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Popliteal lymph node reactions in mice induced by the drug zimeldine
Int. J. lmmunopharmac., Vol. 11, No. 6, pp. 693-702, 1989. Printed in Great Britain.
0192-0561/89 $3.00 + .00 International .Society for Immunopharmacology.
P O P L I T E A L L Y M P H N O D E REACTIONS IN MICE I N D U C E D BY THE DRUG Z I M E L D I N E C. TtlOMAS, J. GROTEN, M. E. KAMMUI.LER, J. M. DE BAKKER, W. SEINEN a n d N. BLOKSMA Department of Toxicology, Section immunotoxicology, University of Utrecht, P.O. Box 80.176, NL-3508 TD Utrecht, The Netherlands (Received 2 July 1988 and in final form 15 December 1988)
Abstract - - The antidepressant drug zimeldine was screened for immune modulating properties using the popliteal lymph node (PLN) assay as a test system in mice. In immunocompetent as well as congenitally athymic nude mice, footpad injection of 1.0 mg zimeldine triggered a bimodal footswelling. A transient oedematous swelling, histologically characterized by mast cell degranulation, was followed by infiltration of polymorphnuelear cells. A dose-dependent PLN enlargement to the agent was observed, which appeared to be more pronounced in immunocompetent mice as compared with athymic nude mice, and in H-2 b mice as compared with !-I-2d mice. After injection of 1.0 mg zimeldine into the footpad of C57BL/10 mice, significant enlargement was already observed by 3 days after injection, was optimal around day 9 and persisted for at least 30 days. Histologically, PLN reactions were characterized by blast transformation of lymphocytes and expansion of paracortical areas prior to germinal center reactions in enlarged follicles. Size of both areas gradually decreased as the medulla filled with plasma cells, 7 - 30 days after injection. The observed reactions could not be transferred with syngeneic lymph node cells after prior exposition to zimeldine in vivo or in vitro. We conclude that zimeldine induces strong and persistent PLN enlargement, blastogenesis and prominent germinal center reactions, immunocompetent T-cells are apparently conducive, but not prerequisite to these reactions, which suggests involvement of multiple mechanisms including those mediated by inflammatory reactions in the foot. It is unlikely that the observed enlargement of PLN can be attributed to a direct chemical modification of leukocyte membranes by zimeldine. The protracted nature of the reaction may indicate that zimeldine somehow interferes with inhibitory feedback mechanisms.
T h e a n t i d e p r e s s a n t zimeldine was w i t h d r a w n f r o m the m a r k e t shortly after its i n t r o d u c t i o n , as it b e c a m e clear t h a t Guillain-Barr8 s y n d r o m e m i g h t occur as a specific, p r o b a b l y immunologically mediated, side effect o f drug t h e r a p y (Nilsson, 1983; Fagius, O s t e r m a n , Sid~n & W i h o l m , 1985). T h e same measure has been t a k e n for o t h e r new drugs f o u n d to induce a u t o i m m u n e - l i k e diseases in m a n (Bournerias & Habibi, 1979; A m o s , 1983; A n o n y m o u s , 1986; H e n r y & M a r t i n , 1987), indicating that current toxicological screening o f drugs is a p p a r e n t l y i n a d e q u a t e to reveal such effects. D e v e l o p m e n t o f valid screening tests, however, is h a m p e r e d by the still scanty knowledge o n m e c h a n i s m s o f druginduced i m m u n e disregulation. During the past years evidence has been presented that d r u g - i n d u c e d a u t o i m m u n i t y m a y have a similar etiology a n d pathogenesis as a u t o i m m u n e diseases associated with graft-versus-host ( G v H ) disease ( G l e i c h m a n n , 1981 ; Gleichmann, Pals, Rolink, Radaszkiewics &
G l e i c h m a n n , 1984). This view has been s u b s t a n t i a t e d by recent data o n drug-induced reactions in the popliteal l y m p h node ( P L N ) assay, originally developed to q u a n t i f y local G v H reactions (Ford, Burr & Simonsen, 1970). A wide variety o f drugs with a n established capacity to incidentally cause a u t o i m m u n e disorders in m a n , a p p e a r e d to induce GvH-like P L N reactions in mice (Nagata, H u r t e n b a c h & G l e i c h m a n n , 1986; H u r t e n b a c h , G l e i c h m a n n , N a g a t a & G l e i c h m a n n , 1987; Kammiiller, P e n n i n k s , De Bakker, T h o m a s , B i o k s m a & Seinen, 1987, Kammiiller & Seinen, 1988). In the present experiments we investigated the effects o f zimeldine in the P L N assay. Various m o u s e strains a n d T-cell deficient mice were usecl to assess possible genetic restriction a n d T-cell d e p e n d e n c e o f the reactions. Histology was p e r f o r m e d to f u r t h e r characterize the n a t u r e of the effects a n d to estimate the c o n t r i b u t i o n o f T- a n d B-lymphocytes. 693
694
C. THOMAS et al.
EXPERIMENTAL PROCEDURES
Animals Female BALB/c mice (H-2 d) were purchased from Harlan-CPB (Zeist, The Netherlands). Female BALB/c nu/nu and BALB/c nu/+ mice were obtained from TNO (Rijswijk, The Netherlands) and DBA/2 mice (H-2 d) from Bomholtgaard (Ry, Denmark). Female C57BL/6 (H-2 b) as well as C57BL/6 nu/nu and male C57BL/10 (H-2 h) mice were obtained from Olac Ltd (Blackthorne, U.K.). Mice arrived at about 6 weeks of age and were used 2 - 6 weeks later. Lab chow and water were provided ad libitum. Materials Zimeldine-dihydrochioride-monohydrate was a gift from ASTRA Alab AB (S6dert/ilje, Sweden). It was dissolved in saline and adjusted to pH 6 - 7 with IN NaOH immediately prior to use. Fresh sheep erythrocytes in Alsever's old solution were washed three times in phosphate-buffered saline (PBS) before injection. Supernatants of monoclonal lgG rat anti-mouse Thy-I (59 AD 2.2) and lgG rat antimouse kappa (R 226) antibodies were kindly provided by Dr W. van Ewijk (Department of Cell Biology and Genetics, Erasmus University of Rotterdam, The Netherlands). Biotinylated rabbit anti-rat lgG antibodies were obtained from Vector Laboratories (Burlingame, CA, U.S.A.), peroxidaseconjugated streptavidin from Sigma Chemical Company (St Louis, MO, U.S.A.), diaminobenzidine from Merck (Darmstadt, F.R.G.) and peroxidase-conjugated PNA (peanut agglutinin from Arachis hypogaea) from Sanbio B.V., Uden, The Netherlands). Determination o f foot swelling Thickness of the feet was determined prior to and at regular intervals after injection using a paw meter according to Bonta & De Vos (1965) with an accuracy of 0.1 mm. Popliteal lymph node assays For each experiment groups of five to six mice were used, unless otherwise indicated. Mice were injected subcutaneously (s.c.) into the right hind footpad with 10/~1 of graded doses of zimeldine using a 27 gauge needle, or with 2 x 107 SRBC in 50/~1 PBS using a 25 gauge needle. The injections were made in the heel~toe direction, as depicted by
Ford (1978). Mice were killed by cervical dislocation at the times indicated. Both PLN were removed, cleaned of adherent fatty tissue, collected on moist filter paper and weighed within 10 min, using a Mettler H20T balance (d = 0.01 mg). The PLN index of individual mice was calculated as the weight ratio of the experimental over the contralateral lymph node. For indirect PLN assays, syngeneic zimeldinetreated leukocytes were injected instead of zimeldine itself. In vivo exposed PLN cells were obtained 2 days after injection of 1.0 mg zimeldine into the footpad. Draining PLN were removed and dissociated in PBS on ice. Cell suspensions were pooled and washed three times in PBS. After counting and assessment of viability by trypan blue exclusion, 10 7 viable cells in 100 tal PBS were injected into the footpad using a 25 gauge needle. Pooled cell suspensions of popliteal, cervical, inguinal and mesenterial lymph nodes from untreated donor mice were used for in vitro treatment with zimeldine. Cell suspensions were prepared as described above, but after washing resuspended in RPMI 1640 medium (Flow Laboratories, Zwanenburg, The Netherlands) supplemented with 5070 heat-inactivated fetal calf serum, 300 mg glutamine/1, 2 g sodium-bicarbonate/l, 2 × 10 ~ M mercaptoethanol and 50 mg gentamycin/l at a density of 5 x 106 viable cells per ml. One ml of these suspensions was transferred to the wells of 24-well flat-bottomed culture plates (Greiner Products, Alphen aan de Rijn, The Netherlands) and supplemented with 1.0 ml PBS containing 0, 5 or 50tag zimeldine. Addition of 100/ag zimeldine proved to be above the cytotoxicity level. Cells were incubated at 37°C and 507o CO2 for 20 h, and subsequently harvested, pooled and washed three times in PBS. 10~ viable cells in a volume of I00/~1 PBS were then injected into paws of recipient mice. PLN indices were determined 7 days after cell transfer.
Histology Immediately after determination of weight, PLN were fixed in 4070 phosphate-buffered formaldehyde and embedded in Historesin (LKB Produkter AB, Bromma, Sweden). Serial sections (3 pm) were stained with methylgreen pyronine. Paws were fixed in 4070 phosphate-buffered formaldehyde for 48 h and successively incubated in 70070 ethanol for 12 h, in a mixture (1:1) of 98070 formic acid and 70070 ethanol for 24 h, and in 7007o ethanol for 24 h, which was refreshed once after 12 h. Subsequently, paws were dehydrated and embedded in Historesin.
Zimeldine-induced Popliteal Lymph Node Reactions Sections (3 ~m) were stained with May-Grtinwald Giemsa.
Determination o f T- and B-lymphocytes in P L N PLN, embedded in Tissue-tek (Miles Laboratories, Naperville IL, U.S.A.), were snap frozen in liquid nitrogen and stored at - 70°C. Serial cryostat sections (6 ~m) were air dried and fixed in acetone for 1 rain. Fc-receptors were blocked by incubation with 1070 normal rabbit serum in PBS for 30 min. To discriminate between T- and B-cells, sections were incubated for 45 rain with, respectively, 1:20 diluted anti-Thy-I or anti-kappa antibodies in PBS supplemented with 1% normal rabbit serum, and subsequently for 30 min with 1:300 diluted biotinylated rabbit anti-rat lgG antibodies in PBS supplemented with 107o normal mouse serum to diminish non-specific staining. Sections were further incubated for 30 min with 1:800 diluted peroxidase-conjugated streptavidin and developed by incubation with 0.05070 diaminobenzidine in 0.05 M T r i s - H C I buffer (pH 7.6), 0.1 M imidazole and 0.01070 H.,O2. Sections were slightly counterstained with haematoxilin, dehydrated and mounted in Depex. For demonstration of germinal centers, acetonefixed cryostat sections were incubated for 45 min with 1:20 diluted peroxidase-conjugated PNA. Sections were developed by incubation with 3-amino9-ethyl carbazole in 0.05 M acetate buffer (pH 5.0) containing 0.01070 H.,O_,, counterstained with haematoxilin and mounted in Aquamount (Gurr, BDH Chemicals, Poolc, U.K.).
Morphometrical analysis" Large pyroninophilic cells, representing immunoblasts were counted in methylgreen pyronine-stained plastic sections at a magnification of 400 x using a 10 × 10 square grid. For each treatment, three to five serial sections of 3 PLN were counted. Data were converted to the average number of large pyroninophilic cells per square mm paracortex, primary follicle and - - if present - - germinal center. Paracortical, follicular and medullary areas were measured in anti-Thy-l- or anti-kappa-stained cryostat sections using a digitizer tablet (Minimop, Kontron Bildanalyse, Eching, F.R.G.). For each treatment four to six serial sections of central parts of 3 PLN were used and areas of follicles and paracortex were expressed relative to the area of the PLN section minus medullary areas. The latter were identified as being kappa positive areas located outside the cortex.
695
Data handling and statistics Data have been expressed as arithmetical mean ___S.E.M. When appropriate, analysis for significance was performed with the two-tailed nonparametric M a n n - W h i t n e y U-test. P-values over 0.05 were considered not significant.
RESULTS
Effects o f s.c. injected zimeldine on f o o t swelling Injection of zimeldine into the foot induced a bimodal foot swelling in C57BL/10 mice (Fig. 1A), C57BL/6 and T-cell deficient C57BL/6 nu/nu mice (Fig. 2) as well as in BALB/c mice and BALB/c nu/nu mice (data not shown). As determined in C57BL/10 (Fig. IA) an immediate, dose-dependent swelling was observed 4 h after injection, which declined within 1 or 2 days. A second dosedependent swelling started around day 3, reached a plateau at day 5 - 7 and declined gradually thereafter. After injection of 1.0 mg zimeldine, control values were not attained until day 50; the presence of crusts often prevented adequate measurements of foot swelling between days 1 4 - 30. A dose of 0.3 mg induced a strong immediate swelling and only a moderate foot swelling around day 3 - 5 , which subsided within the next days (Fig. IA). A dose of 0.1 mg did not induce a significant foot swelling as compared to injection with saline (data not shown). The immediate swelling observed 4 h after injection of 1.0 mg zimeldine in C57BL/10 mice was featured by interstitial oedema and degranulation of mast cells (Fig. 3A). The second swelling, as determined on day 5, was characterized by oedema and massive infiltration of polymorphnuclear cells in the dermis (Fig. 3B) and in the underlying muscle tissue. S.c. injection of 10~ zimeldine-treated syngeneic cells into the footpad elicited a transient foot swelling due to the injected volume of 100 ~1. No second swelling was observed (data not shown).
P L N reactions to zimeldine The kinetics of the PLN response were studied in C57BL/10 mice (Fig. IB). A dose of 0.3 mg induced a moderate but significant PLN enlargement at day 5. PLN indices up till then and afterwards did not differ from the index of saline-treated controls on day 7. Maximal increase of PLN weights after injection of 1.0 mg was found between day 2 and day 5, when indices of 1.5 and 8.0 on an average were calculated, respectively. The highest indices were observed at day 9, although individual
C. THOMAS et al.
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Fig. 1. Kinetics o f zimeldine-induced foot swelling (A) and PLN reaction (B). Groups of six C57BL/10 mice received an injection into the footpad at day 0 with 1.0 mg (©) or 0.3 mg ( 0 ) zimeldine, or with saline (A). The same mice were used to determine foot swelling and PLN indiccs. Vertical bars indicate S.E.M. *P<0.05 as compared to saline-treated controls (A) or saline-treated controls on day 7 (B).
variation was considerable. From day 9 on, PLN indices gradually decreased, but remained s i g n i f i c a n t l y e n h a n c e d u p till d a y 30. A t d a y 50 a f t e r i n j e c t i o n , i n d i c e s w e r e still 2.7 t i m e s t h e c o n t r o l value. R e a c t i o n s to z i m e l d i n e in T-cell d e f i c i e n t B A L B / c n u / n u m i c e were c o m p a r e d w i t h t h o s e in n u / + l i t t e r m a t e s ( T a b l e 1). O n d a y 7 a f t e r i n j e c t i o n o f
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Fig. 2. T-cell dependence of zimeldine-induced foot swelling. Groups o f six mice received an injection into the footpad at day 0 with 1.0 mg zimeldine (C57BL/6: (_3; C57BL/6 n u / n u : O) or saline (C57BL/6: A; C57BL/6 nu/nu: A ) . Foot swelling was determined at times indicated. Vertical bars indicate S.E.M.
Difference with controls
Strain
Treatment
n
P L N index - S.E.M.
Nu/nu Nu/nu
Saline Zimeldine
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62%
Nu/+ Nu/+
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13 15
0.9_.0.1 2.5 ~ 0.2*
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Mean PI.N indices _+ S.E.M. o f n individual mice were determined 7 days after s.c. injection o f 1.0 mg zimeldine in 10 IA solvent or solvent only into the one hind footpad *P<0.05 as compared to saline-treated controls using the Mann - Whitney U-test.
Zimeldine-induced Popliteal Lymph Node Reactions
697
Fig. 3. Histology of paws of C57BL/10 mice, injected s.c. with 1.0 mg zimeldine. Plastic sections were stained with MayGriinwald Giemsa. (A) 4 h after injection. Degranulation of mast cells in the dermis. (B) 5 days after injection. Infiltration of mainly polymorphnuclear cells into the dermis.
C. THOMASet al.
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Fig. 4. Histology of PLN of C57BL/10 mice (A - C, E - F) and BALB/c nu/nu (D), after s.c. footpad injection of 1.0 mg zimeldine. (A, C - F): methyl green pyronine-stained plastic sections; (B): Thy-l-stained cryostat section. (A) Uninjected control. PC, paracortex; F, primary follicle. (B) 20 days after injection. GC, germinal centre; C, follicle corona; PC, paracortex. (C) Detail of germinal center, 30 days after injection, showing mitotic figures (upper arrow), tingible body macrophages (middle arrow) and immunoblasts (lower arrow) (180 × ). (D) Follicle with germinal center in BALB/c nu/nu mouse, 7 days after injection. Scattered immoblasts are present in depleted paracortex (arrows) (150 x ). (E) Medulla, 3 days after injection. Scattered immunoblasts are seen (arrows) among mainly small lymphocytes (210 x ). (F) Medulla, 9 days aftcr injection, showing abundance of plasma cells (400 × ).
Zimeldine-induced Popliteal Lymph Node Reactions 1.0 mg zimeldine, mean P L N indices o f 1.8 and 2.5 were calculated for n u / n u and n u / + mice, respectively. The two B A L B / c strains failed to m o u n t a significant P L N response to doses o f 0.3 mg zimeldine on day 7 after injection (data not shown). Severe necrosis o f the injected paw was f o u n d after injection o f 3.3 rag; therefore no data are included.
M o u s e strain zimeldine
dependence
of PLN
reactions
metrically. Serial cryostat sections o f control P L N stained for Thy-I and k a p p a showed that on an average 60°7o and 30°7o o f the section areas were covered by paracortical and follicular areas, respectively. Whereas this 2:1 ratio did not change u p o n treatment with zimeldine on the times indicated in Table 4, their relative contribution to P L N enlargement changed in time. Enlargement could be
to
P L N responsiveness by 7 days after injection a p p e a r e d to vary a m o n g genetically different mouse strains (Table 2). A dose o f 0.3 mg zimeldine did not elevate the P L N index as c o m p a r e d to control mice in any strain tested. Whereas a dose o f 0.7 mg did not induce a significant reaction in B A L B / c mice, it did in C57BL/10 mice. A significant P L N reaction to 1.0 mg zimeldine was f o u n d in each strain. While D B A / 2 mice showed similar reactivity as B A L B / c mice, the P L N index in C57BL/10 mice was over three times higher.
Indirect P L N reactions to zimeldine D o n o r cell suspensions were prepared f r o m P L N , 2 days after s.c. injection o f 1.0 mg zimeldine or saline. The P L N o f the zimeldine-treated d o n o r s contained twice as many cells as the P L N o f the controls indicating P L N activation by zimeldine. T r a n s f e r o f 10: zimeldine-cxposed cells to syngeneic recipients, however, did not evoke higher P L N responses on day 7 than transfer o f the same n u m b e r o f control cells (Table 3). Likewise, transfer o f 107 syngeneic lymph n o d e cells u p o n incubation with 2.5 ~g or 25/ag z i m e l d i n e / m l for 20 h failed to evoke significantly e n h a n c e d P L N indices in c o m p a r i s o n with control cells.
Table 3. Indirect PLN responses to syngeneic zimeldineexposed lymph node cells Treatment of donor cells
PI.N index _+ S.E.M. on day 7 after cell transfer
n
In vivo Saline Zimcldine
2.3 _+ 0.3 2.3 _+ 0.2
5 l0
In vitro PBS 2.5 ~gr zimeldine/ml 25 /agr zimeldinc/ml
2.9 +- 0.3 2.1 +- 0.2 2.7 _+ 0.3
6 7 7
Groups of n BALB/c mice received 10~ zimeldinetreated or control lymph node cells in 100,1 PBS into one hind footpad. In vivo treated donor cells were obtained 2 days after s.c. injection of 1.0 mg zimeldine or saline into the footpad of BALB/c mice. In vitro treated cells were obtained after incubation of pooled lymph node cell suspensions with PBS or zimeldine for 20 h. Cells were washed three times prior to injection.
Table 4. Number of pyroninophilic cells per square mm in PLN after s.c. zimeldine injection
Histological characterization o f the P L N response to zimeMine P L N o f C 5 7 B L / 6 mice were analysed m o r p h o Table 2. Mouse strain dependence of PI.N reactions to zimeldine
Treatment
BALB/c
PLN index DBA/2
C57B!./10
Saline 0.3 mg zimcldine 0.7 mg zimeldine 1.0 mg zimeldine
1.4 1.5 1.5 2.1
1.0 _+ 0.0 n.d. n.d. 3.0+_0.2*
1.1 _+ 0.1 1.7 ± 0.2 2.5 ± 0.4* 7.6_+ 1.1"
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PLN indices (mean + S.E.M. of five mice per group) were determined 7 days after s.c. injection into the footpad. *P<0.05 as compared with saline-treated controls. n.d., not determined.
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PLN were obtained on various times after injection of 1.0 mg zimeldine into the footpad of C57BI./6 mice. In plastic sections of PLN stained with methyl green pyronine the number of pyroninophilic cells per square mm in five to seven serial sections of three PLN per group was quantified using a grid at magnification 400 ×. Number of pyroninophilic cells per square mm: - - <50; *50- 100; i'100- 500; ~:>500. Germinal centers, if present, all contained over 1000 pyroninophilic cells per square mm. n.d., not determined.
700
C. THOMAS el al.
solely attributed to a proportional expansion of T- and B-cell areas up to day 7, after which the medulla contributed increasingly to PLN enlargement. Medullary expansion attained a maximum around day 20, when the medulla covered on average about 30°7"o ot" the section areas. Histological examination of the F'I.N showed already increased numbers of pyroninophilic cells in the paracortical areas by 2 days after injection ot" 1.0 mg zimeldine (Table 4). In general, further increase was seen until day 7, although considerable individual differences were detected. A lesser, but still substantial number of paracortical immunoblasts was seen from day 9 till day 30. ht follicular areas increased numbers of pyroninophilic cells were not observed until day 7. At that time few secondary follicles, characterized by a densely packed corona surrounding a pyroninophilic germinal center, were present. Germinal centers contained cells in mitosis and many tingiblc body nmcrophages (Fig. 4C). Numbers and size of germinal centers in follicular areas increased until days 2 0 - 3 0 . In the medulla, increased numbers of large pyroninophilic plasma cells were detected between days 9 and 20 after injection (Fig. 4E,F). By day 50, germinal centers had vanished. Blurring of lymph node architecture at this time prevented adequate determination of pyroninophilic cell numbers in the distinct areas. Histological features of PI.N reactions to 0.3 mg zimcldine appeared qualitatively and kinetically similar to those observed after injection of 1.0 rag, but less pronounced (data not shown). In BALB/c n u / n u mice, studied on day 7 after injection of 1.0 mg zimeldine, increased numbers of immunoblasts as compared with controls were seen in the paracortcx, which further appeared to be severely depleted of lymphocytes (Fig. 41)). Secondary follicles were frequently observed, and plasma cells in the medulla were also detected. Presence of germinal center reactions in T-cell deficient and T-cell competent mice was further studied on day 7 after injection using cryostat sections incubated with PNA, specifically staining for germinal center cells (Racdler, Racdler, Arndt & Thicle, 1981; Rcichert, Gallatin, Weissman & Butcher, 1983; Kraal, Hardy, Gallatin, Weissman & Butcher, 1986). in the follicles of PI.N of mice injected with zimeldinc or sheep crythrocytes but not in those of control BALB/c n u / + mice, PNA positive areas were generally detected. Size of germinal centers induccd by sheep erythrocytcs, however, appeared to bc much larger than after zimeldine treatment. In PLN of BAI.B/c n u / n u mice, small PNA-positivc areas were found in many cases (data not shown).
DISCUSSION
Injection of zimeldine into the footpad induced a dose-dependent bimodal foot swelling. The first swelling was recorded by 4 h after injection and subsided within 1 or 2 days. The second swelling usually reached a plateau by day 5 and persisted for 1 or 2 days before subsidence. The bimodal reaction to zimcldinc is indicative of diffcrent underlying mechanisms. This is supported by histological data. The early observed interstitial ocdema, degranulated mast cells and few inflammatory cells indicate that zimeldine-induced release of vasoactive amines from mast cells might have caused the first reaction. The second swelling was characterized by a dense intqammatory infiltrate of mainly polymorphnuclear cells. In mice and rats such infiltrates can be indicative of antigcn specific delayed-type hypersensitivity reactions (Cooper, 1972; Crowle, 1975; Henningsen, Koller, Exon, Talcott & Osborne, 1984). Whereas this is in line with observations that zimeldine could induce contact sensitivity reactions in guinea pigs (Henderson, Edwards, Weston & Dewdncy, 1988), the observations that T-cell deficient nude mice showed macroscopically similar paw reactions to zimeldine indicates that these reactions are rather the result of non-specific inflammation. Next to inflammatory footpad reactions, zimeldine triggered a significant increase of the PLN index in all strains tested. Striking quantitative differences were detected between H-2 ' and 11-2" strains. Experiments with intra-H-2 recombinant strains, however, arc needed to finally assess possible MHC restriction and involvement on non-H-2 genetic factors in PLN reactions to zimeldine. The significant PLN weight increase in n u / n u mice after zimcldine injection is an unexpected phenomenon, because the low mol. wt drugs diphcnylhydantoin (Gleichmann, 1981; Kammi.illcr & Seincn, 1987), D-pcnicillaminc (Hurtenbach et al., 1987) and HgCI, (Stiller-Winkler, Radaszkiewics & Gleichmann, 1988) failed to do so under similar circumstances. PLN activation by zimeldinc in n u / n u mice was confirmed by morphological observations such as increased presence of immunoblasts in the paracortcx and plasma cells in the medulla. Moreover, prelinfinary results have indicated that zimeldinc but not sheep erythrocytes increases the production of IgG by PLN cells from n u / n u mice. The ability of zimcldinc to induce activation of B-cells in the absence of T-cell helper factors suggests that the drug triggers secretion of soluble factors like II.-4 by nmst cells in the footpad as well as in the
Zimeldine-induced Poplitcal Lymph Node Reactions PLN itself, thus inducing T-cell independent stimulation of B-cells (O'Garra, Umland, De France & Christiansen, 1988). Another possibility is that it evokes the secretion of autocrine B-cell-derived growth factors (Muraguchi, Nishimoto, Kawamura, Hori & Kishimoto, 1986), thus bypassing activating signals normally provided by T-cells in immunocompetent mice. T-cells, however, are apparently needcd to induce strong PLN reactions against zimeldine. The T-cell dependent mechanism involved has as yet not been elucidated. The tool. wt of the agent is too low to have immunogenic properties of its own (Benacerraf & Unanue, 1979), and preliminary in vitro data seem to exclude direct mitogenic action or influence on mitogen-induced lymphoproliferation, confirming earlier experiments with lymphoid cells of mice (Henderson et al., 1988) and humans (Kristofferson & Nilsson, 1989). The drug may act as a hapten when covalently bound to a macromolecular carrier, but reactions with amino or thiol groups in vitro could not be demonstrated (Henderson et al., 1988). The resemblance of weight kinetics and morphology of the PLN during the first week after zimeldinc treatment with those induced by diphenylhydantoin (Glcichmann, 1981 ; Gleichmann, Pals & Radaszkicwics, 1983; Kammiiller et al., 1987) and semi-allogeneic lymphoid cells (Ford et al., 1970; Romano, Ponzio & Thorbeckc, 1976; Rolstad, 1985) might point to a GvH-like etiology. It would imply that zimeldine-induced B-cell activation is the consequence of polyclonal helper T-cell activation induced by drug-modified class il determinants of the MHC (Gleichmann et al., 1984). Lymphokines released by these T-cells would provide the second signal to autoantigen recognizing B-cells to start proliferation and antibody production. The amphi-
701
philic nature of zimeldine is in support of this notion, because it is likely to facilitate an interaction with the phospholipid bilayer of cell membranes and association with other membrane determinants (Liillmann, Lullmann-Rauch & Wassermann, 1975; Bockhardt & Liillmann-Rauch, 1980). Further, it is consistent with the observation that reactions to zimeldine are under genetic control and enforced by the presence of immunocompetent T-cells. Several data, however, question whether an "altered self" mechanism is responsible for the zimeldine-induced PLN reactions. First, unlike those reported for diphenylhydantoin by Gleichmann et al. (1981), PLN reactions to zimeldine could not be elicited by transfer of zimeldine-treated cells. Second, PEN enlargement was detected in PLN of nude mice after zimeldine injection, while reactions to diphenylhydantoin and various other drugs have been reported to be strictly T-cell dependent (Gleichmann, 1981; Hurtenbach et al., 1987; Kamm~ller et al., 1987). Third, zimeldine induced extremely prolonged lymph node reactions upon a single s.c. injection, while alloreactive lymphocytes and low mol. wt compounds were found to induce short lasting lymph node reactions with an optimum around day 7 (Gleichmann, 1981; Twist & Barnes, 1973; Kammiiller et al., 1987). The prolonged stimulation suggests that zimeldine (or its metabolites) might be retained at the site of injection or inside the PEN for a long period of time and that it possibly interferes with inhibitory feedback mechanisms. Further experiments are needed to study in detail the production of antibodies by PLN cells stimulated with zimeldine and to elucidate the mode of action of the drug in this system.
REFERENCES
A,,los, H. E. (1983). Immunotoxicology, outline of major problems. In Immunotoxicology (eds Gibson, G. G., Hubbard, R. and Parke, D. V.) pp. l -5. Academic Press, I.ondon. ANONYMOUS(1986). Withdrawal of nomifensine. Lancet, l, 28 I. t]ENACERRAI.,B. & UNANL'F,E. R. (1979). Immunology, principles and phenomenology. In Textbook o f Immunology, pp. 1 - 1I. Williams & Wilkins, Baltimore, MD. BO(KHARDT, H. & Lt)LIMANN-RALI('tl, R. (1980). Zimelidine-induced lipidosis in rats. Acta Pharmac. Toxicol., 47, 45--48. BONrA, I. C. & Dr Vos, C. J. (1965). The effect of estriol-16,17-dihemisuccinateon vascular permeability as evaluated in the rat paw oedema test. Acta Endocrinol., 47, 403- 411. BOUNERIAS, F. & HABIBI, B. (1979). Nomifensine-induced immune hacmolytic anaemia and impaired renal function. Lancet, If, 95- 96. COOPER, M. G. (1972). Delayed-type hypersensitivity in the mouse. I. Induction and elicitation by Salmonella adelaide flagellin and its derivatives. Scand. J. lmmun., l, 167- 178. CROWLE, A. J. (1975). Delayed hypersensitivity in the mouse. Adv. Immun., 20, 197-264.
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FAGIUS, J., OSTERMAN, P. O., SIDI~N, A. ~,¢ WIHOI.M, B.E. (1985). Guillain-Barre syndrome following zimeldine treatment. J. Neur. Neurosurg. Psychiat., 48, 6 5 - 69. FORD, W. I,., BURR, W. & SIIvlt)NSEN, M. (1970). A lymph node weight assay for the Graft-versus-Host activity of rat lymphoid cells. Transplantation, 10, 258- 266. Fop,I), W. L. (1978). Measurement of Graft-versus-Host reactivity. In ltandbook o f Experimental Immunology (ed. Weir, D. M.) pp. 30.1 -30.12. Blackwell, Oxford. Gt.EI(HMANN, H. (1981). Studies on the mechanism of drug sensitization: T-cell-dependent popliteal lymph node reaction to diphenylhydantoin. Clin. Immun. Immure)path., 18, 203- 211. GLEICIIMANN, E., PALS, S . T . , ROIINK, A . G . , RAI)ASZKII!WICS, T. & GI.EICIIMANN, H. (1984). Graft-versus-Host reactions: clues to the etiopathology of a spectrum of immunological diseases. Immun. Today, 5, 324- 332. GIEICIIMANN, U., PAl.S, S. T. 8¢ RADASZKIEWICS,T. (1983). T-cell-dependent B-cell proliferation and activation induced by administration of the drug diphenylhydantoin to mice. ttemat. Oncol., !, 165- 176. HI-~NI)ERSON, D. C., El)WARDS, R. G., WESTON, B. J. & I)EWl)NEY, J. M. (1988). Immunological studies on paroxetine, a novel anti-depressant drng. Int. J. hnmunopharmac., 10, 361 -367. I-h-NMN¢3StiN, G. M., KOt.LER, I.. D., EXON, J. II., TAI LC'o'rT, P. A. & OSaORNL, C. A. (1984). A sensitive delayed-type hypersensitivity model in the rat, for assessing in vivo cell mediated immunity. J. Immun. Meth., 70, 153- 165. HI~NRY, J. A. & MARTIN, A. J. (1987). Thc risk-benefit assessment of antidepressant drugs. Med. Toxicol., 2, 445 - 462. HtJR'rl~NgACll, U., GtEICIIMANN, H., NAOAIA, M. & GIJ~IC:HMA~N, E. (1987). Immunity to D-penicillamine: genetic, cellular and chemical requirements for induction of popliteal lymph node enlargement in the mouse. J. Immun., 139, 411 -416. KAMMUI.I.ER, M. E., PENNINKS, A. H., DE BAKKER, J. M., TrIOMAS, C., BI.OKSMA, N. & St'INEN, W. (1987). An cxperimental approach to chemically induced systemic (auto)immune alterations: the spanish toxic oil syndrome as an example. In Mechanisms o f Cell Injury: ImplicationsJbr lluman Health (ed. Fowler, B. A.) pp. 175- 192. John Wiley, Chichester. KAMM01.IER, M. E. & SEINEN, W. (1988). Structural requirements for hydantoins and 2-thiohydantoins to induce lymphoprolifcrative popliteal lymph node reactions in the mouse. Int. J. Immunopharmac., 10, 997- 1010. KRAAl, G., HARDY, R., GAI LATIN, W. M., WI-:ISSMAN,I. L. & BurCHER, E. C. (1986). Antigen induccd changes in B Cell subsets in lymph nodes: analysis by dual fluorescence flow cytofluorometry. Eur. J. lmmun., 16, 829-834. KRIS'rOI:FERSON, A. & NtLSSOr,,', B. S. (1989). Zimeldinc: febrile reactions and peripheral neuropathy. In AutoimmuniO, and Toxicology. Immune Disregulation Induced by Drugs and Chemicals (cds Kammiiller, M. E., Bloksma, N. and Seinen, W.) pp. 183- 214. Elsevicr Science, Amsterdam. LUI.LMANN, H., LULt.MANN-RAUCH, R. & WASSERMANN, O. (1975). Drug-induced phospholipidosis. CRC crit. Rev. Toxicol., 4, 185-218. MURAGUCHI, A., NISIIIMO'IO, H., KAWAMt;RA,N., HORI, A. & KIStnMOTO, T. (1986). Cell-derived BCGF functions as autocrine growth factor(s) in normal and transformed B lymphocytes. J. hnmun., 137, 179- 185. NAC;ATA, N., HUR rI-:NnAC'H,U. & GI I!ICHMANN,E. (1986). Specific sensitization of Lyt I 2 - T cells to spleen cells modified by the drug D-penicillaminc or a stereo isomer. J. Immun., 136, 136- 142. NII.SSON, B. S. (1983). Adverse reactions in connection with zimeldine treatment: a review. Acta Psych. scand., 308 (Suppl. I), 115-119. O'GARRA, A., UMLAND,S., DF FRANCE,T. & CttRISTIANSEN,J. (1988). B cell factors are plciotropic, lmmun. Today, 9, 45 - 54. RAEI)I ER, A., RAI~DI.FR, E., ARNDT, R. & THIELI-:, H. G. (1981). Centroblasts and centrocytes display receptors for peanut lectin. Irnmun. Lett., 2, 335- 338. RHCHERt, R. A., GALl ATlr',', W. M., WEISSMAN, 1. L. & BUrCHI~R, E. C. (1983). Germinal ccnter B-cells lack homing receptors necessary for normal lymphocyte recirculation. J. exp. Med., 157, 813- 827. ROt.SrAD, B. (1985). The popliteal lymph node Graft-versus-Host (GvH) reaction in the rat: a useful model for studying cell interactions in the immune response? Immun. Rev., 88, 153- 168. ROMANO, T. J., PONZlO, N. M. & TIIORnEC'~F, G. J. (1976). Graft-versus-Host reactions in FI mice induced by parental lymphoid cells: nature of recruited FI cells. J. Irnmun., 116, 1618 - 1623. STILLER-WINKtER, R., RADASZKmWlC'S,T. & GLEXCHMANN, E. (1988). Immunopathological signs in mice treated with mercury compounds. 1. Identification by the poplitcal lymph node assay of responder and nonresponder strains. Int. J. hnmunopharmac., 10, 475- 484. TWIST, V. W. & BARNES, R. D. (1973). Popliteal lymph node weight gain assay for Graft-vcrsus-ilost reactivity in mice. Transplantation, 15, 182-- 185.
0192-0561/89 $3.00 + .00 International .Society for Immunopharmacology.
P O P L I T E A L L Y M P H N O D E REACTIONS IN MICE I N D U C E D BY THE DRUG Z I M E L D I N E C. TtlOMAS, J. GROTEN, M. E. KAMMUI.LER, J. M. DE BAKKER, W. SEINEN a n d N. BLOKSMA Department of Toxicology, Section immunotoxicology, University of Utrecht, P.O. Box 80.176, NL-3508 TD Utrecht, The Netherlands (Received 2 July 1988 and in final form 15 December 1988)
Abstract - - The antidepressant drug zimeldine was screened for immune modulating properties using the popliteal lymph node (PLN) assay as a test system in mice. In immunocompetent as well as congenitally athymic nude mice, footpad injection of 1.0 mg zimeldine triggered a bimodal footswelling. A transient oedematous swelling, histologically characterized by mast cell degranulation, was followed by infiltration of polymorphnuelear cells. A dose-dependent PLN enlargement to the agent was observed, which appeared to be more pronounced in immunocompetent mice as compared with athymic nude mice, and in H-2 b mice as compared with !-I-2d mice. After injection of 1.0 mg zimeldine into the footpad of C57BL/10 mice, significant enlargement was already observed by 3 days after injection, was optimal around day 9 and persisted for at least 30 days. Histologically, PLN reactions were characterized by blast transformation of lymphocytes and expansion of paracortical areas prior to germinal center reactions in enlarged follicles. Size of both areas gradually decreased as the medulla filled with plasma cells, 7 - 30 days after injection. The observed reactions could not be transferred with syngeneic lymph node cells after prior exposition to zimeldine in vivo or in vitro. We conclude that zimeldine induces strong and persistent PLN enlargement, blastogenesis and prominent germinal center reactions, immunocompetent T-cells are apparently conducive, but not prerequisite to these reactions, which suggests involvement of multiple mechanisms including those mediated by inflammatory reactions in the foot. It is unlikely that the observed enlargement of PLN can be attributed to a direct chemical modification of leukocyte membranes by zimeldine. The protracted nature of the reaction may indicate that zimeldine somehow interferes with inhibitory feedback mechanisms.
T h e a n t i d e p r e s s a n t zimeldine was w i t h d r a w n f r o m the m a r k e t shortly after its i n t r o d u c t i o n , as it b e c a m e clear t h a t Guillain-Barr8 s y n d r o m e m i g h t occur as a specific, p r o b a b l y immunologically mediated, side effect o f drug t h e r a p y (Nilsson, 1983; Fagius, O s t e r m a n , Sid~n & W i h o l m , 1985). T h e same measure has been t a k e n for o t h e r new drugs f o u n d to induce a u t o i m m u n e - l i k e diseases in m a n (Bournerias & Habibi, 1979; A m o s , 1983; A n o n y m o u s , 1986; H e n r y & M a r t i n , 1987), indicating that current toxicological screening o f drugs is a p p a r e n t l y i n a d e q u a t e to reveal such effects. D e v e l o p m e n t o f valid screening tests, however, is h a m p e r e d by the still scanty knowledge o n m e c h a n i s m s o f druginduced i m m u n e disregulation. During the past years evidence has been presented that d r u g - i n d u c e d a u t o i m m u n i t y m a y have a similar etiology a n d pathogenesis as a u t o i m m u n e diseases associated with graft-versus-host ( G v H ) disease ( G l e i c h m a n n , 1981 ; Gleichmann, Pals, Rolink, Radaszkiewics &
G l e i c h m a n n , 1984). This view has been s u b s t a n t i a t e d by recent data o n drug-induced reactions in the popliteal l y m p h node ( P L N ) assay, originally developed to q u a n t i f y local G v H reactions (Ford, Burr & Simonsen, 1970). A wide variety o f drugs with a n established capacity to incidentally cause a u t o i m m u n e disorders in m a n , a p p e a r e d to induce GvH-like P L N reactions in mice (Nagata, H u r t e n b a c h & G l e i c h m a n n , 1986; H u r t e n b a c h , G l e i c h m a n n , N a g a t a & G l e i c h m a n n , 1987; Kammiiller, P e n n i n k s , De Bakker, T h o m a s , B i o k s m a & Seinen, 1987, Kammiiller & Seinen, 1988). In the present experiments we investigated the effects o f zimeldine in the P L N assay. Various m o u s e strains a n d T-cell deficient mice were usecl to assess possible genetic restriction a n d T-cell d e p e n d e n c e o f the reactions. Histology was p e r f o r m e d to f u r t h e r characterize the n a t u r e of the effects a n d to estimate the c o n t r i b u t i o n o f T- a n d B-lymphocytes. 693
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C. THOMAS et al.
EXPERIMENTAL PROCEDURES
Animals Female BALB/c mice (H-2 d) were purchased from Harlan-CPB (Zeist, The Netherlands). Female BALB/c nu/nu and BALB/c nu/+ mice were obtained from TNO (Rijswijk, The Netherlands) and DBA/2 mice (H-2 d) from Bomholtgaard (Ry, Denmark). Female C57BL/6 (H-2 b) as well as C57BL/6 nu/nu and male C57BL/10 (H-2 h) mice were obtained from Olac Ltd (Blackthorne, U.K.). Mice arrived at about 6 weeks of age and were used 2 - 6 weeks later. Lab chow and water were provided ad libitum. Materials Zimeldine-dihydrochioride-monohydrate was a gift from ASTRA Alab AB (S6dert/ilje, Sweden). It was dissolved in saline and adjusted to pH 6 - 7 with IN NaOH immediately prior to use. Fresh sheep erythrocytes in Alsever's old solution were washed three times in phosphate-buffered saline (PBS) before injection. Supernatants of monoclonal lgG rat anti-mouse Thy-I (59 AD 2.2) and lgG rat antimouse kappa (R 226) antibodies were kindly provided by Dr W. van Ewijk (Department of Cell Biology and Genetics, Erasmus University of Rotterdam, The Netherlands). Biotinylated rabbit anti-rat lgG antibodies were obtained from Vector Laboratories (Burlingame, CA, U.S.A.), peroxidaseconjugated streptavidin from Sigma Chemical Company (St Louis, MO, U.S.A.), diaminobenzidine from Merck (Darmstadt, F.R.G.) and peroxidase-conjugated PNA (peanut agglutinin from Arachis hypogaea) from Sanbio B.V., Uden, The Netherlands). Determination o f foot swelling Thickness of the feet was determined prior to and at regular intervals after injection using a paw meter according to Bonta & De Vos (1965) with an accuracy of 0.1 mm. Popliteal lymph node assays For each experiment groups of five to six mice were used, unless otherwise indicated. Mice were injected subcutaneously (s.c.) into the right hind footpad with 10/~1 of graded doses of zimeldine using a 27 gauge needle, or with 2 x 107 SRBC in 50/~1 PBS using a 25 gauge needle. The injections were made in the heel~toe direction, as depicted by
Ford (1978). Mice were killed by cervical dislocation at the times indicated. Both PLN were removed, cleaned of adherent fatty tissue, collected on moist filter paper and weighed within 10 min, using a Mettler H20T balance (d = 0.01 mg). The PLN index of individual mice was calculated as the weight ratio of the experimental over the contralateral lymph node. For indirect PLN assays, syngeneic zimeldinetreated leukocytes were injected instead of zimeldine itself. In vivo exposed PLN cells were obtained 2 days after injection of 1.0 mg zimeldine into the footpad. Draining PLN were removed and dissociated in PBS on ice. Cell suspensions were pooled and washed three times in PBS. After counting and assessment of viability by trypan blue exclusion, 10 7 viable cells in 100 tal PBS were injected into the footpad using a 25 gauge needle. Pooled cell suspensions of popliteal, cervical, inguinal and mesenterial lymph nodes from untreated donor mice were used for in vitro treatment with zimeldine. Cell suspensions were prepared as described above, but after washing resuspended in RPMI 1640 medium (Flow Laboratories, Zwanenburg, The Netherlands) supplemented with 5070 heat-inactivated fetal calf serum, 300 mg glutamine/1, 2 g sodium-bicarbonate/l, 2 × 10 ~ M mercaptoethanol and 50 mg gentamycin/l at a density of 5 x 106 viable cells per ml. One ml of these suspensions was transferred to the wells of 24-well flat-bottomed culture plates (Greiner Products, Alphen aan de Rijn, The Netherlands) and supplemented with 1.0 ml PBS containing 0, 5 or 50tag zimeldine. Addition of 100/ag zimeldine proved to be above the cytotoxicity level. Cells were incubated at 37°C and 507o CO2 for 20 h, and subsequently harvested, pooled and washed three times in PBS. 10~ viable cells in a volume of I00/~1 PBS were then injected into paws of recipient mice. PLN indices were determined 7 days after cell transfer.
Histology Immediately after determination of weight, PLN were fixed in 4070 phosphate-buffered formaldehyde and embedded in Historesin (LKB Produkter AB, Bromma, Sweden). Serial sections (3 pm) were stained with methylgreen pyronine. Paws were fixed in 4070 phosphate-buffered formaldehyde for 48 h and successively incubated in 70070 ethanol for 12 h, in a mixture (1:1) of 98070 formic acid and 70070 ethanol for 24 h, and in 7007o ethanol for 24 h, which was refreshed once after 12 h. Subsequently, paws were dehydrated and embedded in Historesin.
Zimeldine-induced Popliteal Lymph Node Reactions Sections (3 ~m) were stained with May-Grtinwald Giemsa.
Determination o f T- and B-lymphocytes in P L N PLN, embedded in Tissue-tek (Miles Laboratories, Naperville IL, U.S.A.), were snap frozen in liquid nitrogen and stored at - 70°C. Serial cryostat sections (6 ~m) were air dried and fixed in acetone for 1 rain. Fc-receptors were blocked by incubation with 1070 normal rabbit serum in PBS for 30 min. To discriminate between T- and B-cells, sections were incubated for 45 rain with, respectively, 1:20 diluted anti-Thy-I or anti-kappa antibodies in PBS supplemented with 1% normal rabbit serum, and subsequently for 30 min with 1:300 diluted biotinylated rabbit anti-rat lgG antibodies in PBS supplemented with 107o normal mouse serum to diminish non-specific staining. Sections were further incubated for 30 min with 1:800 diluted peroxidase-conjugated streptavidin and developed by incubation with 0.05070 diaminobenzidine in 0.05 M T r i s - H C I buffer (pH 7.6), 0.1 M imidazole and 0.01070 H.,O2. Sections were slightly counterstained with haematoxilin, dehydrated and mounted in Depex. For demonstration of germinal centers, acetonefixed cryostat sections were incubated for 45 min with 1:20 diluted peroxidase-conjugated PNA. Sections were developed by incubation with 3-amino9-ethyl carbazole in 0.05 M acetate buffer (pH 5.0) containing 0.01070 H.,O_,, counterstained with haematoxilin and mounted in Aquamount (Gurr, BDH Chemicals, Poolc, U.K.).
Morphometrical analysis" Large pyroninophilic cells, representing immunoblasts were counted in methylgreen pyronine-stained plastic sections at a magnification of 400 x using a 10 × 10 square grid. For each treatment, three to five serial sections of 3 PLN were counted. Data were converted to the average number of large pyroninophilic cells per square mm paracortex, primary follicle and - - if present - - germinal center. Paracortical, follicular and medullary areas were measured in anti-Thy-l- or anti-kappa-stained cryostat sections using a digitizer tablet (Minimop, Kontron Bildanalyse, Eching, F.R.G.). For each treatment four to six serial sections of central parts of 3 PLN were used and areas of follicles and paracortex were expressed relative to the area of the PLN section minus medullary areas. The latter were identified as being kappa positive areas located outside the cortex.
695
Data handling and statistics Data have been expressed as arithmetical mean ___S.E.M. When appropriate, analysis for significance was performed with the two-tailed nonparametric M a n n - W h i t n e y U-test. P-values over 0.05 were considered not significant.
RESULTS
Effects o f s.c. injected zimeldine on f o o t swelling Injection of zimeldine into the foot induced a bimodal foot swelling in C57BL/10 mice (Fig. 1A), C57BL/6 and T-cell deficient C57BL/6 nu/nu mice (Fig. 2) as well as in BALB/c mice and BALB/c nu/nu mice (data not shown). As determined in C57BL/10 (Fig. IA) an immediate, dose-dependent swelling was observed 4 h after injection, which declined within 1 or 2 days. A second dosedependent swelling started around day 3, reached a plateau at day 5 - 7 and declined gradually thereafter. After injection of 1.0 mg zimeldine, control values were not attained until day 50; the presence of crusts often prevented adequate measurements of foot swelling between days 1 4 - 30. A dose of 0.3 mg induced a strong immediate swelling and only a moderate foot swelling around day 3 - 5 , which subsided within the next days (Fig. IA). A dose of 0.1 mg did not induce a significant foot swelling as compared to injection with saline (data not shown). The immediate swelling observed 4 h after injection of 1.0 mg zimeldine in C57BL/10 mice was featured by interstitial oedema and degranulation of mast cells (Fig. 3A). The second swelling, as determined on day 5, was characterized by oedema and massive infiltration of polymorphnuclear cells in the dermis (Fig. 3B) and in the underlying muscle tissue. S.c. injection of 10~ zimeldine-treated syngeneic cells into the footpad elicited a transient foot swelling due to the injected volume of 100 ~1. No second swelling was observed (data not shown).
P L N reactions to zimeldine The kinetics of the PLN response were studied in C57BL/10 mice (Fig. IB). A dose of 0.3 mg induced a moderate but significant PLN enlargement at day 5. PLN indices up till then and afterwards did not differ from the index of saline-treated controls on day 7. Maximal increase of PLN weights after injection of 1.0 mg was found between day 2 and day 5, when indices of 1.5 and 8.0 on an average were calculated, respectively. The highest indices were observed at day 9, although individual
C. THOMAS et al.
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Fig. 1. Kinetics o f zimeldine-induced foot swelling (A) and PLN reaction (B). Groups of six C57BL/10 mice received an injection into the footpad at day 0 with 1.0 mg (©) or 0.3 mg ( 0 ) zimeldine, or with saline (A). The same mice were used to determine foot swelling and PLN indiccs. Vertical bars indicate S.E.M. *P<0.05 as compared to saline-treated controls (A) or saline-treated controls on day 7 (B).
variation was considerable. From day 9 on, PLN indices gradually decreased, but remained s i g n i f i c a n t l y e n h a n c e d u p till d a y 30. A t d a y 50 a f t e r i n j e c t i o n , i n d i c e s w e r e still 2.7 t i m e s t h e c o n t r o l value. R e a c t i o n s to z i m e l d i n e in T-cell d e f i c i e n t B A L B / c n u / n u m i c e were c o m p a r e d w i t h t h o s e in n u / + l i t t e r m a t e s ( T a b l e 1). O n d a y 7 a f t e r i n j e c t i o n o f
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Fig. 2. T-cell dependence of zimeldine-induced foot swelling. Groups o f six mice received an injection into the footpad at day 0 with 1.0 mg zimeldine (C57BL/6: (_3; C57BL/6 n u / n u : O) or saline (C57BL/6: A; C57BL/6 nu/nu: A ) . Foot swelling was determined at times indicated. Vertical bars indicate S.E.M.
Difference with controls
Strain
Treatment
n
P L N index - S.E.M.
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0.9_.0.1 2.5 ~ 0.2*
188o70
Mean PI.N indices _+ S.E.M. o f n individual mice were determined 7 days after s.c. injection o f 1.0 mg zimeldine in 10 IA solvent or solvent only into the one hind footpad *P<0.05 as compared to saline-treated controls using the Mann - Whitney U-test.
Zimeldine-induced Popliteal Lymph Node Reactions
697
Fig. 3. Histology of paws of C57BL/10 mice, injected s.c. with 1.0 mg zimeldine. Plastic sections were stained with MayGriinwald Giemsa. (A) 4 h after injection. Degranulation of mast cells in the dermis. (B) 5 days after injection. Infiltration of mainly polymorphnuclear cells into the dermis.
C. THOMASet al.
698
11]
0.3
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-
-
-
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0.3
.ap.,/~,%
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Fig. 4. Histology of PLN of C57BL/10 mice (A - C, E - F) and BALB/c nu/nu (D), after s.c. footpad injection of 1.0 mg zimeldine. (A, C - F): methyl green pyronine-stained plastic sections; (B): Thy-l-stained cryostat section. (A) Uninjected control. PC, paracortex; F, primary follicle. (B) 20 days after injection. GC, germinal centre; C, follicle corona; PC, paracortex. (C) Detail of germinal center, 30 days after injection, showing mitotic figures (upper arrow), tingible body macrophages (middle arrow) and immunoblasts (lower arrow) (180 × ). (D) Follicle with germinal center in BALB/c nu/nu mouse, 7 days after injection. Scattered immoblasts are present in depleted paracortex (arrows) (150 x ). (E) Medulla, 3 days after injection. Scattered immunoblasts are seen (arrows) among mainly small lymphocytes (210 x ). (F) Medulla, 9 days aftcr injection, showing abundance of plasma cells (400 × ).
Zimeldine-induced Popliteal Lymph Node Reactions 1.0 mg zimeldine, mean P L N indices o f 1.8 and 2.5 were calculated for n u / n u and n u / + mice, respectively. The two B A L B / c strains failed to m o u n t a significant P L N response to doses o f 0.3 mg zimeldine on day 7 after injection (data not shown). Severe necrosis o f the injected paw was f o u n d after injection o f 3.3 rag; therefore no data are included.
M o u s e strain zimeldine
dependence
of PLN
reactions
metrically. Serial cryostat sections o f control P L N stained for Thy-I and k a p p a showed that on an average 60°7o and 30°7o o f the section areas were covered by paracortical and follicular areas, respectively. Whereas this 2:1 ratio did not change u p o n treatment with zimeldine on the times indicated in Table 4, their relative contribution to P L N enlargement changed in time. Enlargement could be
to
P L N responsiveness by 7 days after injection a p p e a r e d to vary a m o n g genetically different mouse strains (Table 2). A dose o f 0.3 mg zimeldine did not elevate the P L N index as c o m p a r e d to control mice in any strain tested. Whereas a dose o f 0.7 mg did not induce a significant reaction in B A L B / c mice, it did in C57BL/10 mice. A significant P L N reaction to 1.0 mg zimeldine was f o u n d in each strain. While D B A / 2 mice showed similar reactivity as B A L B / c mice, the P L N index in C57BL/10 mice was over three times higher.
Indirect P L N reactions to zimeldine D o n o r cell suspensions were prepared f r o m P L N , 2 days after s.c. injection o f 1.0 mg zimeldine or saline. The P L N o f the zimeldine-treated d o n o r s contained twice as many cells as the P L N o f the controls indicating P L N activation by zimeldine. T r a n s f e r o f 10: zimeldine-cxposed cells to syngeneic recipients, however, did not evoke higher P L N responses on day 7 than transfer o f the same n u m b e r o f control cells (Table 3). Likewise, transfer o f 107 syngeneic lymph n o d e cells u p o n incubation with 2.5 ~g or 25/ag z i m e l d i n e / m l for 20 h failed to evoke significantly e n h a n c e d P L N indices in c o m p a r i s o n with control cells.
Table 3. Indirect PLN responses to syngeneic zimeldineexposed lymph node cells Treatment of donor cells
PI.N index _+ S.E.M. on day 7 after cell transfer
n
In vivo Saline Zimcldine
2.3 _+ 0.3 2.3 _+ 0.2
5 l0
In vitro PBS 2.5 ~gr zimeldine/ml 25 /agr zimeldinc/ml
2.9 +- 0.3 2.1 +- 0.2 2.7 _+ 0.3
6 7 7
Groups of n BALB/c mice received 10~ zimeldinetreated or control lymph node cells in 100,1 PBS into one hind footpad. In vivo treated donor cells were obtained 2 days after s.c. injection of 1.0 mg zimeldine or saline into the footpad of BALB/c mice. In vitro treated cells were obtained after incubation of pooled lymph node cell suspensions with PBS or zimeldine for 20 h. Cells were washed three times prior to injection.
Table 4. Number of pyroninophilic cells per square mm in PLN after s.c. zimeldine injection
Histological characterization o f the P L N response to zimeMine P L N o f C 5 7 B L / 6 mice were analysed m o r p h o Table 2. Mouse strain dependence of PI.N reactions to zimeldine
Treatment
BALB/c
PLN index DBA/2
C57B!./10
Saline 0.3 mg zimcldine 0.7 mg zimeldine 1.0 mg zimeldine
1.4 1.5 1.5 2.1
1.0 _+ 0.0 n.d. n.d. 3.0+_0.2*
1.1 _+ 0.1 1.7 ± 0.2 2.5 ± 0.4* 7.6_+ 1.1"
_+ 0.1 _+.0.2 _-,20.2 +_0.1"
699
PLN indices (mean + S.E.M. of five mice per group) were determined 7 days after s.c. injection into the footpad. *P<0.05 as compared with saline-treated controls. n.d., not determined.
Paracortex Primary follicles Presence of germinal centers
0
2
Days after injection 5 7 9 14 20 30
•
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*
n.d.
•
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,
t
t
t
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50
PLN were obtained on various times after injection of 1.0 mg zimeldine into the footpad of C57BI./6 mice. In plastic sections of PLN stained with methyl green pyronine the number of pyroninophilic cells per square mm in five to seven serial sections of three PLN per group was quantified using a grid at magnification 400 ×. Number of pyroninophilic cells per square mm: - - <50; *50- 100; i'100- 500; ~:>500. Germinal centers, if present, all contained over 1000 pyroninophilic cells per square mm. n.d., not determined.
700
C. THOMAS el al.
solely attributed to a proportional expansion of T- and B-cell areas up to day 7, after which the medulla contributed increasingly to PLN enlargement. Medullary expansion attained a maximum around day 20, when the medulla covered on average about 30°7"o ot" the section areas. Histological examination of the F'I.N showed already increased numbers of pyroninophilic cells in the paracortical areas by 2 days after injection ot" 1.0 mg zimeldine (Table 4). In general, further increase was seen until day 7, although considerable individual differences were detected. A lesser, but still substantial number of paracortical immunoblasts was seen from day 9 till day 30. ht follicular areas increased numbers of pyroninophilic cells were not observed until day 7. At that time few secondary follicles, characterized by a densely packed corona surrounding a pyroninophilic germinal center, were present. Germinal centers contained cells in mitosis and many tingiblc body nmcrophages (Fig. 4C). Numbers and size of germinal centers in follicular areas increased until days 2 0 - 3 0 . In the medulla, increased numbers of large pyroninophilic plasma cells were detected between days 9 and 20 after injection (Fig. 4E,F). By day 50, germinal centers had vanished. Blurring of lymph node architecture at this time prevented adequate determination of pyroninophilic cell numbers in the distinct areas. Histological features of PI.N reactions to 0.3 mg zimcldine appeared qualitatively and kinetically similar to those observed after injection of 1.0 rag, but less pronounced (data not shown). In BALB/c n u / n u mice, studied on day 7 after injection of 1.0 mg zimeldine, increased numbers of immunoblasts as compared with controls were seen in the paracortcx, which further appeared to be severely depleted of lymphocytes (Fig. 41)). Secondary follicles were frequently observed, and plasma cells in the medulla were also detected. Presence of germinal center reactions in T-cell deficient and T-cell competent mice was further studied on day 7 after injection using cryostat sections incubated with PNA, specifically staining for germinal center cells (Racdler, Racdler, Arndt & Thicle, 1981; Rcichert, Gallatin, Weissman & Butcher, 1983; Kraal, Hardy, Gallatin, Weissman & Butcher, 1986). in the follicles of PI.N of mice injected with zimeldinc or sheep crythrocytes but not in those of control BALB/c n u / + mice, PNA positive areas were generally detected. Size of germinal centers induccd by sheep erythrocytcs, however, appeared to bc much larger than after zimeldine treatment. In PLN of BAI.B/c n u / n u mice, small PNA-positivc areas were found in many cases (data not shown).
DISCUSSION
Injection of zimeldine into the footpad induced a dose-dependent bimodal foot swelling. The first swelling was recorded by 4 h after injection and subsided within 1 or 2 days. The second swelling usually reached a plateau by day 5 and persisted for 1 or 2 days before subsidence. The bimodal reaction to zimcldinc is indicative of diffcrent underlying mechanisms. This is supported by histological data. The early observed interstitial ocdema, degranulated mast cells and few inflammatory cells indicate that zimeldine-induced release of vasoactive amines from mast cells might have caused the first reaction. The second swelling was characterized by a dense intqammatory infiltrate of mainly polymorphnuclear cells. In mice and rats such infiltrates can be indicative of antigcn specific delayed-type hypersensitivity reactions (Cooper, 1972; Crowle, 1975; Henningsen, Koller, Exon, Talcott & Osborne, 1984). Whereas this is in line with observations that zimeldine could induce contact sensitivity reactions in guinea pigs (Henderson, Edwards, Weston & Dewdncy, 1988), the observations that T-cell deficient nude mice showed macroscopically similar paw reactions to zimeldine indicates that these reactions are rather the result of non-specific inflammation. Next to inflammatory footpad reactions, zimeldine triggered a significant increase of the PLN index in all strains tested. Striking quantitative differences were detected between H-2 ' and 11-2" strains. Experiments with intra-H-2 recombinant strains, however, arc needed to finally assess possible MHC restriction and involvement on non-H-2 genetic factors in PLN reactions to zimeldine. The significant PLN weight increase in n u / n u mice after zimcldine injection is an unexpected phenomenon, because the low mol. wt drugs diphcnylhydantoin (Gleichmann, 1981; Kammi.illcr & Seincn, 1987), D-pcnicillaminc (Hurtenbach et al., 1987) and HgCI, (Stiller-Winkler, Radaszkiewics & Gleichmann, 1988) failed to do so under similar circumstances. PLN activation by zimeldinc in n u / n u mice was confirmed by morphological observations such as increased presence of immunoblasts in the paracortcx and plasma cells in the medulla. Moreover, prelinfinary results have indicated that zimeldinc but not sheep erythrocytes increases the production of IgG by PLN cells from n u / n u mice. The ability of zimcldinc to induce activation of B-cells in the absence of T-cell helper factors suggests that the drug triggers secretion of soluble factors like II.-4 by nmst cells in the footpad as well as in the
Zimeldine-induced Poplitcal Lymph Node Reactions PLN itself, thus inducing T-cell independent stimulation of B-cells (O'Garra, Umland, De France & Christiansen, 1988). Another possibility is that it evokes the secretion of autocrine B-cell-derived growth factors (Muraguchi, Nishimoto, Kawamura, Hori & Kishimoto, 1986), thus bypassing activating signals normally provided by T-cells in immunocompetent mice. T-cells, however, are apparently needcd to induce strong PLN reactions against zimeldine. The T-cell dependent mechanism involved has as yet not been elucidated. The tool. wt of the agent is too low to have immunogenic properties of its own (Benacerraf & Unanue, 1979), and preliminary in vitro data seem to exclude direct mitogenic action or influence on mitogen-induced lymphoproliferation, confirming earlier experiments with lymphoid cells of mice (Henderson et al., 1988) and humans (Kristofferson & Nilsson, 1989). The drug may act as a hapten when covalently bound to a macromolecular carrier, but reactions with amino or thiol groups in vitro could not be demonstrated (Henderson et al., 1988). The resemblance of weight kinetics and morphology of the PLN during the first week after zimeldinc treatment with those induced by diphenylhydantoin (Glcichmann, 1981 ; Gleichmann, Pals & Radaszkicwics, 1983; Kammiiller et al., 1987) and semi-allogeneic lymphoid cells (Ford et al., 1970; Romano, Ponzio & Thorbeckc, 1976; Rolstad, 1985) might point to a GvH-like etiology. It would imply that zimeldine-induced B-cell activation is the consequence of polyclonal helper T-cell activation induced by drug-modified class il determinants of the MHC (Gleichmann et al., 1984). Lymphokines released by these T-cells would provide the second signal to autoantigen recognizing B-cells to start proliferation and antibody production. The amphi-
701
philic nature of zimeldine is in support of this notion, because it is likely to facilitate an interaction with the phospholipid bilayer of cell membranes and association with other membrane determinants (Liillmann, Lullmann-Rauch & Wassermann, 1975; Bockhardt & Liillmann-Rauch, 1980). Further, it is consistent with the observation that reactions to zimeldine are under genetic control and enforced by the presence of immunocompetent T-cells. Several data, however, question whether an "altered self" mechanism is responsible for the zimeldine-induced PLN reactions. First, unlike those reported for diphenylhydantoin by Gleichmann et al. (1981), PLN reactions to zimeldine could not be elicited by transfer of zimeldine-treated cells. Second, PEN enlargement was detected in PLN of nude mice after zimeldine injection, while reactions to diphenylhydantoin and various other drugs have been reported to be strictly T-cell dependent (Gleichmann, 1981; Hurtenbach et al., 1987; Kamm~ller et al., 1987). Third, zimeldine induced extremely prolonged lymph node reactions upon a single s.c. injection, while alloreactive lymphocytes and low mol. wt compounds were found to induce short lasting lymph node reactions with an optimum around day 7 (Gleichmann, 1981; Twist & Barnes, 1973; Kammiiller et al., 1987). The prolonged stimulation suggests that zimeldine (or its metabolites) might be retained at the site of injection or inside the PEN for a long period of time and that it possibly interferes with inhibitory feedback mechanisms. Further experiments are needed to study in detail the production of antibodies by PLN cells stimulated with zimeldine and to elucidate the mode of action of the drug in this system.
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