Interaction of anti-leprosy drugs with the rat serum complement system

lmmunopharmacology, 15 (1988) 143-150 Elsevier 143

IMO 00404

Interaction of anti-leprosy drugs with the rat serum complement system

A r v i n d S a h u 1, K u n a l S a h a 1, A r v i n d K a s h y a p 1 a n d As i t K , C h a k r a b a r t y 2

1 Department of bnmunolo~y, Vallahhbhai Patel Chest Institute. Universi O' o[" Delhi, Delhi, hMia and '-Department 0[" Biochemistry, University College ~?[' Medical S~ iences, Delhi, India

(Received 7 July 1987: accepted 6 January 1988)

Abstract: Dapsone, clofazimine and rifampicin, the three most important constituents of multidrug therapy against leprosy, were studied with respect to their effects on the rat serum complement system, in vitro as well as in vivo. Of the three drugs only dapsone and clofazimine exhibited significant in vitro anti-complement activity and only at a very high, non-therapeutic dose of 0.24 mg/ml. On the contrary, rifampicin could not induce significant in vitro complement consumption. Furthermore, dapsone and clofazimine could reduce rat-serum-mediated rabbit erythrocyte haemolysis in the presence of Mg 2 +-EGTA, indicating that they could also affect the alternative pathway of complement activation. However, the latter pathway of complement consumption by these drugs seems to be insignificant because the factor-B-mediated complement-consumption system is minimal in rat sera. Immunoelectrophoretic study of mixtures of fresh rat sera and anti-leprosy drugs against specific anti-rat-C3 antisera demonstrated that dapsone and clofazimine could not cleave the C3 complement component. In a separate experiment we attempted to reconstitute the haemolytic complement activity consumed by dapsone and clofazimine by adding Cr~t-EDTA sera (a source of C3, C5, C6. C7, C8 and C9), but at most only 12% reconstitution of haemolytic activity could be achieved. We thus conclude that both dapsone and clofazimine could affect the complement system, predominantly through the earlier complement components and at very high, non-therapeutic doses. On the contrary, in-vivo experiments in rats showed that a combination of these three drugs, when given at a therapeutic dose or at 10 times the therapeutic dose for three months, did not afl'ect the complement system.

Key words: Anti-leprosy multidrug therapy: Scrum complement system; Leprosy

Introduction

Mult idrug therapy (MDT) consist ing of dapsone,

r ifampicin and clofazimine was in t roduced as a use-

ful tool in the t rea tment of leprosy (Revankar ,

1986). There are scattered reports on the modula-

t ion of the h u m a n i m m u n e system by the individual

drugs belonging to M D T . Of the 3 drugs, rifampi-

CorresT~ondence: K. Saha, Department of Immunology, Vallabh- bhai Patel Chest Institute, University of Delhi, Delhi I I0 007, India. Abbreviations." ENL, erythema nodosum leprosum; DMSO, dimethyl sulphoxide; GVB, gelatin veronal buffer: MDT, multidrug therapy; Ra/SRBC, rabbit/sheep erythrocyte.

cin has been reported to have an immunosuppres -

sive effect in humans (Vidhidharm et al., 1985),

whereas clofazimine and dapsone inhibit neutrophil

chemotaxis (Van Rensburg et al., 1982; Gar tne r et

al., 1982; Harvath et al., 1986). The complement

system, both classical and alternative, which is an

impor tan t componen t of our immune apparatus, is

known to be modula ted by various life-saving

drugs, such as tetracycline, chloromycetine, ampi-

cillin, s t reptomycin (Chakrabar ty et al., 1981), sul-

phonamides (Zabern et al., 1985), chloroquine

(Chakrabar ty et al., 1985) as well as radiographic

contrast media (Zabern et al., 1983). However, the

action of the above anti- leprosy drugs on the com-

plement cascade has not been reported. In this corn-

0162-3109/88/$03.50 ~ 1988 Elsevier Science Publishers B.V. (Biomedical Division)

144

munication we discuss the effect of these drugs at therapeutic and mega doses on complement depletion in vivo as well as in vitro using rat sera as a source of complement system.

Methods and Materials

Animals

Seventy-seven randomly bred Wistar rats had free access to water and rat chow (Hindustan Lever Co., India). Blood was collected by cardiac puncture under ether anaesthesia.

Drugs

20 mg of dapsone (Wellcome, India) was dissolved in 1 ml of 50% dimethyl sulphoxide (DMSO) in saline; 200 mg of clofazimine (Surhid Geigy Chem- icals and Pharmaceuticals, India), was dissolved in 100 ml of absolute alcohol and further diluted in normal saline; 20 mg of rifampicin (Lupin Labora- tories, India), was dissolved in 1 ml normal saline and 10/tl of 4 N NaOH (Back et al., 1979).

Bu~,rs

Barbital buffer consisted of 22.0 mM 5,5'-diethyl barbituric acid, 9.0 mM sodium 5,5'-diethyl bar- biturate, 2.5 mM CaCI2, 7.0 mM MgCI2 and 0,73 M NaC1, pH 7.4. EDTA (40 mM)-gelatin veronal buffer (GVB) lacking cations and Mg2+-EGTA buffer (10 raM) were made according to Whaley (1985).

Immunological reagents

1% sheep and rabbit erythrocytes (SRBC/RaRBC) were suspended in cold normal saline; anti-rat-C3 antiserum was obtained commercially (Research Plus, U.S.A.); pooled sera from normal rats was used as a C3 reference standard; anti-sheep hae- molysin was procured commercially (Sera-Lab., U.K.); c~at-EDTA, a source of C3, C5, C6, C7, C8 and C9 (Whaley, 1985), was prepared by diluting rat serum in EDTA-GVB.

Consumption of complement by anti-leprosy &ugs in vivo

Rats were divided into three groups:

Group 1 (1 x MDT). Rats were fed daily orally with 2 mg/kg dapsone and 1 mg/kg clofazimine suspended in 1 ml/kg ground-nut oil. A monthly dose of 2 mg/kg dapsone, 6 mg/kg clofazimine and 12 mg/kg rifampicin suspended in 1 ml/kg ground-nut oil was also administered orally.

Group H (10 x MDT). Rats were ted with daily dosages of 20 mg/kg dapsone and 10 mg/kg clofazimine suspended in I ml/kg ground-nut oil. A monthly dose of 20 mg/kg dapsone, 60 mg/kg clofazimine and 120 mg/kg rifampicin suspended in ground-nut oil was also given.

Group Il l (oil control). 1 ml/kg ground-nut oil was administered daily to the control rats.

After three months of drug administration the animals were sacrificed and their serum CH50 (Campbell et al., 1964) and C3 levels (Mancini et al., 1965) were estimated.

In-vitro methods

Complement consumption by anti-leprosy drugs. Complement consumption was studied by titrating total haemolytic complement as described elsewhere (Shulman and Banker, 1969).

The alternative pathway of complement consumption by ant#leprosy drugs. The alternative pathway of complement consumption was studied in vitro using RaRBC (Whaley et al., 1985).

Immunoelectrophoretie study qt~ aetivation q[" C3 by anti-leprosy drugs. Immunoelectrophoresis was carried out in 1% Noble agar (Difco) made in 0.045 M barbital buffer, pH 8.6 on microscopic slides. A current of 8 mA per slide was passed for 1 hour.

Reconstitution o[" drug-depleted complement hae- molytie activity by substituting Cr~t-ED - TA. Dapsone (100 1000 tlg) and clofazimine

(10~40/zg) in 0.1 ml of solvent were added to 0.5

ml of l :6-diluted fresh normal rat serum in different

tubes and incubated at 37°C for 30 min. 0.1-ml ali-

quots were mixed with sensitized SRBC and incu-

bated at 30°C for 30 min for s tudying the format ion

of C3-convertase classical (C1, C4 and C2) or al-

ternative (factor B and D) pathways. Then 0.2 ml

of c ra t -EDTA reagent (1:5 diluted in E D T A - G V B

buffer) was added to the reaction mixture, incubat-

ed at 37°C for 1 hour, and centrifuged cold. Finally,

optical density was read spectrophotometr ical ly at

530 nm.

Results

Action of anti-leprosy drugs on rat serum complement in vivo

The mean normal CH50 levels in rats was about 60

units /ml and the average serum C3 level was about

145

102%/ml of the reference s tandard (Table I). The

control animals which were fed only with ground-

nut oil showed significantly higher serum CH50

levels but, surprisingly, their serum C3 levels re-

mained within normal limits. However, there was

no significant difference between the serum C3

levels of the drug-treated animals and normal rats.

CH50 levels in the animals with the various drug

schedules were also not different from those in nor-

mal rats. Thus, our results showed that serum com-

plement componen ts remained unal tered even when

feeding 10 times the scheduled dose of M D T for

three months . The unexpected effect of g round -nu t

oil in increasing serum CH50 levels is difficult to

unders tand and remains to be studied further.

In-vitro action of anti-lepro,~v drugs on the complement system

Fig. 1 illustrates the dose-dependent depletion of

haemolytic activity of rat serum by dapsone, clo-

TABLE I

In-vivo experiments: total haemolytic complement and serum C3 levels in normal rats and rats fed with anti-leprosy drugs dapsone (DAP), clofazimine (CLO) and rifampicin (RIF)

Group CH50 Serum C3 (n) (units/ml; (% of standard per ml;

mean :1- S.D.) mean + S.D.) (range) (range)

A: Normal rats (12) 59.87 + 27.56 101.82 + 26.54 ( 13.49-109,64) ( 78-172)

B: Drug-treated rats 1: DAP + CLO + R1F + oil (8) 69.37 + 27.55 106.86 ~: 25.24

(36.30 97.72) (81 136)

II: DAP + CLO + RIF + oil (7) 83.15 ± 26.59 108.87 :t- 47.77 (46.77-125.89) (60-210)

Ili: Oil (8) 119.06 ± 26.33 114.0 • 35.30 (81.28-158.49) (48 180)

Statistical evaluation": Avs. B. l p > 0.1 p > 0.1 Avs. B. II 0.1 > p > 0.05 p > 0.1 A vs. B. III p < 0.001 p > 0.l

a There were no significant differences between the mean levels of serum CH50 and C3 in normal and drug-fed rats. However, a significant rise of serum CH50, but not serum C3 level, was observed in rats fed with oil in comparison to normal rats.

I00

kag dopsone and rifompicin added 203 40O 6O0 8O0 I000

I 0 0 I i I I I l i - - - i Rifompicin. I ~ . . . . . . . . 0 0 Dopsone.

8 0 q Clo~zimine.

:~0"

i i 0 I0 20 30 40 50

klg cloFozimine added

Fig. 1. A typical experiment showing depletion o f the COlnple- ment haemolytic activity of a ral serum sample after addition of anti-leprosy drugs. 0.1 ml of solvenl containing difi'erent

amounts of dapsone, clofaziminc or rifampicin were added to

0.5 ml of freshly drawn I:6-diluted rat serum and incubated [\)r 30 rain at 37°C. The remaining complement activity was assayed

by adding 0.1 ml of the reaction mixture to I% sensitized SRBC, and measuring haemolysis spectrophotomelrically at 530 nm. The anti-complement activity is greatest in clol'azm]il]e and least

in rifampicin. Controls showed that these drugs have no direct effects on SRBC haemolysis al the concentrations used in the

lest. This experimenl was pcrl'ormcd 5 limes.

~'60]

4 0 -

fazimine and rifampicin. Of these drugs, rifampicin had the lowest anti-complement activity and clofazimine the highest.

Complement consumption via the alternative pathway by these three anti-leprosy drugs was also studied, and clofazimine was found to be the most reactive drug, while dapsone was also shown to ex- hibit anti-complement activity (Fig. 2). Table II shows that rifampicin was unable to deplete serum complement haemolytic activity significantly by the alternative pathway.

Electrophoretic study

Immunoelectrophoretic studies were conducted with fresh normal rat serum treated with anti-leprosy-drugs. After electrophoresing the above mix-

6 0

/4g dapsone added 2 0 0 4 0 0 6 0 0 8 0 0 IOO0

I I I I I

H Clofazimine

0 0 Dopsone

0

4 0 0 "1"

2 0

146

! i 1 i !

I 0 20 30 40 5o

/4g of clofozimine added

l'ig. 2. The ell'eel on the ahernativc pathway el 'complement con-

sumption of clofazimine and dapsone using RaRBC as an in- dicator. 0.'1 ml of solvent containing graded drug doses were mixed with 1 ml of undiluted fresh rat serum (since rat serum is

delicient in the complement component of the alternative pathway) and incubated at 37°C t\~r 30 min. Thereafter 1-ml aliquots were added to I% RaRBC in Mg 2+-EGTA buffer. Haemolysis

was determined spectropholometrically at 530 nm. CIofazimine

is the most reactive for complement consumption through the alternate pathway. The drugs have no direct effect on RaRBC

haemolysis at the concentrations used in the test. This experiment was done 5 times,

tures, anti-rat-C3 antisera were put in the trough. This study demonstrated that the drug could not split the C3 complement component (Fig. 3) at the concentration at which the drugs showed inhibition of complement haemolytic activity (Figs. 1 and 2).

Reconstitution qf complement activit.l' or rat serum depleted by anti-leprosy dru,~'s hy adding C~"'-EDTA

Fig. 4 shows that the more dapsone and clofazimine were added to the fresh rat serum samples contain-

147

TABLE II

In-vitro study of the effect on the alternative pathway of complement activation by rifampicin using RaRBC and Mg 2 +-EG-

TA buffer

Rifampicin (/lg) Haemolysis (%)

0 32.19 10 31.67

100 29.26

1000 29.76

1 ml undiluted normal fresh rat serum was mixed with different

quantities of rifampicin dissolved in 0.1 ml of solvent and incubated at 37°C for 30 rain. Thereafter a l-ml aliquot was ti-

trated for haemolytic activity using RaRBC in the presence of Mg 2+-EGTA. Values are the means of three experiments. Ri-

fampicin has no direct effect on RaRBC haemolysis at 10 leg,

100 pg and 1000/~g concentrations.

Fig. 3. The effect of clofazimine and dapsone on the rat serum

C3 complement component in vitro. A: The top well contained

fresh normal rat serum mixed with clofazimine (50 itg in 25% alcohol) in the ratio l:l v/v and incubated at 37°C for 1 hour. The bot tom well contained fresh rat serum mixed with alcohol

(25%). The trough contained anti-rat-C3 antisera. No activation was observed. B: The top well contained fresh normal rat serum mixed with dapsone (1000/~g in 50% DMSO) in the ratio of l:l v/v incubated at 37°C for [ hour. The trough contains anti-

rat-C3 antisera. The bot tom well contained fresh normal rat ser-

um mixed with DMSO (50%). No activation was seen. The an- ode is to the right.

ioc

,n

5o

I % hoemolysis before drug oddition

% hoemolysls left offer drug oddition.

% hoemolysis reconstituted offer oddition, o f C TM EDTA.

%hoemolysis which could not be r e c o n s t i t u t e d o f f e r

oddifion of C rot EDTA.

!)ii!i::::i

®

0 IO0 I000

! I

mmmu 200 300 400 500

microgrom dapsone odd~l

J

o Io micro{Fore

®

I

l:: m 20 30

clofozirnine odded

j -OO

-6 E o"

5 0 z

c m u

Fig. 4. Experiment showing the reconstitution of haemolytic complement activity (which was depleted by the drugs) by the addition of Cr"'-EDTA serum (a source of C3 C9 components). (The consumption of haemolytic activity increases with drug concentration. A maximum of 12% haemolytic activity was reconstituted by the addition of Cr"t-EDTA. As the drug concentration increased fu r the r - - from 20 #g in the case of clofazimine and 300 Ftg in the case of dapsone- - the concentration of free drug may have increased, thus

decreasing the percent reconstitution.) This experiment was conducted five times.

148

ing a definitive amount of haemolytic activity, the higher the depletion of total haemolytic activity. Thus, when the dapsone concentration was 100 #g/0.6 ml of the reaction mixture, the haemolytic complement activity dropped from 80% to 25%; when the concentration of dapsone was increased to 1000/tg/0.6 ml, the decrease was from 80% to 0.4%. In the case of clofazimine, the decrease was from 94% to 77% when the drug concentration was 10 #g/0.6 ml and from 97% to 2.7% at a concentration of 40 #g/0.6 ml. When we attempted to reconstitute the complement activity by adding C ra~- EDTA, it was observed that the amount of the reconstituted haemolytic activity never exceeded 12%. When the dapsone concentration was not more than 300 #g or the clofazimine concentration not more than 20 #g, maximum reconstitution was possible. However, when the concentration of these drugs were increased further, the amount of reconstitution of the haemolytic activity was decreased from 12% to 3% (at 1000 #g dapsone)or from 12% to 5% (at 40 t~g clofazimine). Thus, these results indicated that it was not possible to completely reconstitute the haemolytic activity depleted by the drugs by adding terminal complement components (C3 C9). This data further shows that drug concentration was critical for maximum reconstitution; that is, when dapsone was more than 300 /tg and clofazimine was more than 20/Jg there was excess of free drugs, which may have modulated the terminal components and thus reduced the reconstitution of haemolytic activity.

Discussion

The study of the interaction of drugs with the human immune system, and in particular the complement cascade, has attracted the interest of many investigators (Chakrabarty et al., 1981, 1985; Za- bern et al., 1983, 1985). Anti-leprosy multidrug therapy (MDT) had been implemented by the Gov- ernment of India as a National Leprosy Control Measure on which the successful elimination of the this dreadful illness from the globe rests. However, a systematic study of the modulation of the human

immune system by these anti-leprosy drugs has yet to be undertaken.

Ramanathan and Sengupta (1985) demonstrated that chloroquine used to control erythema nodosum leprosum (ENL) in leprosy patient stops the activation of the complement system. Earlier, Saha and his associates (1982) had shown that clofazimine administration in therapeutic doses in leprosy patients failed to change serum C3 levels.

The main purpose of the present study was to investigate the effect of MDT on the complement cascade system. The in-vitro study was designed in such a way so that the effect of excess doses of these drugs could be studied on the serum complement system. The relevance of this in-vitro study to the human situation is that clofazimine is deposited in the tissues of patients, where its concentration reaches as high as 3.30 mg/g tissue (Desikan and Balakrishnan, 1976). Thus it is obvious that it is almost impossible to mimic the tissue condition (effect of high concentration of drug on tissue complement) in-vitro. Our in-vitro study showed that clofazimine and dapsone, when added in large excess to rat sera, could inhibit the complement-mediated lysis of antibody-coated SRBC. It also showed that the drug could inhibit the lysis of RaRBC in the presence of Mg 2 +-EGTA (alternative complement pathway). However, the quantum of complement utilization through the factor-B properdin system scemed to be minimal in rat serum because 80% of hacmolysis of antibody-coated SRBC was obtained with 0.1 ml of 1:6 diluted rat serum; in contrast, as much as 1 ml of undiluted rat sera was required to produce 30 to 55% haemolysis of RaRBC in Mg2+-EGTA buffer. Therefore, the observed depletion of haemolytic activity by these two drugs may occur mostly through the direct pathway. Rifampicin had no significant anti-com- plementary activity, as tested by these two in-vitro methods. These results get some support from the work of Li and Vogt (1982) and Honda et al. (1986), who showed activation of the classical pathway by sugar-cane polysaccharide and of the alternative pathway by an anti-tumour (l --, 3)-/~-l)-glucan from Alcaligenes ./aecalis var. myxogenes re- spectively.

Our immunoelectrophoresis experiment showed that even large amounts of clofazimine and dapsone could not cleave rat C3 (Fig. 3). These results, along with the fact that cr"t-EDTA, a source of C3 C9, could not appreciably reconstitute the haemolytic activity of rat sera lost after addition of the two drugs, indicate that these drugs perhaps could not generally modify the terminal complement components C3, C5, C6, C7, C8 and C9. Also, these drugs may not have been able to modify the complement component belonging to the alternate pathway (Fig. 2 and Table II). Thus we postulate that clofazimine and dapsone possibly act on C1, C4 and C2, the early complement components, a view similar to that expressed by Li and Vogt (1982). An alternative explanation might be that these drugs inhibit the formation of classical C3- convertase, as suggested by the reconstitution experiment (Fig. 4). A similar view has been expressed by Kijlstra and Jeurissen (1982) who showed that human lactoferrin inhibited the formation of C3- convertase.

Finally, we would like to emphasise that our results suggest that MDT, when administered in therapeutic doses both in vivo and in vitro, could not modulate the complement system but, when added to fresh serum in large amounts in vitro, does inhibit the haemolytic complement cascade. Thus it is possible that similar impairment of the tissue-as- sociated complement system may take place in various organs of the patients, where the drugs, and in particular clofazimine, are deposited in large amounts (Desican and Balakrishnan, 1976). This notion lends support to the anti-inflammatory property of clofazimine during ENL episodes. Clo- fazimine deposited in the various organs during le- pra reaction II may inhibit the activation of complement in the tissue, which may in turn explain its anti-inflammatory action during the acute stages of ENL.

Acknowledgement

The authors gratefully acknowledge the financial grant of the Indian Council of Medical Research, New Delhi, to one of the authors (AS).

149

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Interaction of anti-leprosy drugs with the rat serum complement system