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

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I N VIVO EFFECTS OF ANTI-LEPROSY D R U G S ON THE RAT P E R I T O N E A L M A C R O P H A G E S A N D L Y M P H O C Y T E

S U B P O P U L A T I O N S

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 Heal th Organization as a singular effective regimen for leprosy management (WHO, 1988).

The interaction of anti-leprosy drugs with macrophages has been a focus of at tention ever since clofazimine (Conalty & Jackson, 1962) and rifampicin (Johnson, Hand, Francis, King- Thompson & 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). Of the three drugs used in MDT, clofazimine significantly enhances O5 product ion, in vitro, of rat resident peritoneal macrophages in a

dose-dependent manner (Sahu, Saha, Banerjee, Sehgal & Jagga, 1991). Clofazimine is also known to inhibit the proliferation of tymphocytes as well as the migrat ion 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 enhance- ment of prostaglandin synthesis (Anderson, 1985). Since the introduction o f r ifampicin 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).

tAuthor to whom correspondence should be addressed.

721

722 A. SAHU et al.

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 Group (months) MDT schedule

Group I t / IV One Normal diet.* (control)

Group II+/V One (1 x MDT treated)

Group III+/VI (10 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.

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.

* 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.

However , no effect on hum or a l or cellular immuni ty was observed in a control led double-b l ind s tudy ( H u m b e r et al., 1980).

In view of the paucity of i n fo rma t ion concern ing the in vivo effects of ant i - leprosy drugs on the funct ional capaci ty of macrophages and tota l counts of lymphocyte subpopula t ions , the present s tudy was unde r t aken to documen t their effects on (i) rat per i toneal macrophages with special reference to the p roduc t ion of reactive oxygen in termediates , Ia expression and u l t ras t ruc tura l changes , and (ii) b lood lymphocyte subpopu la t ion homeostas is .

EXPERIMENTAL PROCEDURES

Animal treatment. Male Wis tar rats (weighing 2 0 0 g ) were divided into six groups namely, g roup I / I V (control) , group I I / V (1 × MDT) and group I I I / V I (10 x MDT). Ant i - leprosy drugs were mixed with rat feed and adminis te red for 1 m o n t h to rats of g roup I I / V , and group I I I / V I as shown in Table 1 (Sahu, Saha, Kashyap & C hak r aba r t y , 1988). Dapsone , c lofazimine and r i fampic in were

purchased f rom Wellcome, India: Surhid Geigy Chemicals and Pharmaceut ica ls , India and Lupin Labora tor ies , India, respectively. Tap water and ra t feed (Hindus tan Lever Co. , India) were provided ad libitum to all the rats.

Studies on peritoneal macrophages

Eliciting agents. Bacillus Calmet te - Guer in (BCG) suspension (0.1 ml) conta in ing 0.4 x 106 viable organisms was injected in t raper i toneal ly (i.p.) in all the rats of groups I, II and III at day 0. Af te r 20 days 0.2 ml of BCG (0.8 x 106 viable organisms) was reinjected (i.p.) and the above rats were sacrificed on the 30th day. In ano the r exper iment 10 ml of 6°7o sodium caseinate in saline (Central Drug House (P) Ltd, Delhi, India) were given i.p., at day 0, to seven rats. They were sacrificed 3 days af ter in ject ion (Zeller, Buys & Gudewics, 1984).

Isolation o f rat peritoneal macrophages. Per i toneal cells were collected f rom normal or previously injected rats. Sterile H a n k ' s balanced salt solut ion (HBSS) (15 ml) (without phenol red) was injected in t raper i toneal ly into each rat . Per i tonea l exudates conta in ing cells were poured in plastic

Anti-leprosy Drugs and

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 tech- nique using anti-Ia monoclonal antibody (OX-4, Sera Lab, U.K.) and FITC conjugated sheep anti- mouse 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 micro- centrifuge tubes, and pelleted. The glutaraldehyde- treated 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).

the Immune System 723

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, macro- phages), 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-lympho- cytes (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. aureus- induced 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 × 10 6 to 1.2 × 106, a primary response of S. aureus-induced H202 production reached its maximum on the 18th day (8.62 nmols/

724 A. SAHU et al.

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

O£ release H202 release (nmol/60 min/mg (nmol/90 min/mg

Type of protein) protein) OX-4% peritoneal Mean ± SEM (n) Mean ± SEM (n) Mean ± SEM (n) macrophages (range) (range) (range)

A. Resident 53.2 __. 6.7 (10) ND (8) 7.0 ± 1.4 (5) (non-elicited) (28.5 - 84.4) (2.9 - 10.9)

B . BCG-elicited* I. Control

II. 1 × MDT

Ili. 10 × MDT

37.5 - 5.5 (8) 6.88 ± 1.20 (6) 47.2 ± 1.4 (8) (23.1 - 65.9) (4.28 - 11.65) (41.2 - 52.4)

48.3 -+ 13.3 (8) 7.35 _+ 1.72 (6) 44.1 -+ 2.7 (8) ( l 1. l - 124.1) (2.38 - 14.14) (33.1 - 53.4)

70.7 - 7.1 (8) 7.79 + 1.53 (6) 39.7 + 1.7 (8) (47.4- 109.0) (4.12- 14.91) (29.5-43.5)

Statistical evaluation P value B I vs A NS <0.001 <0.001 B II vsB I NS NS NS B I I I vs B I <0.01 NS <0.01

* Two BCG injections were given on day 0 and day 20. Details are described in Table 1. NS : not significant; ND = not detected. A mega dose of MDT treatment of rats showed a remarkable increase of 02- production by BCG-elicited rat peritoneal macrophages. Furthermore, there was a significant decrease in Ia expression on macrophage isolated from l0 × MDT treated rats.

90 m i n / m g prote in) which subsided on the 20th day. A second inject ion of 0.8 x 106 bacter ia resulted in a m a r k e d increase in S. aureus-induced H202 release, which reached its m a x i m u m at day 30 (48.10 n m o l s / 90 m i n / m g protein) . Thus , this initial high dose of BCG should not be used in the present exper iments as it might mask the effect of drugs on the p roduc t ion of reactive oxygen intermediates . For the present exper iments we have used the initial low dose of BCG (0.4 × 106 organisms) . Fu r the rmore , sodium caseinate failed to induce a detectable a m o u n t of H202 release in our rats.

Reactive oxygen intermediates production and la expression by two different populations o f macro- phages vis-/l-vis modulation by anti-leprosy drugs

Table 2 shows tha t per i toneal macrophages f rom M D T unt rea ted rats (resident non-el ici ted macro- phages) p roduced a substant ia l a m o u n t of 02- af ter chal lenging with S. aureus, however, HzOz released could not be detected using the s t andard phenol red assay (the phenol red me thod is unab le to detect less than 1 nmole H202 per ml). The majori ty of these cells were also devoid of Ia antigens, and only 7.0 _+ 1.4°70

of these cells expressed Ia ant igen on their surface. On the o ther hand , in compar i son with resident macrophages , BCG-elici ted macrophages f rom M D T un t rea ted rats p roduced a significant a m o u n t of H202 (6.88 --- 1.20 n m o l e / 9 0 m i n / m g protein) af ter chal lenging with S. aureus, and many cells showed Ia expression on their surface (47.2 _ 1.4°70). Surprisingly, despite the increase of H202 p roduc t ion their Oz releasing capaci ty (37.5 + 5.5 n m o l e / 60 m i n / m g protein) was not increased in compar i son with resident macrophages .

Fu r the rmore , l × M D T t rea tmen t (group II) had no signif icant effect on either S. aureus-induced Oz or H202 p roduc t ion or Ia expression of BCG-elici ted macrophages in compar i son with tha t of BCG controls . In contras t , the higher concen t ra t ion of drugs (10 × MDT, group III) was able to enhance significantly the a m o u n t of S. aureus-induced 02 produc t ion of elicited macrophages (P<0.01, Table 2), a l though it had no significant effect on H202 release by these macrophages . Macrophages isolated f rom l0 x M D T treated rats also showed a signif icant decrease in the M H C class II ant igen expression on their surface (P<0.01, Table 2).

Anti-leprosy Drugs and the Immune System 725

W

Fig. 1. Transmission electron micrographs of peritoneal macrophages isolated from a rat treated with anti-leprosy drugs. The macrophages were stimulated with BCG in vitro. (A) Macrophages obtained from a MDT untreated rat (control, group I) showing scanty cytoplasm with smooth external contour; × 4700. (B) Macrophages obtained from anti-leprosy drug treated rat (10 x MDT, group III) showing many filipodia (F). Bacteria are seen attached to or nearby the cell

membrane. The cytoplasm contains lysosomal granules (L); x 4700.

Anti-leprosy Drugs and

Transmission electron microscopy o f peritoneal macrophages isolated form M D T treated rats

BCG-elicited peritoneal macrophages, obtained from the rats treated with anti-leprosy drugs (10 x MDT, group III) as well as MDT untreated rats (group I) were challenged with BCG in vitro. Electron microscopy of the drug treated macro- phages showed an increase in the cell size with several filipodia on their surface (Fig. 1 B) as compared with the control macrophages which had rounded contours and scanty cytoplasm with very few organdies (Fig. 1A). The macrophages from group III rats also showed lysosomal granules. A number of mycobacteria were attached to the surface of the cell (Fig. 1B).

Alteration o f blood lymphocyte subsets o f rats treated with anti-leprosy drugs

Figure 2 shows the absolute number of peripheral blood lymphocytes and their subsets in controls as well as anti-leprosy drug treated rats. There was no significant difference in the total number of lymphocytes and OX-4 positive B-cells in either group of anti-leprosy drugs treated rats (groups V and VI) as compared to the controls (group IV rats). The mean absolute counts of total T-cells (W3/13 +), T-helper cells (W3/25 +) and T-suppressor cells (OX- 8*) remained unchanged in the 1 x MDT treated group V rats as compared with the controls (group IV rats). However, there was a significant decrease in the mean absolute counts of W3/25 positive cells (P<0.05) and a significant increase in OX-8 positive cells (P<0.05) in the high dose (10 x MDT) treated group VI rats. The mean W3/25 : OX-8 ratio was significantly lower in mega dose treated rats as compared with the controls (P<0.001, Fig. 3).

DISCUSSION

The phagocytosis-induced respiratory burst includes a variety of toxic oxygen metabolites, namely 02, H202, "OH and singlet oxygen (Johnston, 1981). Our recent report (Sahu et al., 1991) and other studies (Wadee, Anderson & Rabson, 1988; Zeis, Savage, O'Sullivan & Anderson, 1990) have demonstrated that anti-leprosy drugs, in particular clofazimine, augment the respiratory burst activity of phagocytes in vitro. On the other hand, the 02- liberated by monocytes of leprosy patients

the Immune System 727

containing M. leprae has been shown to be impaired by superoxide dismutase (Niwa et al., 1984) and the PGL-1 antigen of M. leprae (Neill & Klebanoff, 1988).

In the present study it has been shown that in comparison with rat resident macrophages, rat macrophages immunologically "activated" by BCG infection express more Ia antigen on their surface and produce more HzO2 in response to S. aureus challenge (P<0.001, Table 2). However, it is imperative to point out that they did not show any increase in 02 production (Table 2), a finding similar to that reported by McCabe & Mullins (1990). A similar enhancement in H202 production (Nathan & Root, 1977) and Ia expression (Steinman, 1988) of BCG-elicited mouse peritoneal macrophages has been documented previously.

In vivo treatment of rats (groups II and IIl) with MDT showed that only when group III rats were given mega dose of anti-leprosy drugs (10 x MDT) the number of Ia (OX-4) positive macrophages decreased (P<0.01) (Table 2). These results signified that MDT treatment possibly decreased T-helper cell function leading to the decrease in cytokine levels (IL-4 and y-IFN), which are known to be important for Ia expression on the macrophages (Steinman, 1988). The supporting evidence was further afforded by our observation on the blood lymphocyte subpopulations of the rats. 10 x MDT treatment of group VI rats resulted in a significant decrease in the number of T-helper (W3/25 +) cells (P<0.05) and an increase in the counts of T-suppressor (OX-8 +) cells (P<0.05, Fig. 2) which in turn decreased the W3/ 25:OX-8 ratio (Fig. 3). However, in contast to these results there was a remarkable increase in 02 production by the anti-leprosy drug treated group III rat peritoneal macrophages although, there was no significant increase in H202 production (Table 2). This might be due to possible conversion of H202 into hydroxyl radicals (Niwa et al., 1984). Our results show that inspite of a significant decrease in T-helper (W3/25 +) cells, there was a remarkable increase in the production of Oz by macrophages obtained from the high dose treated rats (10 x MDT). These macrophages also showed development of many filipodia (Fig. 1B), showing thereby that "activation" achieved with the drug is without the help of T-helper cells. Additional support is provided by our previous in vitro experiments, where the resident rat peritoneal macrophages incubated with clofazimine (30 ~g) and stimulated with M. leprae showed a significant increase in the production of superoxide anion. The clofazimine-dependent enhancement of O; production was mediated via

728 A. SAHU et al.

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Fig. 2. A comparative study of blood lymphocyte subpopulations in normal control (group IV) and anti-leprosy drug treated (groups V and VI) rats. Group VI (10 × MDT) rats showed a significant fall in the average number of T-helper (W3/25 ~ ) cells and an increase in the mean count of T-suppressor (OX-8 ~ ) cells. Each point represents the cell count in one

animal.

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Fig. 3. Relative ratio of peripheral blood T-helper and suppressor cells (W3/25 : OX-8) in normal control (group 1V) and anti-leprosy drug treated (groups V and VI) rats. A significant fall in the ratio was observed in group VI (10 x MDT) rats, thereby indicating an altered blood T-cell homeostasis. Each point represents the ratio of one

animal.

a rachidonic acid release (Anderson , Beyers, Savage & Nel, 1988; Sahu e t a l . , 1991). Fur the rmore , when these macrophages were studied under the electron microscope, their cytoplasm showed a dilated endoplasmic ret iculum and a n u m b e r of lysosomes, while the surface m e m b r a n e of the macrophages showed numerous fine f i l ipodia project ing f rom it (unpubl i shed data) . These experiments fur ther suggest tha t " a c t i v a t i o n " can be achieved via a lymphokine- independen t mechan i sm ( Johns ton , 1981).

In conclusion, it may be highl ighted that (i) the enhanced 02 p roduc t ion by macrophages f rom rats t reated with 10 × M D T was possibly due to direct effects of the drugs, in par t icular c lofazimine on macrophages , and was not media ted by T-helper cell funct ion and (ii) admin i s t ra t ion of a mega dose of M D T (10 x MDT) decreased T-helper cells and increased T-suppressor cells, which perhaps in tu rn decreased the la expression on macrophages . It is now well documen ted tha t the high dose of c lofazimine a long with r i fampicin and dapsone is capable of alleviating type II (ENL) react ion in leprosy. Thus , we are p rompted to postula te tha t the a fo re -men t ioned mechan i sm of macrophage

Anti-leprosy Drugs and the Immune System 729

"ac t iva t ion" may help to f ragment the M. leprae in Acknowledgements - - Authors are thankful to the Council the macrophages by O2 product ion and vis-~t-vis it of Scientific and Industrial Research, New Delhi for the may extinguish the aggravated cell mediated financial assistance to the senior author (AS). We are also immunologic struggle against M. leprae during ENL thankful to Mr Arun Jain for his technical assistance.

reaction.

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