR nd CD4 positive cells in splenic tissue on dy 1. CD8 positive cells nd mcrophges were unffected. OX-l9 tretment significntly depleted circulting T cells in rts with nti-gbm GN. Prior to tretment 42 1% of circulting leukocytes (Ox-i positive cells) expressed CD5 nd 37 9% of cells expressed TCR. On dy 5, CD5 positive cells were reduced to % nd TCR positive cells to %. On dy 1, CD5 positive cells were % nd TCR positive cells to %. Tretment with W3/25 produced only minor reduction in circulting T cells (dy 5, CD5 positive cells 27 1%, TCR positive cells 3 5%, dy 1 CD5 positive cells 23 15%, TCR positive cells 22 5%). FACS profiles of CD5, TCR, CD4 nd CD8 expression on circulting lymphocytes in isotype control, OX-19 nd W3/25 treted rts on dy 1 re shown in Figure 2. Effect of T cell depletion of the immune response to sheep globulin Effect of humorl immunity. Rts developing nti-gbm GN demonstrted high serum titers of rt nti-sheep ntibody nd liner deposition of rt globulin long the GBM. Tretment with either nti-cd5 or nti-cd4 ntibodies fter prior sensitiztion did not ffect the circulting titers of rt nti-sheep globulin ntibody. Serum from OX-19, W3/25 or isotype control ntibody treted rts showed identicl curves for the sheep globulin binding cpcity over wide rnge of dilutions (Fig. 3). The deposition of rt immunoglobulin in glomeruli ws unffected by T cell depletion. The end-point titers for detecting rt immunoglobulin by immunofluorescence ws the sme in treted nd control groups. Quntittive nlysis using the ntibody binding ssy demonstrted no difference in glomerulr bound rt immunoglobulin in treted or control rts (isotype control treted, g/2 X l gloms; nti-cd5 treted, g/2 x l gloms; nti-cd4 treted, g/2 X i gloms). Effect on T cell-medited immune responses. Isotype control monoclonl ntibody treted rts with nti-gbm GN demonstrted ntigen specific cutneous DTH to sheep globulin (Fig. 4). Skinfold thickness ws mm t the site of sheep globulin injection compred to mm for horse globulin (P <.1). Norml skin thickness ws mm. Tretment with either nti-cd5 or nti-cd4 ntibody bolished cutneous DTH rections to sheep globulin (nti-cd5 treted mm; nti-cd4 treted mm). Effect of T cell depletion on serum C3 levels Serum C3 levels were higher in rts developing nti-gbm GN thn in norml rts. There were no differences in the serum C3 levels in treted or isotype control treted rts. At serum dilution of 1 in 1, serum C3 in norml rts ws 1.7.5

5 -w -, 3. fl C- Si V.1 V - -' Huwig Ct 1: Mechnisin of glomerulr crescent formtwn s i\' e B 4. Ṡ.-. 7' : I- p ) 4 St 1* 1' % 'S - -% $1 C I I'. -V. C I' -'4-5? --lit 4 -q. 5fj,Sl j.-1._i_ I 'C r 5 C 1%." tt..i '#4' ' & S. -. p 4 V 1 '' As.. (...1 D.9 -'M "-be, _t "S ll # /1 V.,.4 11% ' -., ' : 1 % S p., t 5 -I Er V -I I I i;7? T J i' 1'sc "-.g 5- -V '4 I. ' 4' 'S-k S 'S S I 'V - I' 1. -S - _- 7 - C. ) - ¼r' #4' -- - e*.,45r Vt -.#S I.r _ft,-- 'I Fig. 1. Photomicrogrphs ofglomeruli from rt developing nti-gbm GN, illustrting :1w immunopthologkl fetures of crescentic GN on dy 1. A prolifertive (in with prominent crescent formtion nd MGC ws present in the mjority of glomeruh (A, PAS stin), ssocited with prominent glomerulr mcrophge ccumultion (B, immunoperoxidse using ED-I), nd CD5, CD4 nd CD8 positive cells in glomeruli, (C, immunoperoxidse using OX-l9 D, immunoperoxidse using W3125; E, immunoperoxidse using OX-8 respectively; positive intrglomerulr cells indicted by rrows). NH cells (F, immunoperoxidse using monoelonl ntibody 3.23 demonstrting positive cell in glomerulus) were occsionlly observed but not incresed over thc numbers observed in norml glomcruli (mgnifiction x 4 in ech cse).

6 74 Hung et!: Mechnisms of glomerulr crescent formtion A Isotype control ntibody B Anti-CD5 ntibody C Anti-CD4 ntibody L() L. I,,,.. I I I I I I I I I I I I I I I I I I I Fig. 2. FAGS profiles showing fluorescence intensity (horizontl xis) of CD5 (OX-19), //3TCR (R73), CD4 (W3/25) nd CD8 (OX-8) verses frequency on circulting leukocytes of rts treted with isotype control ntibody, OX-19 or W3/25 on dy 1 of nti-gbm GN Dilution of serum, lix Fig. 3. Rt nti-sheep globulin ntibody demonstrted by ELISA on seril serum dilutions from rts developing nti-gbm GN treted with isotype control ntibody (dots), nti-gd5 ntibody (squres) nd nti- GD4 ntibody (tringles). Titrtion of norml rt serum is lso shown (open circles). rbitry units/1 /.Li (u) compred to u in isotype control treted rts with nti-gbm GN nd nd jx in nti-cds nd nti-cd4 treted rts, respectively. Effect oft cell depletion on nti-gbm GN Both nti-cd5 nd nti-cd4 tretment significntly ttenuted histologicl evidence of glomerulr injury (Fig. 5) nd preserved renl function. Glomerulr crescent formtion, the ppernce of MGC, mcrophge infiltrtion nd proteinuri were significntly reduced compred to isotype control treted rts (Fig. 6). In nti-cds treted rts, crescent formtion occurred in 13 4% of glomeruli (isotype control, 64 1% of glomeruli, P <.1), MGC were reduced to.6.3 c/gcs (isotype control,.4.11 c/gcs, P <.1), nd mcrophge infiltrtion to c/gcs (isotype control c/gcs, P <.1). Proteinuri ws significntly reduced [61 27 mg124 hr (rnge 19.8 to 95.2); isotype control mg/24 hr (rnge 82.4 to 177); P <.5], the rise in serum cretinine ws significntly ttenuted (54 15 j.tm/liter; isotype control xm/liter; P <.1) nd the cretinine clernce ws significntly higher ( mllmin; isotype control.4.1 mllmin, P <.1). This ws ssocited with reduction of CD5, TCR nd CD4 positive cells in glomeruli (CD5 positive cells c/gcs, isotype control c/gcs; P <.1; TCR positive cells c/gcs, isotype control 2.8

7 Hung et l: Mechnisms of glomerulr crescent formtion 75 3 E E U) U) ) -c 2 Cl) 1 Sheep Horse Sheep globulin globulin globulin I OX-19 treted Sensitized rts Sheep globulin W3/25 treted J Sheep globulin Nonsensitized rts Fig. 4. Effect of nti-cd5 nd nti-cd4 ntibody tretment on cutneous DTH responses to the disese inititing ntigen (sheep globulin) in rts developing nti -GBM on dy 1. The response to n irrelevnt ntigen (horse globulin) in sensitized rts treted with isotype control ntibody nd to sheep globulin in non-sensitized rts is lso shown. The shded res represents verge skinfold thickness on the bck of norml WKY rts..43 c/gcs; P <.1; CD4 positive cells c/gcs, isotype control c/gcs; P <.1). CD8 positive cells in glomeruli incresed ( c/gcs, isotype control c/gcs; P <.5; (Fig. 7). NK cells numbers ( c/gcs) were no different from isotype control treted rts (.5.65 c/gcs) (Tble 1). In nti-cd4 treted rts, crescent formtion (13 3% of glomeruli), ppernce of MGC (.4.1 c/gcs) nd mcrophge infiltrtion ( c/gcs, P <.5) were lso significntly reduced compred to isotype treted controls (P < 5; Fig. 6). Proteinuri [68 23 mg124 hr (rnge 43.7 to 94.), P <.5] nd serum cretinine (51 1 sm/liter, P <.1) were significntly reduced nd cretinine clernce (.43.9 mllmin, P <.1) ws significntly higher. CD5 positive cells ( c/gcs), TCR positive cells ( c/gcs) nd CD4 positive cells ( c/gcs) in glomeruli were lso significntly depleted (P <.5), wheres OX-8 positive cells were incresed in number ( c/gcs, P <.5). NK cell numbers ( c/gcs) were the sme s in isotype treted control rts (Tble 1). Discussion The immunopthologicl fetures of crescentic GN show mrked similrities to DTH rections which re dependent on T helper cells. A role for sensitized T cells hs been demonstrted in recruitment of proliferting cells in glomeruli [7] nd development of trnsient proteinuri in rts [9]. Lymphokine production by glomerulr T cells hs lso been demonstrted [25] nd T cells sensitized to GBM cn trnsfer prolifertive GN in chickens [81. These studies provide evidence for the cpcity of T cells to induce glomerulr injury in GN; however, the locl contribution of T helper cells to glomerulr crescent formtion vi DTH-like mechnisms hs not previously been studied. Prominent crescent formtion developed in response to plnted glomerulr ntigen (sheep nti-rt GBM globulin) in WKY rts presensitized by intrderml presenttion of this ntigen in FCA. This presensitiztion resulted in pronounced humorl nd cellulr immune response to sheep globulin. These were quntitted by mesurement of serum titers of rt ntisheep globulin, nd by skin swelling in response to intrderml ntigen chllenge. The prticiption of effectors of humorl nd cellulr immunity in glomeruli of rts developing crescentic GN ws lso demonstrted by the liner deposition of rt immunoglobulin long the GBM, nd the glomerulr locliztion of CD4 positive T cells. Other fetures of this crescentic model of GN which suggested DTH-like cell-medited immunity were the predominnce of CD4 positive T cells over CD8 positive T cells, ccumultion of mcrophges nd the development of

8 76 Hung et!: Mechnisms of glomerulr crescent formtion A.9 Fj là. ft. -1- St YE 4 ''.- c 'I t-!_. -.z,4 - - t _t.. I- -d C' Fig. 7. A photomicrogrph of glomerulus of rt with nti-gbm GN on dy 1 treted with nti-cd5 ntibody, demonstrting incresed number of CD8 positive cells in glomeruli (compre with Figure IE). (Immunoperoxidse, mgnifiction x 4)., 4 & 1* -' S S 9 4 d :,- S Fig. 5. Photomicrogrphs of glomeruli from rts treted with isotype control ntibody (A), nti-cds ntibody (B) nd nti-cd4 ntibody (C) demonstrting the histologicl fetures of GN (PAS, mgnifiction x 4). V A, MGC. Previous studies hve suggested tht the incresed susceptibility of WKY rts to severe glomerulr injury nd crescent formtion is due to incresed NK cell ctivity [26]. However, in the current study NK cells were not prominent in crescentic glomeruli, nd the percentge of circulting NK cells in WKY rts ws low compred to Sprgue-Dwley rts, which re less susceptible to crescentic GN. The role of T helper cells s locl effectors of glomerulr injury nd crescent formtion ws ssessed by in vivo functionl depletion using monoclonl ntibodies. Rts were immunized to sheep globulin nd llowed to develop high titers of specific ntibody nd sensitized T cells. An immune response ws then initited in the glomerulus by plnting this ntigen on the GBM, prior to blocking T cell effector functions. Mesurement of circulting rt nti-sheep globulin nd glomerulr bound rt immunoglobulin nine dys lter confirmed tht this protocol did not lter the humorl immune response to the disese inititing ntigen. Circulting C3 levels were lso unffected. Tretment with OX-19 effectively depleted CD5 positive T cells in the circultion nd the spleen. This ntibody does not bind significntly to B cells [13], nd hs previously been reported to produce prolonged depletion of CDS positive T cells fter single intrvenous dose [19]. Stining with second T cell mrker (TCR) indicted tht this ws not merely due to down-regultion of CD5 ntigen expression. Tretment with W3125 produced miniml depletion CD4 positive T cells. Rts developed DTH skin lesions over the typicl time corse nd with chrcteristic immunohistologicl fetures, fter re-chllenge with the sensitizing ntigen. The bsence of cutneous DTH fter intrderml chllenge in rts treted with OX- 19 nd W3/25 confirmed tht both tretments produced functionl bltion of CD4-dependent cellulr immune responses, consistent with previous reports of the functionl efficcy of in vivo tretment with these ntibodies [ Potentil therpeutic uses of nti-cd4 ntibodies hve recently been reviewed [27].

9 Hung et l: Mechnisms of glomerulr crescent formtion 77. 2o oce E o E.5 ) C Isotype control ntibody Anti-CD4 ntibody Both tretments significntly ttenuted but did not bolish glomerulr crescent formtion. This effect ws ssocited with reduction in the ppernce of CDS nd CD4 positive T cells in glomeruli, fewer gint cells nd reduction of proteinuri. As W3/25 is not n efficient depleting ntibody, it my be nticipted tht resident CD4 lymphocytes recruited to n inflmmtory focus s the result of functionl immunologiclly stimulted response. Their greter reduction in glomeruli of treted rts in comprison to the spleen nd circultion ws consistent with this prediction. Locl T cell-dependent recruitment of mcrophges is typicl feture of DTH. Both nti-cd5 nd nti-cd4 tretments functionlly blocked DTH nd reduced glomerulr mcrophge I I Anti-CD5 ntibody II J Tretment group Fig. 6. Effect of isotype control ntibody, nti-cd5 ntibody nd nti-cd4 ntibody tretment on glomerulr crescent formtion, proteinuri, mcrophge recruitment nd MGC formtion on dy 1 in rts developing nti-gbm GN. Tble 1. Effect of I cell depletion on T cells, I cell subsets, nd NK cells in glomeruli of rts developing nti-gbm GN on dy 1 Tretment group Cell type Isotype control Anti-CD5 Anti-CD4 c/gcs ntibody ntibody ntibody CD5 positive cells TCR positive cells CD4 positive cells CD8 positive cells NK cells Results expressed s men stndrd devition. recruitment nd crescent formtion, suggesting tht these effects re T helper cell dependent. Their filure to completely brogte mcrophge recruitment nd crescent formtion suggests tht other immune meditors such s immunoglobulin lso contribute to these mnifesttions of glomerulr injury. Deposition of utologous ntibody long the GBM hs been demonstrted to recruit mcrophges nd cuse glomerulr injury by Fc dependent mechnisms [28]. The potentil for CD8 positive cells (T cells or NK cells) to contribute to glomerulr injury hs lso recently been demonstrted in similr non-pre-immunized nti-gbm GN model in WKY rts [29]. In tht study, CD8 cell depletion ws induced prior to ntigen sensitiztion, llowing possible effects vi ltertions in ntigen recognition nd T nd B cell sensitiztion s well s the effector phse of the immune response. In the current studies, T cell depletion ws induced fter ntigen sensitiztion to explore only the locl effector role of T helper cells. CD8 positive cells incresed in glomeruli fter tretment with nti-cd5 nd nti-cd4 ntibodies in ssocition with reduced mcrophge recruitment nd injury, indicting tht CD8 positive cells lone do not recruit mcrophges or directly cuse injury. These cells did not stin with specific nti-rt NK cell monoclonl ntibody, suggesting tht they re not NK cells. The explntion for incresed numbers of CD8 bering cells fter tretment with these monoclonl ntibodies is not pprent. In ddition to T helper cells, peritonel mcrophges nd resident tissue mcrophges lso hve the cpcity to express CD4 [15, 23], lthough the functionl role of CD4 on mcrophges is unknown. Some mcrophge popultions in the rt re stined by the monoclonl ntibody W3/25 [23]. In the current studies, lrge numbers of mcrophges were present in glomeruli; however, CD4 bering cells were reltively few in number, indicting tht t lest the mjority of mcrophges recruited to glomeruli in this model were not CD4 positive. The effect of nti-cd4 ntibody on mcrophge function ws studied in model of GN induced by pssive trnsfer of ntibody which results in glomerulr mcrophge recruitment nd injury in the bsence of T cell sensitiztion. In this model nti-cd4 ntibody tretment did not ffect glomerulr mcrophge recruitment or injury, suggesting tht binding of nti-cd4 ntibody to mcrophges is not functionlly significnt in this sitution. In ddition, CD5 is not expressed on mcrophges, but in vivo tretment with nti-cd5 ntibody blocked cutneous DTH nd glomerulr injury to similr extent to nti-cd4 tretment. These observtions suggest tht nti-cd4 tretment does not significntly ffect mcrophge function, nd the

10 78 Hung et!: Mechnisms of glomerulr crescent formtion inhibition of T helper cell effector functions produced by these ntibodies explins their cpcity to inhibit crescent formtion. In conclusion, these studies demonstrte in vivo tretment with monoclonl ntibodies which inhibit T helper cell effector functions ttenute or dely development of glomerulr crescent formtion nd immunopthologicl fetures suggestive of DTH. The reduction of glomerulr mcrophges by both nti- CD5 nd nti-cd4 tretment suggests n importnt role to T helper cells in mcrophge recruitment. These effects were not explined by ltertions in the humorl immune response to the disese inititing ntigen. The observtion tht neither mcrophge recruitment nor crescent formtion could be completely bolished by functionl T helper cell depletion suggests tht glomerulr ntibody deposition (or other immunologicl mechnisms) lso contributes to these fetures of nti-gbm GN. Acknowledgments This work ws supported by grnts from the Ntionl Helth nd Medicl Reserch Council of Austrli nd the Austrlin Kidney Foundtion. The monoclonl nti-rt NK cell ntibody (3.2.2) ws donted by Dr. J. Hiserodt, Pittsburgh, Pennsylvni, USA. Monoclonl nti-rt T cell ntibodies (OX-19, W3/25, nd X8) were donted by the lte Dr. A.F. Willims, Oxford, UK, nd the isotype control monoclonl ntibody by Dr. Xing, Heidelberg, Victori, Austrli. Reprint requests to Dr. P. Tipping, Deprtment of Medicine, Monsh Medicl Centre, Clyton 3168, Victori, Austrli. References 1. BOLTON WK, INNES DJ, STURGILL BC, KAISER DL: T cells nd mcrophges in rpidly progressive glomerulonephritis: Clinicopthologic correltions. Kidney mt 32: , STACHURA I, Si L, WIHTESIDE TL: Mononucler cell subsets in humn idiopthic crescentic glomerulonephritis (ICGN): Anlysis in tissue sections with monoclonl ntibodies. 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