The effect of cyclosporin A on Angiostrongylus cantonensis infection and eosinophilia in mice

THE EFFECT CANTONENSIS K. YOSHIMURA,* Departments

of Parasitology

OF CYCLOSPORIN A ON ANGIOSTRONGYLUS INFECTION AND EOSTNOPHILTA IN MICE H. SUGAYA, K. ISHIDA, W. I. KHAN, T. ABE and K. UNNO~ and tpharmaceutical

Science, Akita Japan

University

School of Medicine,

Hondo,

Akita 010,

(Received 5 April 1993; accepted 24 June 1993) Abstract-YOSHIMURA K., SUGAYA H., ISHIDAK., KHAN W. I., ABET. and UNNO K. 1993. The effect of cyclosporin A on Angiostrongylus cantonensis infection and eosinophilia in mice. International Journal,f~r ParasitologJj 23: 997-1003. Male BALB/c mice were infected with Angiostrongylus cantonensis and at various times p.i. treated with cyclosporin A (CsA) either for 5 days continuously or intermittently, or for 12 16 days on alternate days. They were monitored for peripheral blood eosinophilia and at necropsy examined for CSF and bone marrow eosinophilia, and worm recovery. CsA treatment provoked a transient inhibition of peripheral blood eosinophilia in all groups examined, followed by rebounding eosinophilia. There was no significant difference in bone marrow and CSF eosinophilia between CsA- and vehicle-treated groups. Mice treated with CsA on alternate days yielded lower intracranial worm recovery with small-sized worms at days 7, 21 and 30 p.i. than vehicle-treated groups did. No significant reduction in worm recovery was noted in mice treated for 5 days either continuously or intermittently. although worms, especially female ones, harvested from groups treated with CsA from days - I to 3 or from days 13 to 17 were significantly smaller than those from vehicle-treated groups. CsA-treatment suppressed blastogenic responses of spleen cells against Con A or worm antigen at days 7 and 21 p.i. In vitro treatment of the 3rd stage larvae with CsA did not adversely influence survival of A. can/onensis in mice. These data indicate that CsA exerts antiparasitic effects against A. can/onensis in mice.

INDEX KEY blastogenesis.

WORDS:

Angiosrrongylus

canionensis; cyclosporin

INTRODUCTION THE fungal metabolite

cyclosporin

A

(CsA) has been used as a tool for investigating cellular immune mechanisms in various parasitic infections (Thomson, Smith & Chappell, 1986). The effects of CsA on parasitic infections fall into four categories: Firstly, CsA enhances infections of some parasites e.g. Trypunosoma

cruzi, Giardia muris, Hymenolepis

nuta and Echinococcus

dimi-

multilocularis

(Kierszenbaum, Gottlieb & Budzko, 1983; Belosevic, Faubert & Maclean, 1986; Wastling, Gerrard, Walker & Chappell, 1990; Liance, Bresson-Hadni, Vuitton, Lenys, Carbillet & Houin, 1992). Secondly, CsA acts as an anti-parasitic drug as shown in Schistosoma mansoni, Dipetnlonema

( = Acanthocheilonema)

soides carinii,

Trichinella

H. microstoma,

spiralis,

Mesocestoides

viteae, Litomo-

Strongyloides

ratti,

corti and several other

protozoan parasites (Bueding, Hawkins & Cha, 1981; Bout, Haque & Capron, 1984; Zahner & Schultheiss, 1987; Bolas-Fernandez, Grencis & Wakelin, 1988;

*To whom all correspondence

should

be addressed.

A; BALB/c

mouse;

eosinophilia;

Schad, 1986; Chappell & Thomson, 1988; Chappell, Wastling & Hurd, 1989). Thirdly, CsA exhibits neither anti-parasitic nor immunosuppressive properties as shown in Nematospiroides dubius ( = Heligmosomoides polygyrus) and H. diminuta (Chappell & Thomson, 1988). Fourthly, CsA concurrently displays both antiparasitic and immunosuppressive effects against the same species of parasite, e.g. Paragonimus miyazakii (Hashiguchi & Okamura, 1988). Meanwhile, CsA has been shown to suppress eosinophilic responses in rats immunized with ovalbumin with the previous administration of cyclophosphamide (Thomson, Brown, Simpson, Whiting & Davidson, 1985; Thomson, Milton, Aldridge, Davidson & Simpson, 1986; Mathie, Sewell & Thomson, 1987). It has been also established that nonpermissive hosts (e.g. mice and guinea pigs) infected with Angiostrongylus cantonensis provoke prominent eosinophilia in the bone marrow, peripheral blood and CSF, as opposed to decreasing intracranial worm recovery (Sugaya & Yoshimura, 1988; Yoshimura, Sugaya, Kawamura & Kumagai, 1988; Perez, Capron, Lastre, Venge, Khalife & Capron, 1989). Further-

998

K. YOSHIMURA

more, A. cantonensis infected mice which had been treated with anti-mouse IL-5 monoclonal antibody exhibited neither blood nor CSF eosinophilia, and did show the prolonged survival of intracranial worms with successful worm migration to the lungs in some mice (Sasaki, Sugaya, Ishida & Yoshimura, 1993). From these findings, it seems likely that CsA is capable of suppressing eosinophilic responses of A. cantonensis infected mice, especially in CSF eosinophilia and, in turn, intracranial worm development and survival can be promoted. The current study was thus undertaken to determine the possible effects of CsA on A. cantonensis infection and eosinophilia in mice. MATERIALS AND METHODS Mice. Male BALB/c mice, aged 6 weeks, were purchased from Japan SLC, Hamamatsu, Japan. All animal experiments followed the Guidelines for Animal Experimentation, Akita University School of Medicine. Parasitology. A. cantonensis has been maintained in our laboratory through Biomphalaria glabrata snails and rats. Mice were exposed to 20 third-stage larvae (L,) to provide groups of 5-8 mice as described by Yoshimura & Soulsby (1976). On the day of necropsy, mice under ether anaesthesia were killed and examined for worm recovery from the brain, heart and lungs as described elsewhere (Yoshimura, Aiba & Oya, 1979). Worm measurements were performed as described previously by Yoshimura el al. (1979). Drug treatment. In viva effect of CsA: the commercially available formula, SandimmunR containing 50 mg CsA/ml solvent (Sandoz Ltd., Basel, Switzerland) was used as stock

et al.

solution and diluted 5 times with 0.85% saline solution. Poly(oxyethylene)-castor-oil (Cremophor ERR; Sigma Chemical Co.) added with a final concentration of 1% ethanol was similarly diluted with 0.85% saline and used as vehicle control. Mice were treated with 0.15 ml of CsA solution (50 mg/kg body weight/day) or vehicle alone S.C. for the indicated periods as specified in Fig. 1; groups consisted of 5 continuous daily administrations (groups A-D), 12-16 administrations on alternate days (groups F and G) and 5 intermittent administrations 34 days apart (group E). Additionally, control groups of infected but untreated mice (treated with the same volume of vehicle alone) and uninfected and untreated mice were also provided. In vitro effect of CsA against L,: 20 mg of CsA were dissolved in I ml of ethanol. This solution was further diluted 100, 1000, and 10,000 times with 0.85% saline solutions. The solutions were immediately mixed with an equal volume of saline solution containing 500 L, and then incubated at 25°C for 2 h. Groups of mice were exposed to 30 L, and necropsied at 21 days p.i. for examining worm recovery from the brains. Mice which had been infected with L, in saline solution alone or in saline solution containing 1% ethanol served as controls. Haematology. Mice were bled at appropriate intervals by an opthalmic venous plexus puncture under ether anaesthesia, and examined for nucleated cells and eosinophils in the blood, bone marrow and CSF as described elsewhere (Sugaya & Yoshimura, 1988). Lymphocyte transformation. Lymphocyte transformation assays were performed according to the methods of Yoshimura, Sugaya & Ishigooka (1992) on group G mice and age- and sex-matched noninfected control mice at days 7 and 21 pi.; in this study, however, a predetermined optimum Killing

Killing

Infection a -10

v 30 Day

v

‘111”““““““““““““” B

A

20

10 C

D

-11111 E

Experimental Groups Infection + CsA (50 ms/kg/day) Control Croups Infection + Vehicle No infection, No treatment FIG. 1. Schematic representation of experimental groups for CsA treatment of mice infected with A. canfonensis. Mice were treated with 0.15 ml of CsA solution or vehicle alone s.c., on days - 1 to 3 (group A), on days 7711 (group B), on days 13-17 (group C), on days 2428 (group D) daily, on days - 1,2,6, IO and 14 (group E) intermittently, and on days - 1 to 21 (group G) or to 29 (group F) on alternate days.

Cyclosporin

999

A and A. cantonensis

concentration of 5 pg lipopolysaccharide (LPS) (Difco Lab., Detroit) was also used. The cultures stimulated with LPS were maintained for 48 h and pulsed with )H-thymidine (‘HTdr) 6 h before harvesting. Since the blastogenic responses of spleen cells against Con A and the spontaneous uptake of ‘HTdr by the cells incubated without stimuli were markedly impaired in CsA-treated mice when compared with uninfected and untreated control mice or infected and vehicle-treated mice, results were expressed as % unresponsiveness by the following equation:

% unresponsiveness

=

1i

mean c.p.m. of infected and treated (or untreated) mice mean c.p.m. of inected and untreated mice

x 100.

Statistics. Statistical analyses of the data were performed using the Student’s f-test. P values of < 0.05 were considered to be significant. Time in days

RESULTS

Effects of GA on A. cantonensis when 5 continuous or intermittent injections were given In vivo influence of CsA against A. cantonensis was assessed as shown in Fig. 1; infected mice were treated daily on days - 1 to 3 (group A), days 7-l 1 (group B), days 13-17 (group C), and days 24-28 (group D) p.i. Figure 2 indicates peripheral eosinophihc responses in groups A-D. CsA-treated mice in all groups showed significantly lower eosinophil counts at the end of drug administration than vehicle controls, yielding counts almost equivalent to or less than those of uninfected normal controls; the eosinophil count was extremely low in group A at 1 week p.i. CsA treatment, however, caused a rebounding increase in eosinophil counts as seen in groups A and B (Fig. 2). Blood leukocyte counts tended to decrease gradually until day 21 p.i. in A. cantonensis infected mice regardless of CsA treatment (data not shown). Worm recovery from groups A-E at days 21 and 30 pi. indicated that there was no significant difference between vehicle- and CsA-treated mice in all groups examined (Table 1). Female worms from group A and both male and female worms from group C were significantly smaller in CsA-treated mice than in vehicle-treated mice (data not shown). Effects of CsA on A. cantonensis when 12-16 injections were given on alternate days Continuous or intermittent administration of CsA for 5 days failed to affect worm recovery from the mice. This study was thus undertaken to determine eosinophilic responses and worm recovery in mice

FIG. 2. Mean

eosinophil counts in peripheral blood of various groups of A. cantonensis infected mice which were treated with CsA or vehicle alone at different stages of infection. Mice were treated at days - 1 to 3 [group A: (0) CsA; (O), vehicle], days 7-11 [group B: (+), CsA; (x ),vehicle], days 13-17 [group C: (0) CsA; (B), vehicle] and days 2428 [group D: (a), CsA; (A), vehicle]. (0) Normal control mice; (+) A. cantonensis infection; (1) treatment; ( 1) mouse necropsy. Figures in parentheses denote the number of mice examined. Statistically significant between CsA- and vehicle-treated mice at level of P< 0.05 (*). P< 0.02 (*a) and P< 0.001 (t).

which had been treated with CsA or vehicle on alternate days from days - 1 to 21 (group G) or from days - 1 to 29 p.i. (group F) (Fig. 1). Figure 3 shows that CsA-treated mice in group F produced significantly lower eosinophil counts than those of normal uninfected controls or vehicle-treated controls at 1 week p.i., but then exhibited a steady increase and thus counts were reversed at week 3. Group G mice also showed an identical pattern of eosinophilic responses to that of group F (data not shown). There was no significant difference in CSF and bone marrow eosinophilia between CsA- and vehicletreated mice in groups G and F, except that, in group G, vehicle-treated mice showed significantly higher medullary eosinophilia than CsA-treated mice at 1 week p.i. Worm recovery data indicated that CsA-treated mice in both groups at days 7, 21 and 30 p.i. yielded lower worm burden than vehicle-treated mice (Table 1). Furthermore, both male and female worms recovered from CsA-treated mice of groups F and G

K. YOSHIMURA et al.

1000 TABLE I-RECOVERY

OF A.

canronensis FROM THE BRAINSOF MICE INFECTEDWITH 20

INFECTIVELARVAEAND TREATED

WITH CsA

Group

Worm

Treatment*

I A B C D E G F

Vehicle CsA Vehicle CsA Vehicle CsA Vehicle CsA Vehicle CsA Vehicle CsA Vehicle CsA

alone alone alone alone alone alone alone

Significance

ND-F ND ND ND ND ND ND ND ND ND 10.8f 1.79(S) 6.2f l.30(5) ND ND

recovery 21

6.2f 3.5 f 4.2 f 4.8 f 5.8 f 4.8 f

P
2.28(5)$ 1.29(4) 3.54(6) 3.43(6) I .94(6) 2.64(6) ND ND 4.lf 2.42(6) 5.5 f I .64(6) 7.5 f 2.65(4) 2.0+0.71(5)

ND ND

on day (pi) Significance

NS NS NS

NS

P
30 2.4+ I .82(5)ji 1.0*0.71(5) 2.7 f 2.50(6) 1.8+0.75(6) ND 2.4+??4(5) 3.55 2.43(6) ND ND ND ND 7.2f 3.35(5) 1.0*0.58(7)

Significance

NS NS

NS

PCO.02

*Treatment was given S.C. on days - I to 3 (group A), on days 7 - I I (group B), on days 13-17 (group C), on days 24-28 (group D) daily, on days - I, 2. 6, IO and 14 (group E) intermittently, and on days - I to 21 (group G) or to 29 (group F) on alternate days. Dosage of CsA was 50 mg/kg/day. tNot done $X zlzS.D. (n). $One of 5 mice yielded 4 worms, I male of which was recovered from the lung.

Time in days FIG. 3. Mean eosinophil counts in peripheral blood of A. cantonensis infected mice (group F) which were treated with

CsA or vehicle alone at days - 1 through 29 on alternate days. (0) CsA; (0) vehicle; (V) normal control mice. Figures in parentheses denote the number of mice examined. Statistically significant at level of P< 0.05 (a), P< 0.01 (**) and P
were smaller than those from vehicle-treated mice (data not shown). Lymphocyte transformation assays were performed on spleen cells from group G mice harvested at days 7 and 21 p.i. Blastogenic responses to Con A and worm antigen at day 7 p.i. were found suppressed in CsAtreated mice. Contrarily, vehicle-treated mice provoked enhanced responses to the antigen. It is of interest to note that the spontaneous uptake of JHTdr by spleen cells at day 7 p.i. was quite low in CsAtreated mice at the longer incubation period (96 h) for antigen stimulation, as compared with normal uninfected mice or vehicle-treated mice (Fig. 4). Similar assays at day 21 pi. also revealed the inhibition of spontaneous ‘HTdr uptake by the spleen cells from CsA-treated mice. At this time, however, a comparable degree of inhibition was noted at the incubation periods of 96 h for antigen stimulation and 48 h for Con A or LPS stimulation. There was no significant difference in blastogenic responses against Con A or LPS between CsA- and vehicle-treated mice. By contrast, there was a marked difference in blastogenic response to the worm antigen between CsA- and vehicle-treated mice; although both groups of mice exhibited positive responses to the worm antigen, vehicle-treated mice provoked much higher responses (Fig. 4). In vitro efJ;?ct of CsA against L, Mice were exposed to L, which had been incubated

Cyclosporin

A and A. cantonensis

1001

% Unresponsiveness

FIG. 4. Blastogenic responses stimulated by Con A. LPS and A. cantonensis antigen (Ag) in cultures of spleen cells obtained from A. cantonensis infected mice which were treated with CsA (0) or vehicle alone (W) at days - I through 21 (group G) on alternate days. Med=medium alone without stimuli. Blastogenesis was assessed at days 7 and 21 p.i. Data represent .Y~s.E.M. of 3 (Con A group on day 7)-6 mice. Statistically significant between CsA- and vehicle-treated mice at level of P< 0.05 (*) and P< 0.001 (t).

for 2 h at 25°C with 1, 10 and 100 pg of CsA/ml in saline, or with 1% ethanol in saline or saline alone. Necropsy at day 21 p.i. indicated that there was no significant difference in peripheral leukocyte and eosinophil counts as well as intracranial worm recovery among these 5 groups (data not shown). DISCUSSION We expected initially that the survival of A. canronensis intracranial worms could be prolonged in CsA-treated mice, since eosinophilic responses of rats immunized with ovalbumin have been shown to be suppressed by (LA-treatment (Thomson ef al., 1985, 1986; Mathie et al., 1987) and also since CSF eosinophilia has been shown to be associated with the killing of intracranial worms in mice (Sugaya & Yoshimura, 1988; Yoshimura et al., 1988; Perez et al., 1989; Sasaki et al., 1993). Nevertheless, continuous or intermittent administration of CsA for 5 days at various times after infection with A. cantonensis failed to enhance intracranial worm recovery (Table 1). However, the administration of CsA on alternate days commencing at day - 1caused significant reduction in worm recovery when examined at days 7,21 and 30 p.i. (Table l), suggesting anti-parasitic effects of CsA. Meanwhile, CsA treatment in groups A-G caused a

transient suppression of blood eosinophilic responses, followed by drastic rebounding eosinophilia (Figs. 2 and 3). The degree of the reduction in eosinophil counts was quite high in groups A, F and G, where CsA treatment started at day - 1 pi. Moreover, there was no significant difference in CSF eosinophil levels at necropsy between CsA- and vehicle-treated mice. These findings clearly suggest that anti-parasitic effects of CsA in groups F and G could not be due to CSF eosinophils. As far as we know, there is no information concerning the possible roles of CsA in eosinophilia caused by parasitic infections. On the other hand, eosinophilia induced in rats by administration of cyclophosphamide before immunization with ovalbumin in FCA has been shown to be inhibited by daily administration of CsA (Thomson et al., 1985, 1986; Mathie et al. 1987). The effect of CsA on eosinophilia in murine infection of A. cantonensis, therefore, differs from that seen in rats, immunized with ovalbumin, in that eosinophilia was suppressed only transiently, followed by a prominent rebound. Although the reason for this rebounding eosinophilia remains to be determined, the continuous production of antigens by intracranial worms and their subsequent release may be associated with this phenomenon. Moreover, it is

1002

K. YOSHIMURA et ai.

also conceivable that the rebounding eosinophilia is part of the immune reaction to killed parasites. However, this may be unlikely because the rebounding eosinophilia could be seen even in groups A and B where no significant reduction in worm recovery was noted. The fact that spleen cells from CsA-treated mice at day 7 p.i. showed significantly low blastogenic responses to Con A as compared with the cells from vehicle-treated mice (Fig. 4) is indicative of suppressive effects of CsA on T-cells. The inhibition of spontaneous uptake of ‘HTdr by spleen cells incubated without stimuli in CsA-treated mice also suggests that a T-cell population in splenic cells could have been greatly damaged by CsA treatment. The target and mechanism of anti-A. cantonensis action of CsA still remain to be determined. Since it is established that A. cantonensis L, molt twice in the brain at days 4-6 and 7-9 p.i., respectively (Bhaibulaya, 1975), the targets may be either L1 prior to reaching the brain or worms (L,-L,) developing in the brain; CsA probably damages L3 or L,, since the worm recovery at day 7 p.i. in CsA-treated mice of group G was low and also since the size of female worms recovered from CsA-treated mice of group A was small, although the in vitro damaging effects of CsA on L, could not be demonstrated even at the highest concentration (100 pg/ml) of CsA. Moreover, it seems likely that days 13-l 7 intracranial worms (= L,) could also be vulnerable to CsA, since both male and female worms recovered from group C were smaller in CsAtreated mice than in vehicle-treated mice. As to the killing of intracranial worms, it is of interest to note that a minute amount of CsA (I .9 ng/mg tissue) could be detected in the brain tissues of BALB/c mice which had been inoculated daily with CsA at the dosage of 50 mg/kg/day, and also that this level of CsA in the brain could be maintained throughout the course of drug administration (Atkinson, Boland, Britton & Biggs, 1983). Other investigators have, however, reported that CsA could not be detected in CSF of mice inoculated with this drug (Fazakerley & Webb, 1985). CsA has been shown to affect female reproductive systems of T. spiralis and L. carinii (Bolas-Fernadez et al., 1988; Zahner Jc Schultheiss, 1987). Unfortunately, A. cantonensis cannot reach to sexual maturity in the mouse and, thus, the damaging effects of CsA on the reproductive systems of A. cantonensis are still obscure. Furthermore, the anti-parasite action of CsA has been suggested to be a consequence of modulating the defence mechanisms of the host (Nilsson, Lindblad, Olling & Ouchterlony, 1985; Behforouz, Wenger & Mathison, 1986). Our data on A. cantonensis rather suggests a direct damaging activity of CsA against

certain

developmental

stage(s) of the parasite.

Acknowledgements-We thank Mr S. lshigooka and MS K. Yamashita for technical and secretarial assistance. Thanks arc also due to Sandoz Pharmaceutical Ltd. for a generous gift of “Sandimmun”. This study was supported in part by Grants-in-Aid (Nos. 01480169 and 04670223) for Scientific Research from the Ministry of Education, Science and Culture of Japan.

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