Immunology 1987 60 409-414

Cyclosporin A prevents suppression of delayed-type hypersensitivityin mice immunized with high-dose sheep erythrocytes

L. M. WEBSTER & A. W. THOMSON Immunopathology Laboratory, Department of Pathology, University of Aberdeen,Foresterhill, Aberdeen

Acceptedfor publication 24 October 1986

SUMMARY

When administered intraperitoneally to mice 2 days before immunization with a tolerogenic dose(109) of sheep red blood cells (SRBC), cyclosporin A (CsA; 200 mg/kg) strikingly augmented 4-daydelayed-type hypersensitivity (DTH) footpad reactions. These enhanced responses were similar inmagnitude to those seen in animals sensitized with an immunogenic, low-dose (106) SRBC. The

stimulatory effect of CsA was observed over the dose range of 5-200 mg/kg and was obtained inanimals given the drug in one injection, up to 7 days before sensitization. The augmentation ofDTHwas characterized by footpad swelling, intense mononuclear cell infiltration and increased depositionof '251I-fibrinogen within the challenge site. In addition, increased expression of procoagulant activityby spleen cells in response to antigen was observed. Cell transfer experiments showed that the CsA-enhanced DTH could be adoptively transferred to naive recipients. Additional transfers conducted atthe time ofantigen challenge suggested that, under the conditions described, CsA inhibited the actionof a population of suppressor cells normally effective during DTH reactions.

INTRODUCTION

The capacity of cyclosporin A (CsA) to suppress delayed-typehypersensitivity (DTH) reactions in animals is dependent bothon drug dosage and on the time of its administration in relationto immunization or antigen challenge (Borel et al., 1977;Thomson et al., 1983a; Thomson, Moon & Nelson, 1983b;Mottram et al., 1986). Augmentation of DTH has also beenreported, however, in guinea-pigs given either high dosage CsAsystemically before immunization (Parker, Drossler & Turk,1984; Aldridge & Thomson, 1986a) or short courses of the drugorally, commencing at the time of immunization (Thomson etal., 1983a). Moreover, in rats, enhancement of DTH reactionshas been observed following administration of CsA prior to, orafter, the onset of collagen-induced arthritis (Kaibara et al.,1983). These paradoxical effects have been tentatively attributedto impairment ofsuppressor cell activity (Thomson et al., 1983a;Kaibara et al., 1983; Aldridge & Thomson, 1986b).

In mice, classical DTH reactions to sheep red blood cells(SRBC) can be elicited in footpads challenged with antigen 4days after immunization (Lagrange, Mackaness & Miller,

Abbreviations: Con A, concanavalin A; c.p.m., counts per minute;CsA, cyclosporin A; DTH, delayed-type hypersensitivity; HSA, humanserum albumin; i.p., intraperitoneally; i.v., intravenously; LPS, E. colilipopolysaccharide; PCA, procoagulant activity; PHA, phytohaemag-glutinin; SRBC, sheep red blood cells.

Correspondence: Miss Laura M. Webster, ImmunopathologyLaboratory, Dept. of Pathology, University of Aberdeen, AberdeenAB9 2ZD, U.K.

1974). These and other cutaneous DTH responses are character-ized by mononuclear cell infiltration (Marchal et al., 1978) andfibrin deposition (Colvin et al., 1973; Peters et al., 1986). In thecase of DTH to SRBC, responsiveness can be adoptivelytransferred by splenic T cells to naive recipients (Liew, 1977;Marchal et al., 1978). The magnitude of DTH reactions toSRBC is dependent on the sensitizing dose of antigen; very highdoses (e.g. 109 SRBC) induce the production of specific T-suppressor cells, which reach optimum numbers in the spleen 3-4 days after immunization (Liew, 1977). Since we wished toexamine further the possible influence ofCsA on suppressor cellactivity in vivo, we have investigated the effects of the drug onDTH in animals immunized with a high dose of SRBC.

MATERIALS AND METHODS

MiceClosed colony-bred MF1 mice (8-12 weeks old) were usedthroughout. They were bred in the University Animal Depart-ment, Foresterhill, Aberdeen, maintained in a temperature-controlled environment, and received Oxoid rat and mousebreeding diet with tap water ad libitum.

DrugsCyclosporin A (CsA; batch 83601 Sandoz Ltd, Basle, Switzer-land) was obtained in powder form and dissolved in absoluteethanol to which was added four parts olive oil (Boots PLC Ltd.Nottingham). After thorough mixing, the CsA solution was

409

410 L. M. Webster

administered (01 ml) intraperitoneally (i.p.) in doses rangingfrom I to 200 mg/kg.

ImmunizationSheep red blood cells (SRBC) obtained from blood in Alsever'ssolution (Difco Laboratories, West Molesey, Surrey) werewashed three times in phosphate-buffered saline (PBS), pH 7 2.Mice received either 106 or 109 SRBC in 0-2 ml intravenously(i.v.).

DTH assayMice were challenged 4 days after immunization with 108 SRBCunder one hind footpad; the opposite footpad received PBSalone. Reactions were assessed 24 and 48 hr later by measuringthe increase in dorsoventral thickness of the test over the controlfootpad using a spring-gauge caliper (Schnelltaster, H. C.Kroeplin GMbH, Schluechtern, FRG). All measurements wereconducted by the same individual, and the results wereexpressed as specific increases in footpad thickness (10-' mm,mean+ I SD.

Deposition of J25I-fibrinogen in DTH reaction sitesIodine ('25l)-labelled human fibrinogen ('25l-fibrinogen) andhuman serum albumin ('251-HSA) (isotonic solutions for injec-tion) were obtained from the Radiochemical Centre, AmershamInternational, Amersham, Bucks). One pCi in 0 2 ml wasadministered i.v., either I hr before footpad antigen challenge or18 hr before measurement of DTH reactions at 24 or 48 hr. Atthese latter times, both hind feet were removed at the ankle jointand incorporation of radiolabel estimated in a gamma counter(Wilj Electronics Ltd, Ashford, Kent). Results are expressed asthe difference in counts per minute (mean c.p.m. + 1 SD)between experimental and control feet.

& A. W. Thomson

Manchester). A value of 107 thromboplastin units, U/ml, wasarbitrarily assigned to the stock suspension and linearityobtained over the range of 10 - 104. Since equivalent cellconcentrations were used throughout, the results of PCA areexpressed in thromboplastin units (U) as the mean+ I SD oftriplicate cultures.

StatisticsThe significance of differences between means was calculatedusing the Mann-Whitney U-test, unless otherwise stated(P< 001).

RESULTS

Effect of CsA (200 mg/kg) on DTH reactions

The effect of a single dose ofCsA (200 mg/kg i.p.), given 2 daysbefore immunization, on 4-day DTH footpad reactions ingroups of six mice is shown in Fig. 1. Animals immunized withthe lower antigen dose (106 SRBC) showed good responses,which were not affected significantly by CsA administration. Bycontrast, untreated mice that had been sensitized with the higherantigen dose (109 SRBC) exhibited very weak DTH reactions,which were strikingly increased ( > four-fold; P < 0-01) by CsAtreatment on Day -2. These CsA-enhanced responses weresimilar in magnitude to those observed in animals sensitizedwith 106 SRBC. Histological examination of the DTH reactionsites 24 hr after challenge revealed a very pronounced dermalmononuclear cell infiltrate in animals sensitized with 106 SRBCand in those given CsA 2 days before 109 SRBC (Fig. 2a). Only avery mild inflammatory cell infiltrate, however, was seen in

HistologyFeet were fixed in 10% neutral-buffered formalin, decalcifiedand embedded in paraffin wax using standard procedures.Sections were cut at 5 pm and stained with haematoxylin andeosin (H & E).

Cell transferSpleen cell suspensions were prepared as previously described(McIntosh et al., 1986) in Dulbecco's modified Eagle medium(DMEM; Gibco, Paisley, Renfrewshire) at a concentration of5 x 108/ml. Each mouse received 108 cells i.v., followed immedi-ately by antigen challenge.

Assay of leucocyte procoagulant activityThe procoagulant activity (PCA) of spleen cells (20 x 106/ml;Nunc Minisorb tubes) following 20 hr incubation with antigenor mitogen in RPMI-1640 (Gibco), supplemented with anti-biotics and 10% fetal calf serum (FCS; Gibco) was estimatedusing the method of Morrice et al. (1985). Stimulants used werephytohaemagglutinin (PHA-P; Wellcome, Beckenham, Kent; 5pg/ml), concanavalin A (Con A; Sigma, Poole, Dorset; 2 pg/ml)and SRBC (2% v/v). Briefly, the ability ofthe stimulated washedcells to shorten the recalcification time of normal, pooled,platelet-poor human plasma was determined in a one-stageclotting assay. PCA was quantified by constructing a standardcurve with 10-fold serial dilutions ofhuman brain thromboplas-tin (Manchester Comparative Reagent, Withington Hospital,

12

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o 106 SRBC* CsA + 106 SRBCo 109 SRBC* CsA + 109 SRBC

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4

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0 24 48 72Hr after challenge

Figure 1. Effect of CsA (200 mg/kg) administered 2 days beforeimmunization on DTH responses to SRBC elicited on Day 4. Groups ofsix mice received either the drug vehicle (open symbols) or CsA (closedsymbols) before immunization with 106 (circles) or 109 (squares) SRBC.Results are means+ 1 SD. * Significantly higher (P<0 01) thanuntreated control (109 SRBC).

Cyclosporin A and DTH reactions

Figure 2. Mouse footpads 24 hr after challenge with SRBC. (a) Animal treated with CsA (200 mg/kg) 2 days before immunization with109 SRBC, showing a pronounced mononuclear cell infiltrate. (b) Animal treated with drug vehicle showing only a mild inflammatorycell infiltrate (H & E, magnification x 186).

vehicle-treated mice sensitized with the higher antigen dose(Fig. 2b).

16

14

Effect of CsA on fibrinogen accumulation in DTH reaction sites

Figure 3 shows the extent of '251-fibrinogen or '25I-HSAaccumulated within footpads of CsA-treated mice immunizedwith 109 SRBC and expressing enhanced DTH reactions.Footpad swelling at 24 hr was accompanied by profoundlyincreased deposition of'25I-fibrinogen but not 1251-HSA, indicat-ing that fibrinogen accumulation was specific and not simply theresult of increased vascular permeability. Similar results were

obtained when the isotope-labelled proteins were administered Ihr before footpad challenge or 18 hr before measurement of theDTH reaction. By 48 hr, when footpad swelling had subsided,the amount of '251I-fibrinogen deposited (following i.v. injection30 hr after challenge) did not differ significantly from vehicle-treated controls.

Effect of timing of CsA administration

The influence ofCsA (200 mg/kg) administered at various timesin relation to immunization with 109 SRBC is shown in Table 1.Significant augmentation of 24 hr footpad reactions was

observed in mice treated from Day -7 to Day + 1. However,when CsA was withheld and given between Day 2 and the timeof challenge (Day 4), no significant effect was observed on

footpad reactions.

Effect of CsA dose

The extent ofDTH reactions in mice given various doses ofCsA2 days before immunization with 109 SRBC is shown in Table 2.Doses from 5 to 200 mg/kg significantly enhanced the footpadresponses compared with vehicle-treated controls, although theeffect of 5 mg/kg was significantly less than that obtained withhigher doses.

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CsA Ve*

8-8 1-209 0-5

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CsA Ve

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Figure 3. Effect of CsA (200 mg/kg) administered 2 days beforeimmunization with I09 SRBC on the accumulation of '25I-fibrinogen inDTH reaction sites. Radiolabelled fibrinogen or human serum albumin(HSA) was administered i.v. 18 hr before measurement of DTHreactions at 24 or 48 hr. Results are means + I SD obtained from groups

of six animals. * Significantly higher (P< 0 01) than vehicle control.

Procoagulant activity (PCA) of spleen cells

The PCA of spleen cells removed from mice 4 days afterimmunization (109 SRBC) and stimulated with either mitogenor antigen is shown in Table 3. Whilst both vehicle- and CsA-

411

L. M. Webster & A. W. Thomson

Table 1. Effect of timing ofCsA (200 mg/kg) on DTH inmice immunized with 109

SRBC

24 hr DTH reaction(mm-')

Day CsA Vehicle

-14 2-2+0-4 1-2+0-4- 7 7-0+1-4* 0-5+0-4- 4 8-3+1-2* 07+05- 3 6-2+1-5* 10+05- 2 6-2+1-3* 18+0-5- 1 52+15* 15+07

0 7-8+1 3* 1-5+0-7+ 1 65+1 0* 08+0-6+ 2 1*7+05 15+0-6+ 3 1-2+0-8 1 2+09+ 4 23+08 17+05

Results are means + 1 SDobtained from groups of sixmice.

* Significantly higher thancorresponding vehicle-treatedcontrols (P < 00 1).

Table 2. Effect of dose of CsA (Day -2) onDTH in mice immunized with 109 SRBC

CsA (mg/kg) 24 hr DTH reaction (mm-')

200 6.7+1 2*150 5.0+1 4*100 6-0+0-8*50 5.5+1I0*40 5.6+0 6*30 4-7+1.2*20 4.5+1 2*10 6.0+1 0*5 3.7 ±0.5*t1 118+09

Vehicle 1 6+0-6

Results are means + 1 SD obtained fromgroups of six mice.

* Significantly higher than vehicle-treated controls (P< 0-01).

t Significantly lower than valuesobtained with > 10 mg/kg (P < 00 1).

(200 mg/kg, Day -2) treated groups showed good responses toPHA or Con A, only the latter group exhibited a response toantigen (two-fold increase in PCA).

Transfer of DTH

Table 4 shows the influence on DTH reactions of spleen celltransfer, conducted at the same time as antigen challenge. The

Table 3. Mitogen- and antigen-induced PCA of spleen cells from CsA-treated, immunized mice*

PCA [thromboplastin units (U)]

Treatment (Day -2) Control PHAt Con A SRBC

Vehicle 82+ 10 565+34 344+ 58 62+6CsA (200 mg/kg) 67+ 8 628+26 229+79 134+9t

Results are means + 1 SD obtained from four mice.* Mice immunized with I09 SRBC on Day 0 and spleens removed

on Day 4.t Mitogens were used at concentrations of 5 pg/ml (PHA) or 2 gg/

ml (Con A).t Significantly higher than vehicle-treated controls (P< 0 01 using

Student's f-test).

capacity to express DTH reactions in animals given 200 mg/kg 2days before 109 SRBC was transferred to naive recipients withsome diminution in the extent of reactivity (Groups i and ii). Theenhanced reactivity could also be transferred to untreated miceimmunized with the tolerogenic dose of SRBC (Groups iii andiv). Cells from tolerized donors transferred at the time ofchallenge reduced but did not abrogate DTH reactions in CsA-treated recipients (Groups v and vi). Table 4 also shows thatthere was no evidence of cells present in naive donors capable ofsuppressing the CsA-enhanced DTH responses at the time ofchallenge (Groups vii and viii).

DISCUSSION

Maximal DTH responses to SRBC can be elicited in normalmice sensitized i.v. with low-dose antigen (105 or 106 SRBC),whilst immunization with high-dose SRBC (e.g. 109) inducesminimal or no responsiveness (Lagrange et al., 1974). In theformer instance, CsA (50-100 mg/kg) suppresses DTH whenadministered between immunization and footpad challenge(Thomson et al., 1983b; Shidani et al., 1984). This property canbe attributed to the impairment of T-helper cell activation andlymphokine production (Hess, Tutschka & Santos, 1982;Thomson et al., 1983a, b). There is also evidence to indicate thatwhen given at, or a few hours before, the time of antigenchallenge, CsA is more effective with low, rather than high,doses of eliciting antigen (Shidani et al., 1984; Aldridge &Thomson, 1986a).

In the present study, we have shown that when administered2 days before immunization, CsA (200 mg/kg) strikinglyaugments DTH in animals injected with high-, but not low-doseSRBC. This is evidenced by increases in footpad swelling, and inthe former instance by the infiltration of large numbers ofmononuclear cells into the challenge site and the increaseddeposition of radiolabelled fibrinogen. Moreover, spleen cellsfrom mice exhibiting CsA-enhanced footpad reactions showantigen-induced increases in procoagulant activity in vitro - anindex of DTH in both mice and man (Geczy & Meyer, 1982;Geczy et al., 1983). Our study has further shown that thiscapacity of CsA to prevent the tolerogenic effect of high-doseSRBC can be adoptively transferred by spleen cells to naiverecipients. Furthermore, the action of CsA appears to be

412

Cyclosporin A and DTH reactions 413

Table 4. Effect ofcell transfer at the time ofantigen challenge on DTH in mice immunizedwith 109 SRBC

Donor Recipient

Group CsA 109 SRBC CsA 109 SRBC 24 hr DTH reaction (mm-)

(i) + + - - 45+07*(ii) Vehicle + - - 1-4+09(iii) + + - + 3.8+0.2*(iv) Vehicle + - + 2 0+0 7(v) - + + + 28+ 09*(vi) - + Vehicle + 0-8_+05(vii) - - + + 6.2+1.3*(viii) - - Vehicle + 1 8+0 5

Results are means + 1 SD obtained from groups of six mice.* Significantly higher than vehicle-treated control (P<0 01).

directed against a population of suppressor cells, as is shown bythe data in Table 4. These results suggest that the suppressormechanism affected by CsA may be operative during theafferent phase of the response. This possibility is consistent, inpart, with the observation that cells from tolerized donorstransferred at the time of challenge reduced the DTH reactionsin CsA-treated recipients.

Our findings are consistent with previous observations thathigh-dose CsA (200 mg/kg) given systemically 2 days beforeimmunization can augment 14-day DTH reactions to ovalbu-min in guinea-pigs (Aldridge & Thomson, 1986a). Studies in thislatter species have also shown that the expression of CsA-enhanced DTH can be inhibited by systemic transfer of pooledspleen and peritoneal exudate cells from untreated, immunizeddonors at the time of challenge (Aldridge & Thomson, 1986b).Other anecdotal reports suggest that, under certain conditions,CsA can inhibit the generation of suppressor cell activity in vivo(Kaibara et al., 1983; Parker et al., 1984; Wick, Muller &Schwarz, 1982) or in vitro (Palacios, 1981). Indeed, recently, Li& Nelson (1986) have shown that high-dose CsA inhibits theformation of suppressor T cells regulating the production ofanti-erythrocyte autoantibodies in mice.

It is now well recognized that cytotoxic immunosuppressivedrugs, when applied under the appropriate conditions, canaugment cell-mediated immunity. The most studied of these iscyclophosphamide, which when given to mice in high dosageprior to immunization enhances DTH responses to SRBC(Askenase, Hayden & Gershon, 1975), ovalbumin (Thomson,Nelson & Moon, 1983) or picryl chloride (Zembala & Asherson,1976). Similar potentiating effects on T-cell function have beendescribed for methotrexate, cycloheximide and other cytotoxicagents (Orbach-Arbouys & Castes, 1979; Goto et al., 1981;Orbach-Arbouys, Andrade-Mena & Mathe, 1983; Parker &Turk, 1984). The augmentory effect of cyclophosphamide onDTH to SRBC in the mouse has been attributed to eliminationof the precursors of T-suppressor cells (Liew, 1977). It seemslikely that, under similar experimental conditions, CsA mayselectively inhibit the activity of such cells, perhaps by prevent-ing the release of soluble suppressor factor (Asherson &Zembala, 1975). The remarkable capacity of CsA to inhibit theinduction ofunresponsiveness to high-dose SRBC, when admin-

istered up to 7 days prior to immunization, may also reflect itsretention in (and slow release from) various tissues after drugwithdrawal (Atkinson et al., 1984). Further work is in progressto identify the cell populations affected by CsA and themechanisms underlying the augmentation of DTH in thisexperimental model.

ACKNOWLEDGMENTS

L. M. Webster is in receipt of a SERC-CASE postgraduate researchstudentship. We thank Drs M. K. Jasani and R. F. Peters, Ciba-GeigyResearch Centre, Horsham, West Sussex, for valuable discussions, andMrs I. M. Watson for typing the manuscript.

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