Int. J. lrnmunopharmac., Vol. 14, No. 4, pp. 7 2 1 - 7 3 0 , 1992. Printed in Great Britain.

0 1 9 2 - 0 5 6 1 / 9 2 $5.00 + .00 Pergamon Press Ltd. © 1992 International Society for lmmunopharmacology.

I N VIVO EFFECTS OF A N T I - L E P R O S Y D R U G S ON THE RAT PERITONEAL MACROPHAGES AND LYMPHOCYTE SUBPOPULATIONS ARVIND SAHU,* KUNAL SAHA, *t ASHOK MUKHERJEE t and VIRENDRA N. SEHGAL§ *Department of Immunology, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi-110007; *Institute of Pathology, Indian Council of Medical Research, P.O. Box 4909, Safdarjang Hospital Campus, New Delhi-110016; and ~Department of Dermatology, STD, and Leprology, Lady Hardinge Medical College, New Delhio110001, India (Received 19 June 1991 and in final form 17 October 1991)

Abstract - - The present study describes the in vivo effects of anti-leprosy drugs on rat peritoneal

macrophages and T-cell homeostasis. It was observed that BCG-elicited rat peritoneal macrophages produced more H:Oz and expressed more Ia antigen on their cell surfaces compared with resident peritoneal macrophages. Furthermore, elicited macrophages isolated from rats administered multidrug therapy (MDT), consisting of dapsone, clofazimine and rifampicin in high dose (10 × MDT) released more O,-. On the contrary, there was a significant decrease in the Ia antigen expression on these macrophages. Anti-leprosy drug treatment in high dose (10 × MDT) decreased the total number of blood T-helper (W3/25 ÷) cells and increased the total number of blood T-suppressor (OX-8 +) cells which resulted in a significant decrease in a W3/25 : OX-8 ratio. Electron microscopy of elicited macrophages isolated from 10× MDT treated rats showed development of many filipodia compared with control macrophages. These data show that 10 × MDT treatment in rats for 1 month alters the homeostasis of blood T-cell subpopulations which perhaps decreases the Ia expression on macrophages. However, the increase in O_~ production and the appearance of filipodia on the macrophages is due to a direct effect of drugs on the macrophages. MDT treatment for 1 month in a therapeutic dose has no effect on the above-mentioned parameters.

Multidrug therapy (MDT), comprising dapsone and rifampicin for paucibacillary leprosy or dapsone, clofazimine and rifampicin for multi-bacillary leprosy, has been recommended by the World Health Organization as a singular effective regimen for leprosy management ( W H O , 1988). The interaction of anti-leprosy drugs with macrophages has been a focus of attention ever since clofazimine (Conalty & Jackson, 1962) and rifampicin (Johnson, Hand, Francis, KingT h o m p s o n & Corwin, 1980) were reported to accumulate in macrophages. In lepromatous leprosy, macrophages contain an enormous number of Mycobacterium leprae, even then the production o f 0 2 and "OH is inadequate (Niwa, Sakane, Miyachi & Ozaki, 1984). O f the three drugs used in M D T , clofazimine significantly enhances O5 production, in vitro, o f rat resident peritoneal macrophages in a

tAuthor to whom correspondence should be addressed. 721

dose-dependent manner (Sahu, Saha, Banerjee, Sehgal & Jagga, 1991). Clofazimine is also known to inhibit the proliferation of tymphocytes as well as the migration of polymorphonuclear leucocytes, whereas dapsone potentiates the afore-mentioned properties (Anderson, 1985), due to their respective pro- and anti-oxidant properties (Anderson et al., 1981). Furthermore, clofazimine and dapsone exerted mutually antagonistic properties towards enhancement of prostaglandin synthesis (Anderson, 1985). Since the introduction o f rifampicin in clinical use, many reports have been published on the effect of this antibiotic on cell-mediated and humoral responses. In some studies rifampicin suppressed both humoral and cellular responses to various antigens (Paunescu, 1970; Nilsson, 1971; Grassi & Pozzi, 1972; Mukerjee, Schuldt & Kasik, 1973).

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Table 1. Drug schedule in six groups of rats. Animals of groups I, I1, and III were used for the macrophage study, while of groups IV, V and VI were used for the lymphocyte study Duration (months)

MDT schedule

Group I t/IV (control)

One

Normal diet.*

Group II+/V (1 x MDT treated)

One

Daily diet contained 0.00435% (w/w) dapsone, and 0.00217% (w/w) clofazimine. Once a month their diet was mixed with 0.00435% (w/w) dapsone, 0.01304% (w/w) clofazimine and 0.0261% (w/w) rifampicin.

Group III+/VI (10 x MDT treated)

One

Daily diet contained 0.0435% (w/w) dapsone and 0.0217% (w/w) clofazimine. Once a month their diet was mixed 0.0435% (w/w) dapsone, 0.1304% (w/w), clofazimine and 0.261% (w/w) rifampicin.

Group

* All rats had free access to water and rat chow (Hindustan Lever Co., India). Rat chow mixed with anti-leprosy drugs was protected from light while preparation and storage. The average intake of chow per rat was 11.5 g. 0.1 ml (0.4 x 106 viable organisms) and 0.2 ml (0.8 x l0 s viable organisms) of BCG were injected intraperitoneally on days 0 and 20, respectively, in all animals of groups I, II and III. Drugs were administered from day zero.

H o w e v e r , n o effect o n h u m o r a l or cellular i m m u n i t y was o b s e r v e d in a controlled d o u b l e - b l i n d study ( H u m b e r et al., 1980). In view o f the paucity o f i n f o r m a t i o n c o n c e r n i n g the in vivo effects o f anti-leprosy drugs on the f u n c t i o n a l capacity of m a c r o p h a g e s a n d total counts o f l y m p h o c y t e s u b p o p u l a t i o n s , the present study was u n d e r t a k e n to d o c u m e n t their effects o n (i) rat peritoneal m a c r o p h a g e s with special reference to the p r o d u c t i o n o f reactive oxygen intermediates, Ia expression a n d u l t r a s t r u c t u r a l changes, a n d (ii) b l o o d l y m p h o c y t e s u b p o p u l a t i o n homeostasis.

EXPERIMENTAL PROCEDURES

Animal treatment. Male W i s t a r rats (weighing 2 0 0 g ) were divided into six groups namely, g r o u p I / I V (control), g r o u p I I / V (1 × M D T ) a n d g r o u p I I I / V I (10 x M D T ) . Anti-leprosy drugs were mixed with rat feed a n d a d m i n i s t e r e d for 1 m o n t h to rats o f g r o u p I I / V , a n d g r o u p I I I / V I as s h o w n in T a b l e 1 (Sahu, Saha, K a s h y a p & C h a k r a b a r t y , 1988). D a p s o n e , clofazimine a n d rifampicin were

p u r c h a s e d f r o m Wellcome, India: Surhid Geigy Chemicals a n d P h a r m a c e u t i c a l s , India a n d L u p i n L a b o r a t o r i e s , India, respectively. T a p water a n d rat feed ( H i n d u s t a n Lever Co., India) were provided ad libitum to all the rats.

Studies on peritoneal macrophages Eliciting agents. Bacillus C a l m e t t e - G u e r i n (BCG) suspension (0.1 ml) c o n t a i n i n g 0.4 x 106 viable o r g a n i s m s was injected i n t r a p e r i t o n e a l l y (i.p.) in all the rats of groups I, II a n d III at day 0. A f t e r 20 days 0.2 ml o f B C G (0.8 x 106 viable organisms) was reinjected (i.p.) a n d the a b o v e rats were sacrificed o n the 30th day. In a n o t h e r experiment 10 ml o f 6°7o s o d i u m caseinate in saline (Central Drug H o u s e (P) Ltd, Delhi, India) were given i.p., at day 0, to seven rats. They were sacrificed 3 days after injection (Zeller, Buys & Gudewics, 1984).

Isolation

of

rat

peritoneal

macrophages.

Peritoneal cells were collected f r o m n o r m a l or previously injected rats. Sterile H a n k ' s balanced salt solution (HBSS) (15 ml) (without p h e n o l red) was injected intraperitoneally into each rat. P e r i t o n e a l exudates c o n t a i n i n g cells were p o u r e d in plastic

Anti-leprosy Drugs and the Immune System Petri-dishes and kept at 37°C for an hour. Finally, non-adherent cells were removed by washing the plates three times with HBSS. The viability of cells was determined by the trypan blue exclusion technique which was always more than 95%. Superoxide anion (O2) estimation. Of released by resident non-elicited or BCG-elicited rat peritoneal macrophages was estimated by a previously described method based on the superoxide dismutase inhibitable reduction of ferricytochrome c (Sahu et al., 1991). Hydrogen peroxide estimation. Each dish containing 3 × 10~'resident (taken from normal rats) or BCG-elicited macrophages was covered with 0,5 ml phenol red solution. Thereafter, 0.5 ml of Staphylococcus aureus suspension (made in phenol red solution) containing 3 × 107 bacteria was added to each dish and the amount of H202 liberated was calculated by the method of Pick & Keisari (1980). Cell protein was estimated by employing the method of Lowry, Rosenbrough, Farr & Randall (1951). The results were expressed as nanomoles of H202 released/90 min/mg protein.

Immunostaining of Ia antigen on macrophages. Peritoneal cells were allowed to adhere on a microscopic glass slide at 37°C for 1 h, in a humid atmosphere, rinsed three times with HBSS, air dried and fixed with cold acetone. Immunostaining was performed by indirect immunofluorescence technique using anti-Ia monoclonal antibody (OX-4, Sera Lab, U.K.) and FITC conjugated sheep antimouse IgG (Sera Lab, U.K.) (Rambukkana, Saha, Sahu & Chopra, 1988). Macrophage counts were undertaken on 200 cells using a fluorescent microscope (Nikon Optiphot, Japan). Electron microscopy. Elicited macrophages, 1 × 104, obtained from rats of groups I and IlI were incubated with 10 x 106 BCG in HBSS for 1 h at 37°C. After 1 h cells were washed three times with HBSS. Thereafter, cells were fixed in situ with 2% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.3, and kept at 4°C for 4 h. They were then removed from the Petri-dishes with a rubber policeman, transferred to polypropylene microcentrifuge tubes, and pelleted. The glutaraldehydetreated pellet was then washed with 0.2 M sucrose buffer, (pH 7.2), post-fixed in 2% osmium tetroxide for 90 min, dehydrated in ethanol and embedded in EPON resin in a Beem capsule. The ultra thin sections were cut with a glass knife, stained with uranyl acetate and lead citrate and examined under a Jeol 100CS II electron microscope (Jeol, Japan).

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Studies on peripheral blood lymphocytes In this study no eliciting agents were used. Rats were divided into three groups, i.e. group IV (normal control), group V (1 × MDT) and group V1 (10 × MDT). The drug schedule used has been described in Table 1.

Isolation and immunophenotypic analysis of blood lymphocytes. After 1 month of MDT treatment animals were bled under light ether anaesthesia. The total and differential leucocyte counts were performed using the standard technique, and lymphocytes were separated using Ficoll (Lymphoprep, Nyegaard & Co., Norway; density 1.077 g/ml). Lymphocyte subsets were quantified by the indirect immunofluorescence technique using appropriate anti-rat monoclonal antibodies (Sera Lab, U.K.) as described earlier (Rambukkana et al., 1988). The monoclonal antibodies used were: W3/13 (Pan T), W3/25 (T-helper cells, macrophages), OX-8 (T-cytotoxic/suppressor cells, natural killer cells), OX-4 (Ia antigen, B-cells), as well as affinity purified FITC conjugated sheep anti-mouse IgG. Monoclonal antibody OX-4 binds to Ia antigen present on macrophages and B-cells. Since activated T-lymphocytes are rarely found in peripheral blood of rats, OX-4 was used as a marker for B-lymphocytes (Thompson, Bowen & Burton, 1987).

Statistical analysis The results were grouped and statistically evaluated by performing Student's t-test.

RESULTS

Determination of optimal dose of BCG for eliciting peritoneal macrophages Two sets of experiments were performed to study the H202 response in BCG-elicited macrophages. In the first experiment, 0.1 ml (0.4 × 106 organisms) and 0.2 ml (0.8 × 106 organisms) of BCG were injected intraperitoneally (i.p.) at the 0 and 20th day, respectively. A detectable amount of S. aureusinduced H202 release was recorded 5 days after the second injection (8.87 nmols/90 min/mg protein). This response persisted up to 34 days. In the second experiment, when the initial dose of BCG was increased from 0.4 × 1 0 6 to 1.2 × 106, a primary response of S. aureus-induced H202 production reached its maximum on the 18th day (8.62 nmols/

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Table 2. Effect of anti-leprosy drugs on 02 and HzO2 release and la (OX-4) expression of rat peritoneal macrophages

Type of peritoneal macrophages

O£ release (nmol/60 min/mg protein) Mean ± SEM (n) (range)

H202 release (nmol/90 min/mg protein) Mean ± SEM (n) (range)

A. Resident (non-elicited)

53.2 __. 6.7 (10) (28.5 - 84.4)

ND (8)

7.0 ± 1.4 (5) (2.9 - 10.9)

37.5 - 5.5 (8) (23.1 - 65.9)

6.88 ± 1.20 (6) (4.28 - 11.65)

47.2 ± 1.4 (8) (41.2 - 52.4)

II. 1 × MDT

48.3 -+ 13.3 (8) ( l 1. l - 124.1)

7.35 _+ 1.72 (6) (2.38 - 14.14)

44.1 -+ 2.7 (8) (33.1 - 53.4)

Ili. 10 × MDT

70.7 - 7.1 (8) (47.4- 109.0)

7.79 + 1.53 (6) (4.12- 14.91)

39.7 + 1.7 (8) (29.5-43.5)

NS NS