Intracellular growth of an obligatory parasite Mycobacterium leprae. Host bacterial interactions

BIOCHIMIE, 1974, 56, 231-237.

Intracellular growth of an obligatory parasite Mycobacterium leprae. Host bacterial interactions. G. P. TALWAR, A. D. KRISHNAN, P. JHA and V. MF.HRA.

ICMR-WHO Research and Training Centre in Immunology. Department of Biochemistry, All-India Institute of Medical Sciences, N e w Delhi-110016, India.

In p a y i n g h o m a g e to M. Macheb.oeuf, w e have c o n s i d e r e d it a p p r o p r i a t e to discuss a p i e c e of w o r k , xvhieh r e c a l l s i n d i r e c t l y his early i n t e r e s t a n d c o n t r i b u t i o n s in the field of M y c o b a c t e r i a l chemistry. L e p r o s y is an i n t r i g u i n g disease. Subjects suffer i n g f r o m this m a l a d y p r e s e n t a w i d e s p e c t r u m of the f o r m in w h i c h the disease is manifest. In t u b e r e u l o i d l e p r o s y patients, the skin lesions are r e s t r i c t e d to l o c a l i z e d patches. T h e r e a r e few or no b a c t e r i a p r e s e n t in the lesions. In l e p r o m a t o u s l e p r o s y , on the o t h e r h a n d , the lesions are w-idely d i s s e m i n a t e d w i t h v e r y high b a c t e r i a l load. In b e t w e e n these t w o p o l a r forms lies a c o n t i n u o u s s p e c t r u m w i t h v a r i a b l e degree of host r e s i s t a n c e to infection. A c h a r a c t e r i s t i c feature of the t u b e r e u l o i d l e p r o s y is t h e p r e s e n c e in a b u n d a n c e of l y m p h o c y t e s a r o u n d giant cells, w h e r e a s the ¢ small cell >> infiltration is a b s e n t in l e p r o m a t o u s l e p r o s y lesions (fig. 1). The reciprocal relationship between lymphocyte i n f i l t r a t i o n a n d the n u m b e r of b a c t e r i a in lesions suggests that l y m p h o c y t e s m a y have an i m p o r t a n t role in b a c t e r i a l p r o l i f e r a t i o n . Var i a b l e degree of b a c t e r a e m i a does not seem to d e p e n d on the relative v i r u l e n c e of the i n f e c t i n g o r g a n i s m , as no d e t e c t a b l e differences have been found in the i n f e c t i v i t y of M y e o b a c t e r i a d e r i v e d from v a r i o u s p a r t s of the w o r l d in e x p e r i m e n t a l test mice (Rees 1973).

Host h n m u n e Responses. (a) Humoral immunity : Serum i m m u n o g l o b u lins a r e in.variably r a i s e d in l e p r o m a t o u s l e p r o s y p a t i e n t s (table I). The c h a n g e is p a r t i c u l a r l y n o t i c e a b l e in I g G ; u s u a l l y IgA or IgM are also i n c r e a s e d . High i m m u n o g l o b u l i n s in spite of low n u t r i t i o n a l status, could be due to the well recognized a d j u v a n t a c t i o n of M y c o b a c t e r i a p r e s e n t in large a m o u n t s in these patients. It could also be due to the c o n t i n u o u s e x p o s u r e of the p a t i e n t s to a v a r i e t y of antigens because of the u n h y g i e n i c

c o n d i t i o n s in w h i c h most l e p r o s y p a t i e n t s live. The p r e s e n c e of c r y o g l o b u l i n s a n d i m m u n e complexes h a s also been r e p o r t e d in p a t i e n t s u n d e r c h e m o t h e r a p y (Wager, 1969; T u r k a n d Waters, 1969). L e p r o m a t o u s l e p r o s y p a t i e n t s a r e able to elicit n o r m a l r e s p o n s e on s e c o n d a r y i m m u n i z a tion w i t h v a c c i n e s such as TAB (fig. 2). (b) Celt mediated Immune Response (CMI) : A d e p r e s s i o n of CMI in l e p r o m a t o u s l e p r o s y (LL) p a t i e n t s is i n d i c a t e d b y a n u m b e r of observations : 1) T h e y fail to s h o w d e l a y e d h y p e r s e n s i t i v i t y r e a c t i o n to l e p r o m i n . 2) The blast t r a n s f o r m a t i o n of p e r i p h e r a l leuk o e y t e s w i t h p h y t o h a e m a g g l u t i n i n (PHA) is d i m i n i s h e d (table II). 3) T r a n s f o r m a t i o n w i t h specific antigens e.g. M. leprae is p o o r (table III). 4) L y m p h o e y t e s f r o m L L p a t i e n t s w h e n cultur e d w i t h M. leprae are deficient in g e n e r a t i o n of b i o l o g i c a l l y active p o l y p e p t i d e s , influencing the migration and aggregation of m a e r o p h a g e s ( T a l w a r et al., 1972 ; Katz et al., 1971 ; Myrvang, 1972). The p r o p o r t i o n of ¢ B ~ cells c a r r y i n g i m m u n o globulin d e t e r m i n a n t s on the surface, that a r e p r e s u m a b l y d e s t i n e d to b e c o m e a n t i b o d y f o r m i n g cells, is i n c r e a s e d in l y m p h nodes of l e p r o m a t o u s l e p r o s y p a t i e n t s (table IV). On the o t h e r h a n d , a d e c r e a s e in T cells p o p u l a t i n g p a r a e o r t i e a l a r e a s of l y m p h nodes has been r e p o r t e d (Turk a n d W a t e r s , 1968). B cells a r e also i n c r e a s e d in p e r i p h e r a l c i r c u l a t i o n ( D w y e r et al., 1973; GajlP e e z a l s k a et al., 1973 ; T a l w a r et al., 1973). The n o r m a l r a t i o of B a n d T cells is t h e r e f o r e a l t e r e d b o t h in c i r c u l a t i o n as w e l l as in s e c o n d a r y l y m p h o i d organs. The shift could be due to a n y one or c o m b i n a t i o n of the f o l l o w i n g factors : 1) E n h a n c e d p r o d u c t i o n of B cells

G. P. T a h v a r , A. D. K r i s h n a n , P. J h a a n d V. M e h r a .

232

2) D i m i n i s h e d generation or maturation of T cells 3) C h a n g e s i n s u r f a c e c h a r a c t e r i s t i c s of T and or B cells

influencing their << l l o m i n g >> a n d circulation patterns. Homing characteristics o f .5lCr t a g g e d lymphoeytes a r e a l t e r e d i n m a n y , b u t n o t all, cases of untreated lepromatous leprosy (Talwar

Fro. l a . - - Lepromatous leprosy. The dermis has sheets of foamy macrophayes Jwith indistinct cell outlines. L y m p h o c y t e s a r e f e w a n d n o g i a n t cells a r e

Tuberculoid leprosy shoqwing granulomaFIG. l e . tous infiltration in the dermis of the akin and the presence of abundant lymphocytes. (H. a n d E. s t a i n

seen. H. a n d E. S t a i n (X 180).

× 180).

FIG. l b . - - Lepromatous leprosy. Higher magnification sho,ws the character of foamy macrophages (leprae cells), ,with abundance of inlracellular M. l e p r a e . S c a r c i t y of l y m p h o c y t e s is to be n o t e d . ( Z i e h l N e e l s e n s t a i n × 1800).

FI6. ld. - - Tubereuloid leprosy, sho~wing details of epilheloid cells, giant cell and lymphocytes which form the cellular infiltrate in the dermis. (H. a n d E. s t a i n × 700).

TABLE I.

l m m u n o f l o b u l i n s in sera of leprosy patients. Category of patients

No. of

lgG

lgA

IgM

1 4 . 3 5 _____0 . 0 5 P ~ 0.001

2 . 7 7 ______0 . 0 5 P <~ o.001

1.23 + 0.03 N.S.

9 . 7 --t- 0 . 0 4 P <. 0.001

1 . 0 7 ~___0 . 1 0

1.2 ~ 0.10 N.S.

7 . 9 5 -I- 0 . 1 1

1.71 +

eases

studied 1.

Lepromatous l e p r o s y (LL)

2.

Tubereuloid leprosy

3.

Normal

30

10

( D a t a f r o m J h a , T a l w a r a n d B h u t a n i , 1971).

BIOCHIMIE, 1974, 56, n ° 2.

M.S. 0.16

1 . 0 5 "+" 0 . 0 6

Intracellular g r o w t h of M y c o b a c t e r i u m l e p r a e . et al., 1972). In o t h e r studies i n d i r e c t e v i d e n c e has been o b t a i n e d for the p r e s e n c e of l y m p h o c y t o t o x i c factors in sera of LL patients (Balak r i s h n a n , 1973).

e.g. c o r d f a c t o r is b e l i e v e d to cause w a s t a g e disease. S i m i l a r l y g r a n u l o m a s can be i n d u c e d in h e a l t h y animals by m y c o b a c t e r i a l extracts. It may, h o w e v e r , be stated that the p r e c i s e reason for d e p r e s s i o n of CMI in l e p r o m a t o u s l e p r o s y patients is not k n o w n . Does it p r e e x i s t a n d r e n d e r a subject exposed to this i n f e c t i o n to develop the disease or is it a c o n s e q u e n c e of the b a c t e r i a l load ? T h e r e is p e r h a p s both a specific and a p p a r e n t l y a non-specific type of d e p r e s s i o n of the T cell function. T h e r e s p o n s i v e n e s s to PHA is a m e l i o r a t e d in LL patients after t r e a t m e n t of the subjects w i t h Dapsone, a d r u g that r e d u c e s the b a c t e r i a l load (table II). Thus n o n r e s p o n s i veness of p e r i p h e r a l l e u k o c y t e s to p h y t o m i t o g e n s m a y be r e l a t e d to the massive p r e s e n c e of bacteria. It m a y also reflect a d i m i n u t i o n in the n u m b e r of PHA r e a c t i n g t h y m u s d e r i v e d cells in circulation.

3:>0~ 280 240

7 :'

200

l!, hJ 1 6 0 if' tI- I2C

~l

4J .-

;:q

',:1 ::1

iil :;d

0 FIG. 2.

TN -

-

AH

233

BH

Titers of antibodies against Salmonella

antigens. N : normal subjects; L : lepromatous le-

prosy patients ; T : Tubercutoid leprosy patient ; TH :

S. typhi cH>> antigen; A H : S. paratyphi A <( H>> antigen; BH : S. paratyphi B <> antigen. (Data from Jha et al. 1971).

It has been suggested that patients w i t h lepromatous l e p r o s y have a specific t o l e r a n c e or inability to r e s p o n d to M. leprae antigens (Godal, 1971). Is this failure genetic or a c q u i r e d ? This is a question that r e m a i n s u n a n s w e r e d . C h a k r a v a r t i and Vogel (1972) h a v e p u b l i s h e d r e c e n t l y t h e i r studies on about 100 h o m o z y g o u s and h e t e r o z y g o u s t w i n s in a l e p r o s y e n d e m i c

TABLE II.

Mitotic response of peripheral leucocytes from different categories of leprosy patients to PHA. Treatment

Plasma (serum) used

Percent relative response(*) (PRR) Mean ~ SEM

Lepromatous leprosy

untreated

Lepromatous leprosy

untreated

Lepromatous leprosy

treated

Tuberculoid leprosy

untreated

Tuberculoid leprosy

treated

Aut ologous plasma Standard AB serum Autologous plasma Autologous plasma Autologous plasma Autol ogous plasmas

46.0 -t- 7.4 (12) p ~ 0,001 46.6 -P 4.6 (12) p ~ 0.001 130.3 -P 7.8 (17) p ~ 0.001 87.1 ~__ 13.0(6) p ~ O.O5 103.4 + 13.1 (6) p > O.05 113.6 -+- 27.0 (6) p ~ 0.05

Category

Contacts

CPM in DNA per 2 X 106 cells of leprosy patients or contact cultured in autologous plasma (or standard AB serum) CPM in DNA per 2 X 106 cells of normal controls cultured in autologous plasma (or standard AB serum) (Data from Mehra, Talwar, Balakrishnan and t~hutani, 1972).

(*) PRR :

It is not i n c o n c e i v a b l e that m y c o b a c t e r i a l products m a y h a v e an effect on some of these changes

BIOCHIMIE, 1974, 56, n ° 2.

× lO0

area. The i n c i d e n c e of l e p r o s y a m o n g s t both siblings was h i g h e r in i d e n t i c a l than ill n o n - i d e n -

234

G. P . T a l w a r , A. D. K r i s h n a n , P. J h a a n d V. M e h r a . TABLB I I I .

Blast transformation of peripheral leukocytes [rom lepromatous and tuberculoid leprosy patients with M. l e p r a e .

Subject

ClinicaI status

BS

LL

Histopathology

LL

Bacteriological and morphological indices

++++++

Mitsuda reaction

Translormation (')

- -

ve

1.45

- -

ve

J

- -

ve

1.61

75 p. cent

KB

LL

LL

++++++

.34

60 p. cent

LL

RB

LL

++

5 p. cent TT TT TT

MS

KP DS

(*) E x p r e s s e d as

TT TT TT

--

---

-~- ve 7t- ve -~- ve

4.53 2.41 2.76

CPM in DNA p e r 1 X 106 cells c u l t u r e d in t h e p r e s e n c e of M. leprae CPM in DNA p e r 1 X 106 cells c u l t u r e d w i t h o u t M. leprae.

Fro. 3a.

Fro. 3c. - - Z i e h l - N e e l s e n slain of a cultivated macrophage infected Iwith M. l e p r a e on day 25 ('2500 ×). Note t h e e l o n g a t i o n of m y c o b a c t e r i a l rods.

FIo. 3 (a) a n d (b). - - Radioautographs of monocgle derived macrophage infected 'with M. l e p r a e (a) and noninfected (b) after labelling with 3H-methyl thgmidine (2500 X). The m a c r o p h a g e s in b o t h sets w e r e c u l t i v a t e d f r o m t h e s a m e p a t i e n t a n d the t i m e of ex poSure to a H - t h y m l d i n e w a s e q u a l .

BIOCHIMIE, 1974, 56, n ° 2.

Fro. 3d. - - Electron microscopic autoradiograph of an infected cell after 3H-thymidine palse (25000 × ) . The radioactioity grains were present uniquely in the bacterial rods.

Intracellular growth of

Mycobacterium leprae.

235

TABLE IV.

Lymphocytes bearing immunoglobulin determinants in lymph nodes from normal and leprosy patients.

A.

1.

2.

3.

4.

5.

6.

N'. of total cells counted

Percentage o[ fluorescent cells

Mesenteric

152

20.3

Mesenteric

188

31.4

Traeheobronehlal

167

17.9

Mesenteric

145

21.4

Mesenteric

214

22.9

Mesenteric

248

26.6

Mean

23.4

Supratrochlear

118

52.5

Inguinal

180

31.0

Supra! rochlear

161

41.6

Supratrochlear

251

33.4

Inguinal

244

61.0

Supratroehlear

201

34.5

Lymph node examined

Subject - Age - Sex

Normal lymph nodes

K.P. 46 yrs M B.L. 54 yrs M S.K. 12 yrs M S.L. 42 yrs M R.D. 38 yrs F V.K. 2 yrs M

B.

Leprosy patients [

1.

2.

3.

V.D. 45 yrs M S.P. 40 yrs M S.D. 64 yrs M

4.

R.B. 47 yrs M

5.

6.

B.L. 35 yrs M Chand 40 yrs M

Mean

42.3

(Data f r o m Varma, Balakrishnan, Vasudevan and Talwar, 1971).

t i c a l t w i n s s u g g e s t i n g t h e p o s s i b i l i t y of g e n e t i c s u s c e p t i b i l i t y as a p r e d i s p o s i n g f a c t o r to t h e disease. No definite c o n c l u s i o n can, h o w e v e r , be d r a w n as t h e t y p e o f l e p r o s y a m o n g s t s o m e i d e n tical t w i n s w a s of a n o n e o i n c i d e n t type.

BIOCHIMIE, 1974, 56, n ° 2.

Impact of defective T-lymphocyte function on bacterial growth. of

A k e y q u e s t i o n t h a t a r i s e s is a b o u t t h e n a t u r e the cellular mechanisms that prevent the

G. P. Tahvar, A. D. Krishnan, P. Jha and V. Mehra.

236

growth of bacteria in t u b e r c u l o i d leprosy patients. It is p r e s u m a b l y t h e i r i m p a i r m e n t that p e r m i t s extensive bacterial p r o l i f e r a t i o n in lepromato,us leprosy cases. To s t u d y these, we have developed a simple method to cultivate m a e r o p h a g e s from p e r i p h e r a l blood monoeytes ( K r i s h n a n a n d T a l w a r , 1973). About 15 Leighton tube cultures are o b t a i n e d from 20 ml of blood. The cultures can be m a i n -

phocytes on e n c o u n t e r w i t h the bacteria. Sensitized l y m p h o c y t e s may a l t e r n a t i v e l y have a direct <> action on the bacteria. F u r t h e r research should give some a n s w e r s to these questions.

Concluding Comments. Patients suffering from leprosy p r e s e n t a wide s p e c t r u m of the disease w i t h variable degree of reistance to the infection. There is no depression

TABLE V.

Mycobacterial multiplication in cultivated macrophoges derived [rom peripheral blood monocyles of leprosy patients. CPM aH-thymidine incorporated per 5 × t0 ~ phagocytic cells N*"

]Clinical status

Macrophages + lymphocyies + M. leprae

Macrophages + M. leprae

36.458 53 929 52.354 6.332 32 381

45.628 59.596 83.476 54.969 78.447 26.260

i

1. 2. 3.

LI,

: I !

4.

TT

I

6.

TT

5.

LL LL "FT

!

t a i n e d for p r o l o n g e d periods (up to 2 m o n t h s or more) in s e r u m rich media. The cultures are infected in vitro with freshly p r e p a r e d M. leprae from h u m a n biopsies. The growth of phagocytosed bacteria is assessed by a highly q u a n t i t a t i v e method e m p l o y i n g the i n c o r p o r a t i o n of 3H-thym i d i n e into DNA. Macrophages b e i n g t e r m i n a l cells, do not divide in vitro. The r a d i o a c t i v e precursor measures therefore selectively, the DNA synthesis in the phagocytosed bacteria w i t h o u t i n t e r f e r e n c e of the host cell. This c o n c l u s i o n is s u p p o r t e d by data i n fig. 3. An interesting, though p r e l i m i n a r y finding on a few cases studied so fa r , is that the p r o l i f e r a t i o n of b a c t e r i a is better in m a c r o p h a g e s cultivated a n d infected in the absence of l y m p h o c y t e s (table V). The presence of l y m p h o e y t e s has a m o d u l a t i n g effect on i n t r a c e l l u l a r growth of M. leprae. F u r t h e r m o r e , there are i n d i c a t i o n s that l y m p h o c y t e s from t u b e r c u l o i d leprosy cases are more competent than those from l e p r o m a t o u s l e p r o s y patients, i n r e d u c i n g the p r o l i f e r a t i o n of the bacteria (table V). The m e c h a n i s m by w h i c h l y m p h o c y t e s exercise this effect is not k n o w n . It m a y be through the m u l t i t u d e of biologically active p o l y p e p t i d e s (lymphokines) elaborated by the sensitized lym-

BIOCHIMIE, 1974, 56, n ° 2.

of h u m o r a l i m m u n e responses. Serum i m n m n o g l o b u l i n s are i n v a r i a b l y raised in l e p r o m a l o u s leprosy patients. The p r o p o r t i o n of <> lyinphocytes as c o m p a r e d to <> cells is elevated in l y m p h nodes and in circulation. Lepromatous leprosy (LL) patients can elicit n o r m a l response (antibody titres) on s e c o n d a r y i m m u n i z a t i o n with vaccines such as TAB. Cell mediated i m m u n e responses are h o w e v e r i m p a i r e d in LL cases. The blast t r a n s f o r m a t i o n of p e r i p h e r a l leukocytes w i t h PHA and w i t h M. leprae antigens is depressed. There is a d i m i n i s h e d f o r m a t i o n of biologically active p o l y p e p t i d e s i n f l u e n c i n g the m i g r a t i o n and aggregation of macrophages. Bacterial gro~,th has been m e a s u r e d in vitro i n m a c r o p h a g e s derived from p e r i p h e r a l blood of t u b e r e u l o i d a n d lepromatous leprosy patients. M. leprae proliferate better in cultures devoid of lymphocytes. In tuberculoid leprosy cases, the p r e s e n c e of l y m p h o cytes reduces significantly the i n t r a c e l h d a r growth of t h e bacteria.

Aekno~wledgment. This work has received support from the Indiah Council of Medical Research and The World Health Organization (Leprosy and Immunology Divisions), and a PL 480 grant (No. N00014-70-C-0179) of the Offlee of Naval Research.

Intracellular growth of Mycobacterium leprae. REFERENCES. 1. Balakrishnan, K. (1973) In : Proceedings of the s y m p o s i u m on I m m u n o l o g y of leprosy held by I n d i a n Council of Medical Research, New Delhi. (In Press). 2. Chakravarti, M. R. ~ Vogel, F. (1972) A t w i n study on leprosy. Topics in H u m a n Genetics 1, George Thieme. Publ. Stuttgart. 3. Dwyer, J. M., Bullock, W. D. ~ Fields, J. P. (1973) N~w Eng. J. Med., 288, 1036. 4. Gajl-Peczalska, K. J., Lira, S. D., Jackson, R. R. Good, R. A. (1973) New Eng. J. Med., 288, 1033. 5. Godal, T., Myklestad, B., Samuel, D. R. ~ Mvrvang, B. (1971) Clin. exp. i m m u n o l , 9, 821. ~ " 6. Jha, P., Balakrishanan, K., Talwar, G. P..~ Bhutani, L. K. (1971) Int. J. Leprosg~, 39, 14. 7. Ktz, S. I., Bebetz, B. H. ~ Zaias, N. (1971) Arch. Derm., 103, 358. 8. Krishnan, A. D. & Talwar, G. P. (1974) Ind. J. Med. Res. (accepted for publication). 9. Mehra, V. L., TaIwar, G. P., Balakrishnan, K. Bhntani, L. K. (1972) Clin. exp. Immunol., 12, 205.

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