- Email: [email protected]
Modulation of human lepromatous monocyte-macrophage functions in vitro by Tuftsin
Int. J. Immunopharmac., Vol. 12, No. 8, pp. 847-858, 1990. Printed in Great Britain.
0192-0561/90 $3.00 + .00 Pergamon Press pie. International Society for lmmunopharmacology.
MODULATION OF H U M A N LEPROMATOUS M O N O C Y T E - MACROPHAGE FUNCTIONS I N VITRO BY TUFTSIN RAVl R. IYER, *t H. K. PRASAD,* L. K. BHUTANI § a n d D. N. RAO *H *Department of Biochemistry, *Department of Biotechnology and ~Department of Dermatovenereology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110 029, India
(Received 14 July 1989 and in final form 18 April 1990)
-Human peripheral blood monocytes/macrophages derived from normal donors, patients of tuberculoid leprosy (BT/TT) and lepromatous leprosy (BL/LL) were assayed for stimulated phagocytic responses to the potent macrophage stimulator "Tuftsin" (NH2 - Thr - Lys - Pro - Arg - OH) after varying periods (6 h to 14 days) of culture in vitro. The assays consisted of visual scoring of ingested Mycobacterium leprae and radiometric measurement of ingested ~4C-acetate labelled Staphylococcus aureus and Myeobacterium tuberculosis (H37Ra). While normal and BT/TT macrophages showed a progressively increasing ability for tuftsin-stimulated phagocytosis with increasing age of culture in vitro, BL/LL macrophages showed the opposite response so that 14-day cultures were refractory to a stimulatory dose of up to 7.0 ~M (10 to 20 times the optimal dose for normal and BT/TT macrophages). The 14-day BL/LL macrophage cultures were, however, responsive to 35 ~M tuftsin (100 times the optimal dose for normal macrophages). Analysis of the dose - response curves also indicates that BT/TT cultures despite exhibiting an apparent similarity to normal macrophages demonstrate a rightward shift for a maximal stimulated phagocytosis. Finally SEM photo-micrographs of 14-day macrophage cultures of the three groups revealed that while normal and BT/TT cultures demonstrated an increase in membrane ruffling and filopodia on stimulation with 0.8 ~M tuftsin, BL/LL cultures exhibited none of the features associated with stimulation. From the above findings, we conclude that lepromatous macrophages may display an aberrant differentiation profile leading to a terminal state of unresponsiveness and that the defect may possibly lie at the level of tuftsin receptor expression or transmembrane signal transduction.
Abstract
Leprosy is a p o l y m o r p h i c disease caused by Mycobacterium leprae. T h e bacillus has a predeliction to stay within cells o f the m o n o c y t e - m a c r o p h a g e (M+) series a n d in the S c h w a n n cells o f peripheral nerves. This results in a g r a n u l o m a w h i c h in the l e p r o m a t o u s leprosy (LL), shows intracellular bacilli within M+. A l t h o u g h the exact causes o f the defective cellm e d i a t e d i m m u n i t y in leprosy are still unclear (Godal, 1978; K a p l a n & C o h n , 1984), the failure o f the M+ to kill or inhibit intracellular g r o w t h o f M. leprae is a c o n s p i c u o u s characteristic of the l e p r o m a t o u s f o r m o f the disease. It is also i m p o r t a n t to k n o w w h e t h e r the M+ in LL patients is a m e n a b l e to i m m u n o p h a r m a c o l o g i c a l m a n i p u l a t i o n a n d if so, w h a t are the kinetics a n d profile o f such stimulated responses, l m m u n o m a n i p u l a t i o n s o f the phagocytic
system would help in the clinical m a n a g e m e n t o f infectious diseases in view o f our increased k n o w ledge in this field. In this study, we investigated the effect o f the potent, m u l t i m o d a l , M+ stimulator, " T u f t s i n " o n the phagocytic f u n c t i o n o f h u m a n peripheral b l o o d m o n o c y t e s / m a c r o p h a g e s derived f r o m n o r m a l , t u b e r c u l o i d leprosy ( B T / T T ) a n d l e p r o m a t o u s leprosy ( B L / L L ) individuals, after the cells have been cultured in vitro for varying periods (6 h to 14 days). T h e present study is designed to address the following issues: (1) the o p t i m a l c o n c e n t r a t i o n o f tuftsin needed to give m a x i m a l phagocytic response; (2) analysis o f phagocytic response vis-gl-vis the d u r a t i o n o f culture in vitro; (3) the difference in
tpresent address: Molecular Genetics Laboratory, M. G. H. Cancer Centre, Bldg. 149, 13th Street, Charles Town, Massachusetts, U.S.A. IIAuthor to whom correspondence should be addressed. Abbreviations used in this paper: M+, macrophage; RPMI-1640, Rosewell Park Memorial Institute-1640 Medium. 847
R. R. IYERet al.
848
phagocytic pattern, if any, of Staphylococcus aureus, live avirulent Mycobacterium tuberculosis (H37RD and M. leprae; (4) the role of serum in stimulating the effect of tuftsin on phagocytosis; (5) effect of tuftsin on opsonin-based stimulatory effect; and (6) objective parameter to study the stimulation of phagocytic response.
Viability and purity o f cultures The average yield and viability of PBMC after F i c o l l - Paque isolation were 1 - 2 x 1 0 6 PBMC/ml and 98% respectively. Approximately 10% of the PBMC were adherent M+. The presence of nonspecific esterase activity was 95% after 6 h and 99% after 7 days of culture. The number of adherent M+ per well was adjusted to 1_+0.3 × 105 and 2 _+ 0.1 × 10; cells/well in all experiments.
EXPERIMENTAL PROCEDURES
Patients A total of five healthy, normal, 15 B T / T T and 20 B L / L L individuals, classified according to their clinical and histopathologic findings (Ridley & Jopling, 1966), were investigated for the ability of their M~ to be stimulated by tuftsin for enhanced phagocytic function. The patients were registered at the Leprosy Clinic, Department of Dermato-Venereology, A.I.I.M.S., New Delhi. They received antiHansen's chemotherapy for less than 6 months in most cases, while a few were fresh untreated cases. Patients with a history of a "Reactional Episode" or in an "Active Reactional state" (showing Type I and Type II lepra reaction) were not included in this study. Isolation and culture o f peripheral blood monocytes Peripheral blood mononuclear cells (PBMC) were isolated by density gradient (Boyum, 1968) separation on F i c o l l - P a q u e (Pharmacia, Upsaala, Sweden). Blood (30 ml) was mixed with an equal volume of minimal essential medium (MEM) (GIBCO-BIOCULT, Irvine, Scotland) containing 40 U/ml of preservative free heparin (Upjohn & Co., Kalamazoo, U.S.A.) layered on 15 ml of F i c o l l Paque in a conical polypropylene centrifuge tube and centrifuged at 400 × g for 30 rain at 18°C. The interphase cells were collected, washed thrice in cold MEM (4°C) and resuspended in 50% human AB serum (heat-inactivated) enriched with RPMI-1640 medium (GIBCO-BIOCULT, Irvine, Scotland) containing 50 U / m l penicillin (GIBCO) and 2 mM L-glutamine, at a concentration of 1 - 2 x 106 cells/ml. This was then distributed equally (1 ml/well) into 24-well plates (Linbro, Flow Labs, Irvine, Scotland) and incubated for 6 h at 37°C in a moist atmosphere containing 5% COz. The non-adherent cells were removed by washing with pre-warmed MEM (37°C) and the monocyte-enriched monolayers were then cultured in RPMI 1640- 50% AB serum for varying periods (6 h to 14 days) before assaying for stimulated phagocytic function.
Culture and radiolabelling o f S. aureus and M. tuberculosis (i) S. aureus. S. aureus (Cowan 1 strain) was grown in nutrient broth (DIFCO, Detroit, MI) for 4 h with continuous stirring. When the bacteria were in the last quarter of the log phase of growth, they were harvested by centrifugation (3000 x g for 30 min), washed twice with normal saline and incubated in 10 ml of MEM containing 5% glucose and universally labelled sodium [~4C] acetate (10 ~Ci/ml; sp. act. 60.3 mCi/mmol; Radiochemical Division, Bhabha Atomic Research Centre, Trombay, Bombay) for 3 h at 37°C. The culture was terminated by adding 10 ml of a mixture of sodium chloride (0.15 M) and sodium acetate (1.0M), centrifuged for 30 rain at 3000 x g, washed thrice with cold normal saline. The pellet was resuspended in 5 ml of phosphate-buffered saline (0.1 M; pH 7.2) and stored at 4°C until use. Serial dilutions of labelled S. aureus (1 x 108 to 1 × 105 bacteria/ml) were precipitated onto glass fibre discs by trichloroacetic acid (TCA), and radioactivity incorporated measured in a liquid scintillation counter (LKB-Rack beta, LKB-Wallace, U.K., Counter efficiency = 53%). The radioactivity incorporated was 2 x 104 counts/rain/1 x 1 0 6 bacteria. The concentration of labelled bacteria in the stock suspension was determined by plating 5 ~1 of volume of serial dilutions onto 8 mm diameter ring slides. The smears were flame-fixed and stained for gram-positive cocci. The number of bacteria per ml of the stock suspension was calculated by counting the bacteria in 20 high power fields (HPF) and expressed according to the formula: {[(Number of bacteria per HPF) x number of HPF per 8 mm ring slide] × dilutionfactor} + 5 × 103 = bacteria/ml. The concentration of labelled S. aureus in the suspension was adjusted to 1 × 108 cocci/ml. (ii) M. tuberculosis (H37R,). M. tuberculosis (H37Ra) [obtained from Dr P. S. Murthy, Department of Biochemistry, UCMS, New Delhi] was grown for 4 weeks as a stationary, pellicle culture on
Modulation of Human Lepromatous Monocyte-M~, Functions by Tuftsin
849
PI-IAGOCYTOSlS ASSAY PROTOCOL Me monolayer culture (1-2 x 10S/well) in RPMI.1640 + 51)% AB serum / for 6 brs, 3,7 & 14 days On appropriate day remove medium, add fresh RPMI +/- Tuftsin +/14 C- labelled Bacteria bacteria : M¢ --- 10:1) +/- 30% AB serum I
incubate 30 min/37°C
1 ¥ Remove infective medium, wash x 3 with MEM, strip Mtl with 0.4% Xylocaine in MEM (4°C/6 hrs.)
harvest and precipitate on glass fibre discs
/
T
Count CPM on B counter ~ C P M (test) PHAGOCYTIC INDEX (P.I.) = CPM (control) x 100% Fig. 1. Protocol for the radiometric assay of phagocytosis by macrophage monolayer cultures.
Middlebrook 7H9 broth (MB7H9; DIFCO, Detroit, MI) containing 20°7o glucose. The bacteria were harvested after centrifugation (2000 × g for 20 min), resuspended in 20 ml of fresh MB7H9 containing Tween 80 (0.5 g/litre), 20°7o glucose and 10 to 20 glass beads and stirred at 37°C for 8 h to disperse bacterial clumps. The dispersed bacilli were reharvested (5000 × g for 30 min) and suspended in fresh MB7H9 (30 ml) containing Tween-80, 10°70 glucose and sodium [14C] acetate (10/aCi/ml), incubated for 72 h at 37°C with constant stirring and were processed as above. The bacteria were finally stored in PBS (10 ml) containing Tween-80 at 4°C until use. The radioactivity incorporated (1 x 104 counts/min/1 x 10 6 bacilli) and concentration of bacilli (1 × 107 AFB/ml) in the stock suspension were done as described above.
1solution of M. leprae from human skin biopsies M. leprae, free of tissue debris were extracted (Prasad & Nath, 1981) from skin biopsies of two untreated LL patients with 6 + bacteriological and high morphological indices with multiple nodular lesions scattered throughout the body. A part of isolated M. leprae were heat-killed by autoclaving (15 Psi; 30 min) while the remainder was stored live at 4°C as a suspension in RPMI-1640 until use.
Tuftsin Tuftsin was synthesized in our laboratory (Rao, Iyer, Prasad & Nath, 1987), purified, dissolved in PBS to a concentration of 1 mg/ml and stored at - 2 0 ° C until use. A notable feature of the tuftsin synthesis adopted by us was that arginine was added separately to the tripeptide Thr - L y s - Pro, thereby eliminating the contamination of inhibitory tripeptide, Lys - Pro - Arg.
Assay of phagocytosis by measurement of ingested radiolabelled bacteria Figure 1 gives the protocol for radiometric assay of phagocytosis. The following points deserve emphasis: (a) since the assay depends on the measurement of the uptake of radiolabelled bacteria by the entire Md~ population within a particular culture well, care was taken that: (a) Md~ cell density was maintained at 1 _+ 0.3 x 105 to 2 _+ 0.1 x 10~ cells/well; (b) non-phagocytosed bacteria were removed at the end of the phagocytic period by repeated washings. Tuftsin was used as follows: (c) Presented along with bacteria to the Mdp culture throughout the 30 min phagocytic period; (d) bacteria were first treated with tuftsin, washed and then added to the Md~ monolayers without AB serum;
850
R. R. IYER et al. Table 1. D o s e - response phagocytic profile of 7 & 14 day culture of macrophage from five normal donors to tuftsin (0 - 7.0/aM) stimulation assayed by the ingestion of 14C-acetate labelled S. aureus in the presence of 30% AB serum (heat-inactivated) 7-day cultures Counts/min + S.D.
CONTROL TUFTS|N (MM) 0.08 0.166 0.33 0.66 3.5 7.0 Negative Control (Background)
14-day cultures
Phagocytic index
243.97 _+ 14.8
100.0 _+ 6%
N.D. N.D. 430.93 _+ 4.4 391.13 +_ 14.34 27.50 _+ 2.29 232.15 _+ 30.6 45 _+ 8
N.D. N.D. 176.63 _+ 2% t 160.63_+6%* 110.87+0.009 NS 95.15 _+ 12% NS --
Counts/min _+ S.D. 305.7 _+ 63.3 733.5 698.5 895.0 891.0 673.5 502.0 32
_+ 16.5 _+ 88.5 ___ 96.4 _+ 44 _+ 150.5 _+ 73.24 _+ 2
Phagocytic index 100.0 _+ 20.7 239.9 228.5 293.0 291.5 220.3 164.21
_+ 2.25% t _+ 12.67%* _+ 10.8%* _+ 4.9%* _+ 22.3%* _+ 14.6%*
Mean +_ S.D. of triplicate experiments; *P<0.05%; +P<0.01; *P<0.001. NS = Not significant; N.D. = not determined.
(e) h u m a n A B serum (heat inactivated) was either used at 30°7o c o n c e n t r a t i o n or absent in all the assays; (f) all experiments were d o n e in triplicate.
Assay of phagocytosis ofM. leprae by v&ual scoring of ingested bacter& M o n o c y t e s were cultured for 7 a n d 14 days o n 12 m m d i a m e t e r sterile glass cover slips (Blue Star, New Delhi) in 24-well culture plates (Linbro) as described earlier. M. leprae (heat-killed/live) o b t a i n e d f r o m h u m a n skin biopsies, were interacted with the M+ m o n o l a y e r in a M+ : b a c t e r i a ratio o f l:10+tuftsin (at c o n c e n t r a t i o n o f 0 . 8 # M a n d 35.0 ~M) for 30 m i n in the absence of 30% A B serum. N o n - p h a g o c y t o s e d b a c t e r i a were r e m o v e d by washing thrice with p r e w a r m e d M E M , the cover slips were fixed with 2°7o g l u t a r a l d e h y d e a n d stained with Ziehl - N e e l s e n ' s stain prior to microscopic e x a m i n a tion. T h e phagocytic index (PI) was calculated by r a n d o m c o u n t i n g of 300 m a c r o p h a g e s a n d expressed as the n u m b e r o f M+ c o n t a i n i n g >-3 acid fast bacilli per 100 M+ (expressed as percentage).
f r o m three d o n o r s u n d e r serum free assay conditions a n d the phagocytic assay c o n d u c t e d as in Fig. 1, but w i t h o u t any f u r t h e r a d d i t i o n o f tuftsin. Simultaneously, controls in triplicate were assayed for phagocytosis of " t u f t s i n u n t r e a t e d " radiolabelled b a c t e r i a u n d e r identical conditions.
Scanning electron microscopy (SEM) of macrophages treated with tuftsin M a c r o p h a g e s derived f r o m n o r m a l (3), B T / T T (5) a n d BL/LL (8) individuals were cultured for 14 days a n d pulsed with 0.8/~M tuftsin for 30 min, washed with RPMI-1640, a n d t h e n fixed with a mixture o f 2 . 5 % g l u t a r a l d e h y d e a n d 1% p a r a f o r m a l d e h y d e in s o d i u m cacodylate b u f f e r (0.1 M , p H 7.3) for 2 h at 4°C. The cells were washed a n d d e h y d r a t e d in a graded e t h a n o l series (30 to 100%) followed by two changes o f amyl acetate. The cover slips were dried in liquid CO2 using a critical-point drying a p p a r a t u s , s p u t t e r - c o a t e d with gold ions a n d visualized in a Joel scanning electron microscope.
Stat&tical analys& Assay of opsonic effects in the activity of tuftsin on macrophages Radiolabelled S. aureus a n d M. tuberculosis (H37Ra) (1 × 107 bacilli/ml) in n o r m a l saline c o n t a i n i n g 0.8 ~M tuftsin were i n c u b a t e d at 37°C for 1 h with c o n s t a n t agitation. The bacteria were t h e n pelleted by c e n t r i f u g a t i o n (3000 x g for 30 min) a n d w a s h e d thrice with fresh n o r m a l saline to remove u n b o u n d tuftsin. The tuftsin " o p s o n i z e d " b a c t e r i a were t h e n a d d e d to 14 day cultures of n o r m a l M+
D a t a are analysed by the one-way analysis o f variance, a n d the levels o f significance, if any, are s h o w n in the results.
RESULTS
T h e present study showed t h a t o p t i m a l results were o b t a i n e d by using M~ : b a c t e r i a in the ratio o f 1:10, with a phagocytic period o f 30 m i n a n d with
Modulation of Human Lepromatous M o n o c y t e - M ~ Functions by Tuftsin
851
350 300- ~ 250,
,~ _z 200150O
°°"r
0_
1002
t Controls
50
t
.
r e c e i v e d no T u f t s i n
(P.I. = 100-'S.D.)
0
,
o
o~5
~.0
/~
Normol
~
TT M 6
:
: LL M ~
Me,
,
3.5
TUFTSIN CONCENTRATION
7:0 IN ~ J M )
Fig. 2. Dose-response profile of tuftsin stimulated phagocytosis of ]4C-acetate labelled S. aureus by peripheral blood monocyte- macrophages from normal, B T / T T and B L / L L individuals (five subjects each) cultured in vitro for 14 days prior to the assay. The response of normal macrophages given in Table 1 has been superimposed on the B T / T T and B L / L L curve for comparison. Table 2. Phagocytosis of '4C-acetate labelled S. aureus by 14-day cultures of macrophages from three B L / L L patients over a period of 60 min in the presence of 0.8/aM tuftsin and 30°70 AB serum (heat-inactivated) Control (M~ + bacteria + 30o70 AB serum) Counts/rain _+ S.D. 675.34 -4- 28.87 779 _+ 129.4 1275.5 1481
_+ 107.5 _+ 160.54 -
-
-1991.5 _+ 157.5 2054 _+ 182.8
Test (Mdp + bacteria + 30% AB serum + 0.8/~M tuftsin) Time 8 18 38 25 36 32 45 50 53 60
min min s min min s min min min min
Counts/min _+ S.D.
Statistical significance
703.67 _+ 140.48 1093.34 + 130.07
NS NS
1280 + 130 1694.67 _+ 217.42
NS NS
2521.0 -+ 248 2377.34 _+ 117.11 -2355.34 _+ 162.9
---NS
Mean +_ S.D. of triplicate experiments; NS = not significant; Md~ = macrophages. bacteria suspended in a m i n i m a l v o l u m e o f 0.3 ml o f m e d i a (Tables l , 2 a n d 3). These o b s e r v a t i o n s a p p e a r to be related to the f r e q u e n c y o f p h a g o c y t e b a c t e r i a e n c o u n t e r s or hits, a n d s u p p o r t s earlier findings (Lehnert & M o r r o w , 1985). A progressive increase in phagocytic response to the labelled S. a u r e u s with increasing c o n c e n t r a t i o n o f tuftsin was seen in 7 a n d 14 day old M~ cultures f r o m n o r m a l subjects (Table 1). M a x i m a l phagocytic activity was at 0 . 3 3 / a M tuftsin a n d 14-day n o r m a l M~ cultures displayed a p p r o x i m a t e l y d o u b l e the
increase in phagocytic activity with 0.33/aM tuftsin a n d c o n t i n u e d at the same m a x i m a l level up to 0.66/aM tuftsin. H i g h e r doses o f tuftsin (7.0/aM) decreased the response to a value s h o w n by u n s t i m u lated controls. W h e n 14-day cultures f r o m the five B T / T T a n d B L / L L patients (Fig. 2) were tested u n d e r identical conditions, the p a t t e r n o f response s h o w e d some interesting features w h e n c o m p a r e d with n o r m a l Md~ cultures. In B T~ T T cultures m a x i m a l response was at 0.66/aM tuftsin a n d persisted up to 3.5/aM tuftsin
852
R. R. IYERet al. 400-
300 O:
C~ z ¢_)
m I-
200
~ S e r u m free
0 0 -IO-
--
I\
100
30% Serum
' C o n t r o l s
50
•
,
i
3
,
,
~
.
.
.
.
7
.
.
i
14
DAYS O F C U L T U R E
Fig. 3. Relationship between age of culture and the response to 0.8/aM tuftsin stimulated phagocytosis of ~4C-acetate labelled M. tuberculosis (H37Ra) by blood monocyte-macrophages from five normal donors under 30% AB serum and serum free assay conditions. The macrophages were pretreated with tuftsin for 10 min prior to interaction for 30 min with bacteria.
which is 5 to 10 times the dose optima for 14-day normal M~. The response also showed a lag period compared to normal Md~. The phagocytic response with 0.66/aM and 3.5/aM tuftsin were higher by 28% and 50% (in P.I. units) respectively compared to that of normal macrophages. The response curve thus appears to have shifted to the right in 14-day B T / T T cultures. In contrast, 14-day B L / L L cultures showed no statistically significant stimulation of phagocytic activity over the entire range of tuftsin concentration up to 7/aM. This lack of response exhibited by B L / L L Mdp was not due to the inadequate time of 30 rain allowed for phagocytosis. In a second experiment using 14-day cultures from three B L / L L patients, the monolayers were incubated with labelled S. aureus for 60 min with/without 0.8/aM tuftsin but were refractory to tuftsin stimulation (Table 2). Subsequent experiments were done using a tuftsin concentration of 0.8/aM which was in the optimal range and a phagocytic period of 30 min. Since the 14-day normal M~ cultures responded more in terms of phagocytic stimulation than 7-day old cultures, the phagocytic response profile was assessed with duration of culture age. The role of 30% AB serum in terms of synergism in phagocytic activity if any, was also evaluated.
Normal macrophages cultured for 6 h to 14 days were stimulated with 0.8/aM tuftsin in the presence of labelled M. tuberculosis (H37Ra) (Fig. 3, Table 3). Further, 6 h old normal M~ showed a high degree of basal phagocytic activity (counts/min = 2796 _+ 10; Table 3) while the increment in terms of phagocytic stimulation by tuftsin was small. However, the basal activity falls in 3, 7 and 14-day old cultures, but the ability to respond in phagocytic activity on tuftsin stimulation shows a progressive increase. Also the difference in the basal activity between 3, 7 and 14-day cultures is only about 200 counts/min (Table 3) while the increase in 14-day old normal M+ is nearly 3 to 4 times on 3-day cultures. Finally there was no statistically significant increase in phagocytic activity contributed by 30% human AB serum. While 7 and 14-day BT/TT Md~cultures from eight patients showed a similar response as described above under serum free assay conditions, the 14-day old cultures of B L / L L Md~ of eight patients tested exhibited a decreased response to levels that are statistically insignificant (Fig. 4). Since the increase in phagocytosis on tuftsin stimulation was seen to vary directly in the case of normal and B T / T T M~ and inversely in the case of B L / L L M+ with duration of culture age, all further experiments on phagocytosis were conducted on M~b cultured at two different time periods (7 days and 14 days). Table 4 gives the results of the phagocytic response to tuftsin by 7 and 14-day old cultures of normal, B T / T T and B L / L L M+ from 5, 8 and 8 subjects respectively when assayed for the ingestion of heatkilled and live M. leprae. Normal 7-day cultures showed a 1.6 and 1.8 times greater phagocytosis than controls with heat-killed and live M. leprae respectively on stimulation with 0.8/aM tuftsin. By day 14 of culture, the response increased to 2.36 and 2.16 times for heat killed and live M. leprae respectively. Similarly, 7 day B T / T T cultures showed 1.98 and 1.95 times phagocytic stimulation by 0.8/aM tuftsin which increased to 2.3 and 2.57 times in 14-day cultures for heat-killed and live M. leprae respectively. On the contrary, 7-day B L / L L tuftsin stimulated cultures showed phagocytic increase of 2.3 and 2.35 times for heat-killed and live M. leprae respectively, but were refractory to 35/aM tuftsin. By day 14, the cultures became refractory to 0.8 taM tuftsin, but being stimulated at 35/aM tuftsin with an increase of 2.09 and 2.08 times greater phagocytosis for heat-killed and live M. leprae respectively. The phagocytosis stimulating effect to tuftsin is not dependent on a non-specific charge based opsonic effect (tuftsin is highly basic; PI = 8.9).
Modulation of Human Lepromatous Monocyte-Mdp Functions by Tuftsin
853
Table 3. Relationship between age of culture and the phagocytic response to 0.8/aM tuftsin of macrophages from five normal donors assayed by the ingestion of ~4C-acetate labelled M. tuberculosis (H37Ra) in the presence and absence of 300/0 serum (heat-inactivated) Normal Macrophages Age of
Serum free assay
culture
Control counts/min
2796
P.I. counts/min
30o/o AB serum containing assay
Test (0.8/aM tuftsin)
Control 2675
Test (0.8/aM tuftsin)
_ 10
3705.5 _+ 334.5
_+ 250
3287.5 + 92.5
100 756
+ 0.3% _+ 86
132.5 _+ 9% (NS) 1326.5 _+ 232.5
100 930
_+ 9.3% _+ 67
122.8 _+ 2.8% (NS) 1942 _+ 170
P.I. counts/min
100 636
_+ 11.40/0 _ 17
175.5 + 17.5°70* 1793.7 _ 78.1
100 ___ 7.2070 684.6 _+ 13.5
208.8 _ 8.750/0* 1666 + 163.2
P.I. counts/min
100 _+ 2.7°70 816.5 _+ 26.2
282 3141
100 _+ 2°70 870.4 _+ 21.85
243.4 _+ 9.8O7ot 2607.7 _+ 76.84
P.I.
100
6h
3 days
7 days _+ 4.35% t -4- 120
14 days _+ 3.2O/o
384.7 _+
3.8O7o*
100
+ 0.25o/o
299.6 _+
0.09o/o*
Mean _.+ S.D. of triplicate experiments; */:'<0.05; tp<0.01; *P<0.001. NS = Not significant.
"~
-:
300.
200.
~ ".
o-.--o LL M~
"-
Q
"-
,j
",
I--
~,
0
<
lOO, ---// Controls
l "t---O
0
receive
no T u f t s i n
P.I,=100 *-- S.D. i
7
I/4 DAYS OF CULTURE
Fig. 4. Relationship between age of culture and the phagocytic response to 0.8/aM tuftsin by B T / T T and B L / L L macrophages from eight patients each as measured by the ingestion of ~4C-acetate labelled M. tuberculosis (H37Ra) under serum free assay conditions. The macrophages were pretreated with tuftsin for 10 min prior to interaction for 30 min with bacteria. T u f t s i n treated b a c t e r i a were n o t p h a g o c y t o s e d at significantly greater degrees t h a n u n t r e a t e d bacteria (Table 5). F u r t h e r Mdp p r e t r e a t e d with 0 . 8 / a M tuftsin
for 10 m i n followed by a 30 m i n i n t e r a c t i o n with labelled bacteria exhibited phagocytic s t i m u l a t i o n to the same degree as when tuftsin was present with b a c t e r i a for the entire 30 m i n phagocytic period. F u r t h e r , scanning electron m i c r o g r a p h s clearly d e m o n s t r a t e d two features o f the effect o f tuftsin (Fig. 5): (1) while control m o n o l a y e r s showed the typical fried egg m o r p h o l o g y o f u n s t i m u l a t e d m a c r o p h a g e s with a central ruffled cell b o d y a n d a large spread-out peripheral cytoplasmic area with m i n i m a l ruffling, 0 . 8 / a M tuftsin treated 14-day old n o r m a l a n d B T / T T Mdp cultures showed a d r a m a t i c increase in fllopodia a n d k n o b b e d processes giving the cells sea-urchin or porcupine-like a p p e a r a n c e . T h e peripheral cytoplasm was t h r o w n out as filop o d i a a n d the cell a t t a c h e d to the s u b s t r a t u m by long retraction fibrils. (2) B L / L L Mdp cultures showed m i n i m a l u l t r a s t r u c t u r a l alterations after tuftsin t r e a t m e n t which included a slight increase in filopodia with small retraction fibrils at the cell periphery b u t was otherwise very similar in external m o r p h o l o g y to u n t r e a t e d controls. DISCUSSION Tuftsin is a well d o c u m e n t e d stimulator o f m a c r o phage phagocytic a n d microbicidal functions (Najjar, 1983). O u r results show t h a t 0.66 to 0.8/aM tuftsin is the o p t i m a l range for phagocytic stimulation which is within the observed range (Flemming, 1985). W e have quantitatively assayed phagocytosis by m a c r o p h a g e s using: (a) visual scoring o f ingested
854
R . R . IVE~ et al.
Table 4. The phagocytic response to tuftsin by 7- and 14-day-macrophage cultures from three normal, eight B T / T T and B L / L L subjects assayed by visual counting of the number of macrophages containing >13 M. leprae per 100 macrophages under serum free assay conditions (a) NormalMacrophages Heat killed M. leprae Age of culture
Control (P.I. in %)
7 days 14 days
48.3 _+ 5% 40.4_+8.2%
7 days 14 days
44.1 _+ 1.6% 41.8 _+ 0.9%
Heat killed M. leprae
Live M. leprae
Live M. leprae
Test Control (0.8 taM tuftsin) (P.I. in %) (P.I. in %) 78.2 _+ 3.4%** 45.6 _+ 6.8% 95.6_+ 1.8%** 45 _+ 3.1% (b) B T / T T Macrophages
Test (0.8 taM tuftsin) (P.I. in %) 82.4 _+ 5.6% t 97.2 + 3.01% t
85.6 +_ 2.7%* 40.1 _+ 5.2% 96.7 _+ 1.9%** 36.7 + 3.6% (c) B L / L L Macrophages
78.5 _+ 2%** 94.6 + 2.7%**
Control Tuftsin (0.8 taM) Tuftsin (35 taM) Control Tuftsin (0.8 taM) Tuftsin (35 taM)
7 days (P.I.) 35.8 _+ 8.2% 82.4 _+ 2.1°70** 32.9 _+ 2.6% (NS) 38.1 _+ 5.7% 89.91 _+ 3.6%** 40.2 _+ 4.1 (NS)
14 days (P.I.) 40.8 + 2.3% 38.6 + 4.7% (NS) 85.6 + 5.1%** 44.1 _+ 5.6% 41.3 _+ 6.4% (NS) 92.1 _+ 1.9%**
Mean + S.D. of triplicate experiments; *P<0.05; NS = not significant; tp<0.01. bacteria in stained specimens under the light microscope (Stossel, 1975); (b) measurement of ingested radiolabelled bacteria or particles (Michell, Pancake, Noseworthy & Karnovsky, 1969; Suzuki, Booth & Grecz, 1971); and (c) lysis of macrophages followed by plating on agar. The d o s e - r e s p o n s e profile of phagocytosis of S. aureus and M . tuberculosis (H3vRa)clearly demonstrates two points - - (a) at the optimal tuftsin concentration, 14-day old normal M+ cultures show a greater increase in phagocytosis than 7-day old cultures; and (b) 14-day old B T / T T cultures exhibit a shift to the right in the d o s e - r e s p o n s e curve for S. aureus. In the analysis of r e c e p t o r - l i g a n d binding curves, a shift to the right indicates an increased dissociation of the complex and decreased binding of the ligand to the receptor. The refractoriness of 14-day old B L / L L M+ to the range of tuftsin used (0.8 ~M to 7.0/~M) for assaying the phagocytosis of S. aureus and M . tuberculosis at 0.8 ~M tuftsin respectively, may be explained as an extreme shift to the right of the above d o s e - response curve, raising the possibility that - - (a) B L / L L M+ (14-day) have a decreased number of tuftsin receptors or receptors with altered binding affinities; or (b) the decreased response at higher concentrations may be due to the production of a significant amount of the inhibitory tripeptide, L y s - P r o - A r g due to enzymatic cleavage of tuftsin by macrophage membrane aminopeptidases (Najjar, 1983).
The fact that B L / L L M+ display a differential response at 7 and 14 days of culture to 0.8/aM and 35.0taM tuftsin as measured by ingestion of M . leprae strengthens the earlier inference of altered tuftsin receptor expression (either in number or in affinity for ligand) in these cells with increasing age of culture. The response of 14-day old B L / L L cultures at 35/aM tuftsin may indicate expression of tuftsin receptors with a lowered affinity for its ligand in the older cultures. Multiple receptors with varying affinities for an identical or similar ligand have been documented for Fc r e c e p t o r - I g G interactions in murine and human systems (Diamond & Yelton, 1981; Unkeless, Fleit & Mellman, 1981; Tax, Hermes, Willems, Capel & Koene, 1984; Looney, A b r a h a m & Anderson, 1986). The existence of dual receptors with varying affinity for tuftsin and related peptides was postulated (Bar-Shavit, Terry, Blumberg & Goldman, 1982; Segal, Tzehoval & Feldman, 1983). The expression of these two receptor forms may vary in concentration on different phagocytic cells and stages of differentiation. The isolation of a tuftsin binding protein (Bump, Lee, Wleklik, Reichler & Najjar, 1986) from rabbit peritoneal neutrophil membranes which exists as a dominant 500,000 mol. wt form and a subordinate 250,000 mol. wt form supports the above postulation. In the present study the results point to a decreased ability of B L / L L M+ to exhibit tuftsin-stimulated phagocytosis with increasing age of culture, though
Modulation of Human Lepromatous M o n o c y t e - M ~ Functions by Tuftsin
M~ alone
855
M~ + Tuftsin
Normal
BT/TT
BL/LL
Fig. 5. Morphologic changes induced by 0.8/~M tuftsin treatment of 14-day macrophage cultures derived from normal, B T / T T and B L / L L individuals (three each) as visualized by SEM.
Modulation of Human Lepromatous Monocyte- M~b Functions by Tuftsin
857
Table 5. Assay of opsonic effect in the stimulatory response to tuftsin by 14-day normal macrophage cultures Untreated radiolabelled bacteria S. aureus
Counts/min + S.D. Phagocytic index
1425 + 115.4 100 + 8.09°/0
Tuftsin treated radiolabelled bacteria
H37Ra
S. aureus
H37Ra
2678 _ 33.8 100 _+ 1.3%
1398 _+ 211.8 98.1 _+ 0.0%
3019 _ 412 112.7 + 15.4% (NS)
Mean _+ S.D. of triplicate experiments; NS = not significant.
the basal phagocytic ability o f the u n s t i m u l a t e d M~ cultures was m o r e or less the same in all the three groups. Thus, while B L / L L patients m a y exhibit a d e q u a t e non-specific i m m u n e defences to various b a c t e r i a at a basal level, they a p p e a r u n a b l e to e n h a n c e these f u n c t i o n s o n stimulation. T h e o b s e r v a t i o n t h a t t u f t s i n - t r e a t e d S. a u r e u s a n d M . t u b e r c u l o s i s were p h a g o c y t o s e d to the same extent as u n t r e a t e d b a c t e r i a u n d e r serum free assay c o n d i t i o n s rules o u t the m e c h a n i s m o f non-specific opsonic e f f e c t s / c h a r g e b a s e d p h e n o m e n a for tuftsin o n M~ phagocytic f u n c t i o n . Finally, the S E M p h o t o g r a p h s establish the effect o f tuftsin o n the surface m e m b r a n e o f n o r m a l a n d B T / T T cultures (14-day), while B L / L L cultures lack a similar effect. P h a g o c y t i c activity o f m o n o c y t e / m a c r o p h a g e s in leprosy as a f u n c t i o n of d u r a t i o n o f culture age has
n o t been r e p o r t e d so far. O u r d a t a suggest t h a t the peripheral b l o o d m o n o c y t e s in B L / L L patients exhibit a c h a n g e towards a n end stage t h a t is relatively refractory to activation by agents like tuftsin. O u r results showing a differential response to tuftsin with varying age in culture indicate t h a t the level of d i f f e r e n t i a t i o n o f the M~ p o p u l a t i o n has to be a c c o u n t e d for in studies investigating i m m u n o modulatory approaches. W e are currently studying the possibilities raised in this study by investigating serum tuftsin levels a n d tuftsin receptor characteristics. Acknowledgements - - We thank Prof. I. Nath, Depart-
ment of Biotechnology for providing laboratory facilities and valuable guidance. The assistance of Dr S.N. Upadhya, National Institute of Immunology, New Delhi, during scanning electron microscopy of macrophage cultures is gratefully acknowledged.
REFERENCES
BAR-SHAVIT, Z., TERRY, S., BLUMBERG,S. • GOLDMAN, R. (1982). Neurotensin-macrophage interaction: specific binding and augmentation of phagocytosis. Neuropeptides, 2, 325 - 335. BOYUM, A. (1968). Separation of leukocytes from blood and bone marrow. Scand. J. clin. Lab. Invest., 97, 7 7 - 8 9 . BUMP, N. J., LEE, J., WLEKLIK, M., REICHLER, J. & NAJJAR, V. A. (1986). Isolation and subunit composition of the tuftsin receptor. Proc. natn. Acad. Sci. U.S.A., 83, 7187-7191. DIAMOND, B. & YELTON, D. E. (1981). A new Fc receptor on mouse macrophages binding IgG3. J. exp. Med., 153, 514-519. FLEMMING, K. B. P. (1985). Stimulation and depression of the RES by pharmacological agents. In The Reticuloendothelial System: A Comprehensive Treatise (eds Hadden, J. W. and Szentivanyi, A.) Vol. 8, pp. 247-284. Pharmacology, Plenum Press, New York. GODAL, Z. (1978). Immunological aspects of leprosy - - present status. Prog. Allergy, 25, 211-242. KAPLAN, G. 8z COHN, Z. A. (1984). The immunology of leprosy. Int. Rev. exp. Path., 28, 45 - 7 8 . LEHNERT, B. E. & MORROW, P. E. (1985). The initial lag in phagocytic rate by macrophages in monolayer is related to particle encounters and binding. I m m u n . Invest., 14, 5 1 5 - 521. LOONEY, R. J., ABRAHAM, G. N. 8z ANDERSON, C. L. (1986). Human monocytes and U937 cells bear two distinctive Fc receptors for IgG. J. I m m u n . , 136, 1641 - 1647. MICHELL, R. H., PANCAKE, S. J., NOSEWORTHY, J. & KARNOVSKY, M. (1969). Measurement of rates of phagtocytosis: the use of cellular monolayers. J. cell. Biol., 40, 216-224. NAJJAR, V. A. (1983). Tuftsin, a natural activator of phagocytic cells: an overview. Ann. N . Y . Acad. Sci., 419, I - 11. PRASAD, H. K. & NATH, I. (1981). Incorporation of 3H-thymidine in M. leprae residing in differentiated human macrophages. J. Med. Microbiol., 14, 279-293. RAO, O. N., IYER, R. R., PRASAD, H. K. & NATH, I. (1987). A novel synthetic route to the phagocytosis stimulation tetrapeptide, "tuftsin". Med. Sci. Res., 15, 1199-1201.
858
R . R . IYER et al.
RIDLEY, D. S. & JOPLING, W. H. (1966). Classification of leprosy according to immunity. A five group system. Int. J. Lepr., 34, 2 5 5 - 273. SEGAL, S., TZEHOVAL, E. & FELDMAN, M. (1983). The function of tuftsin and similar sequences in other proteins. Ann. N.Y. Acad. Sci., 419, 168-178. SmLEY, D. L. & KRAHENBUHt, J. L. (1987). Mycobacterium leprae-burdened macrophages are refractory to activation by gamma interferon. Infect. Immun., 55, 446- 450. STOSSEL, T. P. (1975). Phagocytosis: recognition and ingestion. Semin. Hemat., 12, 8 3 - 116. SuzuKh J. B., BOOTH, R. R. & GRECZ, N. (1971). Evaluation of phagocytic activity by ingestion of labelled bacteria. J. Infect. Dis., 123, 9 3 - 96. TAX, W. J. M., HERMES, F. F. M., WILLEMS, R. W., CAPEL, J. A. • KOENE, R. m. P. (1984). Fc receptors for mouse lgGl on human monocytes: polymorphism and role in antibody-induced T cell proliferation. J. Immun., 133, 1185- 1189. UNKELESS, J. C., FLEIT, H. & MELLMAN, I. S. (1981). Structural aspects of the heterogeneity of immunoglobulin Fc receptors. Adv. Immun., 31, 247.
0192-0561/90 $3.00 + .00 Pergamon Press pie. International Society for lmmunopharmacology.
MODULATION OF H U M A N LEPROMATOUS M O N O C Y T E - MACROPHAGE FUNCTIONS I N VITRO BY TUFTSIN RAVl R. IYER, *t H. K. PRASAD,* L. K. BHUTANI § a n d D. N. RAO *H *Department of Biochemistry, *Department of Biotechnology and ~Department of Dermatovenereology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110 029, India
(Received 14 July 1989 and in final form 18 April 1990)
-Human peripheral blood monocytes/macrophages derived from normal donors, patients of tuberculoid leprosy (BT/TT) and lepromatous leprosy (BL/LL) were assayed for stimulated phagocytic responses to the potent macrophage stimulator "Tuftsin" (NH2 - Thr - Lys - Pro - Arg - OH) after varying periods (6 h to 14 days) of culture in vitro. The assays consisted of visual scoring of ingested Mycobacterium leprae and radiometric measurement of ingested ~4C-acetate labelled Staphylococcus aureus and Myeobacterium tuberculosis (H37Ra). While normal and BT/TT macrophages showed a progressively increasing ability for tuftsin-stimulated phagocytosis with increasing age of culture in vitro, BL/LL macrophages showed the opposite response so that 14-day cultures were refractory to a stimulatory dose of up to 7.0 ~M (10 to 20 times the optimal dose for normal and BT/TT macrophages). The 14-day BL/LL macrophage cultures were, however, responsive to 35 ~M tuftsin (100 times the optimal dose for normal macrophages). Analysis of the dose - response curves also indicates that BT/TT cultures despite exhibiting an apparent similarity to normal macrophages demonstrate a rightward shift for a maximal stimulated phagocytosis. Finally SEM photo-micrographs of 14-day macrophage cultures of the three groups revealed that while normal and BT/TT cultures demonstrated an increase in membrane ruffling and filopodia on stimulation with 0.8 ~M tuftsin, BL/LL cultures exhibited none of the features associated with stimulation. From the above findings, we conclude that lepromatous macrophages may display an aberrant differentiation profile leading to a terminal state of unresponsiveness and that the defect may possibly lie at the level of tuftsin receptor expression or transmembrane signal transduction.
Abstract
Leprosy is a p o l y m o r p h i c disease caused by Mycobacterium leprae. T h e bacillus has a predeliction to stay within cells o f the m o n o c y t e - m a c r o p h a g e (M+) series a n d in the S c h w a n n cells o f peripheral nerves. This results in a g r a n u l o m a w h i c h in the l e p r o m a t o u s leprosy (LL), shows intracellular bacilli within M+. A l t h o u g h the exact causes o f the defective cellm e d i a t e d i m m u n i t y in leprosy are still unclear (Godal, 1978; K a p l a n & C o h n , 1984), the failure o f the M+ to kill or inhibit intracellular g r o w t h o f M. leprae is a c o n s p i c u o u s characteristic of the l e p r o m a t o u s f o r m o f the disease. It is also i m p o r t a n t to k n o w w h e t h e r the M+ in LL patients is a m e n a b l e to i m m u n o p h a r m a c o l o g i c a l m a n i p u l a t i o n a n d if so, w h a t are the kinetics a n d profile o f such stimulated responses, l m m u n o m a n i p u l a t i o n s o f the phagocytic
system would help in the clinical m a n a g e m e n t o f infectious diseases in view o f our increased k n o w ledge in this field. In this study, we investigated the effect o f the potent, m u l t i m o d a l , M+ stimulator, " T u f t s i n " o n the phagocytic f u n c t i o n o f h u m a n peripheral b l o o d m o n o c y t e s / m a c r o p h a g e s derived f r o m n o r m a l , t u b e r c u l o i d leprosy ( B T / T T ) a n d l e p r o m a t o u s leprosy ( B L / L L ) individuals, after the cells have been cultured in vitro for varying periods (6 h to 14 days). T h e present study is designed to address the following issues: (1) the o p t i m a l c o n c e n t r a t i o n o f tuftsin needed to give m a x i m a l phagocytic response; (2) analysis o f phagocytic response vis-gl-vis the d u r a t i o n o f culture in vitro; (3) the difference in
tpresent address: Molecular Genetics Laboratory, M. G. H. Cancer Centre, Bldg. 149, 13th Street, Charles Town, Massachusetts, U.S.A. IIAuthor to whom correspondence should be addressed. Abbreviations used in this paper: M+, macrophage; RPMI-1640, Rosewell Park Memorial Institute-1640 Medium. 847
R. R. IYERet al.
848
phagocytic pattern, if any, of Staphylococcus aureus, live avirulent Mycobacterium tuberculosis (H37RD and M. leprae; (4) the role of serum in stimulating the effect of tuftsin on phagocytosis; (5) effect of tuftsin on opsonin-based stimulatory effect; and (6) objective parameter to study the stimulation of phagocytic response.
Viability and purity o f cultures The average yield and viability of PBMC after F i c o l l - Paque isolation were 1 - 2 x 1 0 6 PBMC/ml and 98% respectively. Approximately 10% of the PBMC were adherent M+. The presence of nonspecific esterase activity was 95% after 6 h and 99% after 7 days of culture. The number of adherent M+ per well was adjusted to 1_+0.3 × 105 and 2 _+ 0.1 × 10; cells/well in all experiments.
EXPERIMENTAL PROCEDURES
Patients A total of five healthy, normal, 15 B T / T T and 20 B L / L L individuals, classified according to their clinical and histopathologic findings (Ridley & Jopling, 1966), were investigated for the ability of their M~ to be stimulated by tuftsin for enhanced phagocytic function. The patients were registered at the Leprosy Clinic, Department of Dermato-Venereology, A.I.I.M.S., New Delhi. They received antiHansen's chemotherapy for less than 6 months in most cases, while a few were fresh untreated cases. Patients with a history of a "Reactional Episode" or in an "Active Reactional state" (showing Type I and Type II lepra reaction) were not included in this study. Isolation and culture o f peripheral blood monocytes Peripheral blood mononuclear cells (PBMC) were isolated by density gradient (Boyum, 1968) separation on F i c o l l - P a q u e (Pharmacia, Upsaala, Sweden). Blood (30 ml) was mixed with an equal volume of minimal essential medium (MEM) (GIBCO-BIOCULT, Irvine, Scotland) containing 40 U/ml of preservative free heparin (Upjohn & Co., Kalamazoo, U.S.A.) layered on 15 ml of F i c o l l Paque in a conical polypropylene centrifuge tube and centrifuged at 400 × g for 30 rain at 18°C. The interphase cells were collected, washed thrice in cold MEM (4°C) and resuspended in 50% human AB serum (heat-inactivated) enriched with RPMI-1640 medium (GIBCO-BIOCULT, Irvine, Scotland) containing 50 U / m l penicillin (GIBCO) and 2 mM L-glutamine, at a concentration of 1 - 2 x 106 cells/ml. This was then distributed equally (1 ml/well) into 24-well plates (Linbro, Flow Labs, Irvine, Scotland) and incubated for 6 h at 37°C in a moist atmosphere containing 5% COz. The non-adherent cells were removed by washing with pre-warmed MEM (37°C) and the monocyte-enriched monolayers were then cultured in RPMI 1640- 50% AB serum for varying periods (6 h to 14 days) before assaying for stimulated phagocytic function.
Culture and radiolabelling o f S. aureus and M. tuberculosis (i) S. aureus. S. aureus (Cowan 1 strain) was grown in nutrient broth (DIFCO, Detroit, MI) for 4 h with continuous stirring. When the bacteria were in the last quarter of the log phase of growth, they were harvested by centrifugation (3000 x g for 30 min), washed twice with normal saline and incubated in 10 ml of MEM containing 5% glucose and universally labelled sodium [~4C] acetate (10 ~Ci/ml; sp. act. 60.3 mCi/mmol; Radiochemical Division, Bhabha Atomic Research Centre, Trombay, Bombay) for 3 h at 37°C. The culture was terminated by adding 10 ml of a mixture of sodium chloride (0.15 M) and sodium acetate (1.0M), centrifuged for 30 rain at 3000 x g, washed thrice with cold normal saline. The pellet was resuspended in 5 ml of phosphate-buffered saline (0.1 M; pH 7.2) and stored at 4°C until use. Serial dilutions of labelled S. aureus (1 x 108 to 1 × 105 bacteria/ml) were precipitated onto glass fibre discs by trichloroacetic acid (TCA), and radioactivity incorporated measured in a liquid scintillation counter (LKB-Rack beta, LKB-Wallace, U.K., Counter efficiency = 53%). The radioactivity incorporated was 2 x 104 counts/rain/1 x 1 0 6 bacteria. The concentration of labelled bacteria in the stock suspension was determined by plating 5 ~1 of volume of serial dilutions onto 8 mm diameter ring slides. The smears were flame-fixed and stained for gram-positive cocci. The number of bacteria per ml of the stock suspension was calculated by counting the bacteria in 20 high power fields (HPF) and expressed according to the formula: {[(Number of bacteria per HPF) x number of HPF per 8 mm ring slide] × dilutionfactor} + 5 × 103 = bacteria/ml. The concentration of labelled S. aureus in the suspension was adjusted to 1 × 108 cocci/ml. (ii) M. tuberculosis (H37R,). M. tuberculosis (H37Ra) [obtained from Dr P. S. Murthy, Department of Biochemistry, UCMS, New Delhi] was grown for 4 weeks as a stationary, pellicle culture on
Modulation of Human Lepromatous Monocyte-M~, Functions by Tuftsin
849
PI-IAGOCYTOSlS ASSAY PROTOCOL Me monolayer culture (1-2 x 10S/well) in RPMI.1640 + 51)% AB serum / for 6 brs, 3,7 & 14 days On appropriate day remove medium, add fresh RPMI +/- Tuftsin +/14 C- labelled Bacteria bacteria : M¢ --- 10:1) +/- 30% AB serum I
incubate 30 min/37°C
1 ¥ Remove infective medium, wash x 3 with MEM, strip Mtl with 0.4% Xylocaine in MEM (4°C/6 hrs.)
harvest and precipitate on glass fibre discs
/
T
Count CPM on B counter ~ C P M (test) PHAGOCYTIC INDEX (P.I.) = CPM (control) x 100% Fig. 1. Protocol for the radiometric assay of phagocytosis by macrophage monolayer cultures.
Middlebrook 7H9 broth (MB7H9; DIFCO, Detroit, MI) containing 20°7o glucose. The bacteria were harvested after centrifugation (2000 × g for 20 min), resuspended in 20 ml of fresh MB7H9 containing Tween 80 (0.5 g/litre), 20°7o glucose and 10 to 20 glass beads and stirred at 37°C for 8 h to disperse bacterial clumps. The dispersed bacilli were reharvested (5000 × g for 30 min) and suspended in fresh MB7H9 (30 ml) containing Tween-80, 10°70 glucose and sodium [14C] acetate (10/aCi/ml), incubated for 72 h at 37°C with constant stirring and were processed as above. The bacteria were finally stored in PBS (10 ml) containing Tween-80 at 4°C until use. The radioactivity incorporated (1 x 104 counts/min/1 x 10 6 bacilli) and concentration of bacilli (1 × 107 AFB/ml) in the stock suspension were done as described above.
1solution of M. leprae from human skin biopsies M. leprae, free of tissue debris were extracted (Prasad & Nath, 1981) from skin biopsies of two untreated LL patients with 6 + bacteriological and high morphological indices with multiple nodular lesions scattered throughout the body. A part of isolated M. leprae were heat-killed by autoclaving (15 Psi; 30 min) while the remainder was stored live at 4°C as a suspension in RPMI-1640 until use.
Tuftsin Tuftsin was synthesized in our laboratory (Rao, Iyer, Prasad & Nath, 1987), purified, dissolved in PBS to a concentration of 1 mg/ml and stored at - 2 0 ° C until use. A notable feature of the tuftsin synthesis adopted by us was that arginine was added separately to the tripeptide Thr - L y s - Pro, thereby eliminating the contamination of inhibitory tripeptide, Lys - Pro - Arg.
Assay of phagocytosis by measurement of ingested radiolabelled bacteria Figure 1 gives the protocol for radiometric assay of phagocytosis. The following points deserve emphasis: (a) since the assay depends on the measurement of the uptake of radiolabelled bacteria by the entire Md~ population within a particular culture well, care was taken that: (a) Md~ cell density was maintained at 1 _+ 0.3 x 105 to 2 _+ 0.1 x 10~ cells/well; (b) non-phagocytosed bacteria were removed at the end of the phagocytic period by repeated washings. Tuftsin was used as follows: (c) Presented along with bacteria to the Mdp culture throughout the 30 min phagocytic period; (d) bacteria were first treated with tuftsin, washed and then added to the Md~ monolayers without AB serum;
850
R. R. IYER et al. Table 1. D o s e - response phagocytic profile of 7 & 14 day culture of macrophage from five normal donors to tuftsin (0 - 7.0/aM) stimulation assayed by the ingestion of 14C-acetate labelled S. aureus in the presence of 30% AB serum (heat-inactivated) 7-day cultures Counts/min + S.D.
CONTROL TUFTS|N (MM) 0.08 0.166 0.33 0.66 3.5 7.0 Negative Control (Background)
14-day cultures
Phagocytic index
243.97 _+ 14.8
100.0 _+ 6%
N.D. N.D. 430.93 _+ 4.4 391.13 +_ 14.34 27.50 _+ 2.29 232.15 _+ 30.6 45 _+ 8
N.D. N.D. 176.63 _+ 2% t 160.63_+6%* 110.87+0.009 NS 95.15 _+ 12% NS --
Counts/min _+ S.D. 305.7 _+ 63.3 733.5 698.5 895.0 891.0 673.5 502.0 32
_+ 16.5 _+ 88.5 ___ 96.4 _+ 44 _+ 150.5 _+ 73.24 _+ 2
Phagocytic index 100.0 _+ 20.7 239.9 228.5 293.0 291.5 220.3 164.21
_+ 2.25% t _+ 12.67%* _+ 10.8%* _+ 4.9%* _+ 22.3%* _+ 14.6%*
Mean +_ S.D. of triplicate experiments; *P<0.05%; +P<0.01; *P<0.001. NS = Not significant; N.D. = not determined.
(e) h u m a n A B serum (heat inactivated) was either used at 30°7o c o n c e n t r a t i o n or absent in all the assays; (f) all experiments were d o n e in triplicate.
Assay of phagocytosis ofM. leprae by v&ual scoring of ingested bacter& M o n o c y t e s were cultured for 7 a n d 14 days o n 12 m m d i a m e t e r sterile glass cover slips (Blue Star, New Delhi) in 24-well culture plates (Linbro) as described earlier. M. leprae (heat-killed/live) o b t a i n e d f r o m h u m a n skin biopsies, were interacted with the M+ m o n o l a y e r in a M+ : b a c t e r i a ratio o f l:10+tuftsin (at c o n c e n t r a t i o n o f 0 . 8 # M a n d 35.0 ~M) for 30 m i n in the absence of 30% A B serum. N o n - p h a g o c y t o s e d b a c t e r i a were r e m o v e d by washing thrice with p r e w a r m e d M E M , the cover slips were fixed with 2°7o g l u t a r a l d e h y d e a n d stained with Ziehl - N e e l s e n ' s stain prior to microscopic e x a m i n a tion. T h e phagocytic index (PI) was calculated by r a n d o m c o u n t i n g of 300 m a c r o p h a g e s a n d expressed as the n u m b e r o f M+ c o n t a i n i n g >-3 acid fast bacilli per 100 M+ (expressed as percentage).
f r o m three d o n o r s u n d e r serum free assay conditions a n d the phagocytic assay c o n d u c t e d as in Fig. 1, but w i t h o u t any f u r t h e r a d d i t i o n o f tuftsin. Simultaneously, controls in triplicate were assayed for phagocytosis of " t u f t s i n u n t r e a t e d " radiolabelled b a c t e r i a u n d e r identical conditions.
Scanning electron microscopy (SEM) of macrophages treated with tuftsin M a c r o p h a g e s derived f r o m n o r m a l (3), B T / T T (5) a n d BL/LL (8) individuals were cultured for 14 days a n d pulsed with 0.8/~M tuftsin for 30 min, washed with RPMI-1640, a n d t h e n fixed with a mixture o f 2 . 5 % g l u t a r a l d e h y d e a n d 1% p a r a f o r m a l d e h y d e in s o d i u m cacodylate b u f f e r (0.1 M , p H 7.3) for 2 h at 4°C. The cells were washed a n d d e h y d r a t e d in a graded e t h a n o l series (30 to 100%) followed by two changes o f amyl acetate. The cover slips were dried in liquid CO2 using a critical-point drying a p p a r a t u s , s p u t t e r - c o a t e d with gold ions a n d visualized in a Joel scanning electron microscope.
Stat&tical analys& Assay of opsonic effects in the activity of tuftsin on macrophages Radiolabelled S. aureus a n d M. tuberculosis (H37Ra) (1 × 107 bacilli/ml) in n o r m a l saline c o n t a i n i n g 0.8 ~M tuftsin were i n c u b a t e d at 37°C for 1 h with c o n s t a n t agitation. The bacteria were t h e n pelleted by c e n t r i f u g a t i o n (3000 x g for 30 min) a n d w a s h e d thrice with fresh n o r m a l saline to remove u n b o u n d tuftsin. The tuftsin " o p s o n i z e d " b a c t e r i a were t h e n a d d e d to 14 day cultures of n o r m a l M+
D a t a are analysed by the one-way analysis o f variance, a n d the levels o f significance, if any, are s h o w n in the results.
RESULTS
T h e present study showed t h a t o p t i m a l results were o b t a i n e d by using M~ : b a c t e r i a in the ratio o f 1:10, with a phagocytic period o f 30 m i n a n d with
Modulation of Human Lepromatous M o n o c y t e - M ~ Functions by Tuftsin
851
350 300- ~ 250,
,~ _z 200150O
°°"r
0_
1002
t Controls
50
t
.
r e c e i v e d no T u f t s i n
(P.I. = 100-'S.D.)
0
,
o
o~5
~.0
/~
Normol
~
TT M 6
:
: LL M ~
Me,
,
3.5
TUFTSIN CONCENTRATION
7:0 IN ~ J M )
Fig. 2. Dose-response profile of tuftsin stimulated phagocytosis of ]4C-acetate labelled S. aureus by peripheral blood monocyte- macrophages from normal, B T / T T and B L / L L individuals (five subjects each) cultured in vitro for 14 days prior to the assay. The response of normal macrophages given in Table 1 has been superimposed on the B T / T T and B L / L L curve for comparison. Table 2. Phagocytosis of '4C-acetate labelled S. aureus by 14-day cultures of macrophages from three B L / L L patients over a period of 60 min in the presence of 0.8/aM tuftsin and 30°70 AB serum (heat-inactivated) Control (M~ + bacteria + 30o70 AB serum) Counts/rain _+ S.D. 675.34 -4- 28.87 779 _+ 129.4 1275.5 1481
_+ 107.5 _+ 160.54 -
-
-1991.5 _+ 157.5 2054 _+ 182.8
Test (Mdp + bacteria + 30% AB serum + 0.8/~M tuftsin) Time 8 18 38 25 36 32 45 50 53 60
min min s min min s min min min min
Counts/min _+ S.D.
Statistical significance
703.67 _+ 140.48 1093.34 + 130.07
NS NS
1280 + 130 1694.67 _+ 217.42
NS NS
2521.0 -+ 248 2377.34 _+ 117.11 -2355.34 _+ 162.9
---NS
Mean +_ S.D. of triplicate experiments; NS = not significant; Md~ = macrophages. bacteria suspended in a m i n i m a l v o l u m e o f 0.3 ml o f m e d i a (Tables l , 2 a n d 3). These o b s e r v a t i o n s a p p e a r to be related to the f r e q u e n c y o f p h a g o c y t e b a c t e r i a e n c o u n t e r s or hits, a n d s u p p o r t s earlier findings (Lehnert & M o r r o w , 1985). A progressive increase in phagocytic response to the labelled S. a u r e u s with increasing c o n c e n t r a t i o n o f tuftsin was seen in 7 a n d 14 day old M~ cultures f r o m n o r m a l subjects (Table 1). M a x i m a l phagocytic activity was at 0 . 3 3 / a M tuftsin a n d 14-day n o r m a l M~ cultures displayed a p p r o x i m a t e l y d o u b l e the
increase in phagocytic activity with 0.33/aM tuftsin a n d c o n t i n u e d at the same m a x i m a l level up to 0.66/aM tuftsin. H i g h e r doses o f tuftsin (7.0/aM) decreased the response to a value s h o w n by u n s t i m u lated controls. W h e n 14-day cultures f r o m the five B T / T T a n d B L / L L patients (Fig. 2) were tested u n d e r identical conditions, the p a t t e r n o f response s h o w e d some interesting features w h e n c o m p a r e d with n o r m a l Md~ cultures. In B T~ T T cultures m a x i m a l response was at 0.66/aM tuftsin a n d persisted up to 3.5/aM tuftsin
852
R. R. IYERet al. 400-
300 O:
C~ z ¢_)
m I-
200
~ S e r u m free
0 0 -IO-
--
I\
100
30% Serum
' C o n t r o l s
50
•
,
i
3
,
,
~
.
.
.
.
7
.
.
i
14
DAYS O F C U L T U R E
Fig. 3. Relationship between age of culture and the response to 0.8/aM tuftsin stimulated phagocytosis of ~4C-acetate labelled M. tuberculosis (H37Ra) by blood monocyte-macrophages from five normal donors under 30% AB serum and serum free assay conditions. The macrophages were pretreated with tuftsin for 10 min prior to interaction for 30 min with bacteria.
which is 5 to 10 times the dose optima for 14-day normal M~. The response also showed a lag period compared to normal Md~. The phagocytic response with 0.66/aM and 3.5/aM tuftsin were higher by 28% and 50% (in P.I. units) respectively compared to that of normal macrophages. The response curve thus appears to have shifted to the right in 14-day B T / T T cultures. In contrast, 14-day B L / L L cultures showed no statistically significant stimulation of phagocytic activity over the entire range of tuftsin concentration up to 7/aM. This lack of response exhibited by B L / L L Mdp was not due to the inadequate time of 30 rain allowed for phagocytosis. In a second experiment using 14-day cultures from three B L / L L patients, the monolayers were incubated with labelled S. aureus for 60 min with/without 0.8/aM tuftsin but were refractory to tuftsin stimulation (Table 2). Subsequent experiments were done using a tuftsin concentration of 0.8/aM which was in the optimal range and a phagocytic period of 30 min. Since the 14-day normal M~ cultures responded more in terms of phagocytic stimulation than 7-day old cultures, the phagocytic response profile was assessed with duration of culture age. The role of 30% AB serum in terms of synergism in phagocytic activity if any, was also evaluated.
Normal macrophages cultured for 6 h to 14 days were stimulated with 0.8/aM tuftsin in the presence of labelled M. tuberculosis (H37Ra) (Fig. 3, Table 3). Further, 6 h old normal M~ showed a high degree of basal phagocytic activity (counts/min = 2796 _+ 10; Table 3) while the increment in terms of phagocytic stimulation by tuftsin was small. However, the basal activity falls in 3, 7 and 14-day old cultures, but the ability to respond in phagocytic activity on tuftsin stimulation shows a progressive increase. Also the difference in the basal activity between 3, 7 and 14-day cultures is only about 200 counts/min (Table 3) while the increase in 14-day old normal M+ is nearly 3 to 4 times on 3-day cultures. Finally there was no statistically significant increase in phagocytic activity contributed by 30% human AB serum. While 7 and 14-day BT/TT Md~cultures from eight patients showed a similar response as described above under serum free assay conditions, the 14-day old cultures of B L / L L Md~ of eight patients tested exhibited a decreased response to levels that are statistically insignificant (Fig. 4). Since the increase in phagocytosis on tuftsin stimulation was seen to vary directly in the case of normal and B T / T T M~ and inversely in the case of B L / L L M+ with duration of culture age, all further experiments on phagocytosis were conducted on M~b cultured at two different time periods (7 days and 14 days). Table 4 gives the results of the phagocytic response to tuftsin by 7 and 14-day old cultures of normal, B T / T T and B L / L L M+ from 5, 8 and 8 subjects respectively when assayed for the ingestion of heatkilled and live M. leprae. Normal 7-day cultures showed a 1.6 and 1.8 times greater phagocytosis than controls with heat-killed and live M. leprae respectively on stimulation with 0.8/aM tuftsin. By day 14 of culture, the response increased to 2.36 and 2.16 times for heat killed and live M. leprae respectively. Similarly, 7 day B T / T T cultures showed 1.98 and 1.95 times phagocytic stimulation by 0.8/aM tuftsin which increased to 2.3 and 2.57 times in 14-day cultures for heat-killed and live M. leprae respectively. On the contrary, 7-day B L / L L tuftsin stimulated cultures showed phagocytic increase of 2.3 and 2.35 times for heat-killed and live M. leprae respectively, but were refractory to 35/aM tuftsin. By day 14, the cultures became refractory to 0.8 taM tuftsin, but being stimulated at 35/aM tuftsin with an increase of 2.09 and 2.08 times greater phagocytosis for heat-killed and live M. leprae respectively. The phagocytosis stimulating effect to tuftsin is not dependent on a non-specific charge based opsonic effect (tuftsin is highly basic; PI = 8.9).
Modulation of Human Lepromatous Monocyte-Mdp Functions by Tuftsin
853
Table 3. Relationship between age of culture and the phagocytic response to 0.8/aM tuftsin of macrophages from five normal donors assayed by the ingestion of ~4C-acetate labelled M. tuberculosis (H37Ra) in the presence and absence of 300/0 serum (heat-inactivated) Normal Macrophages Age of
Serum free assay
culture
Control counts/min
2796
P.I. counts/min
30o/o AB serum containing assay
Test (0.8/aM tuftsin)
Control 2675
Test (0.8/aM tuftsin)
_ 10
3705.5 _+ 334.5
_+ 250
3287.5 + 92.5
100 756
+ 0.3% _+ 86
132.5 _+ 9% (NS) 1326.5 _+ 232.5
100 930
_+ 9.3% _+ 67
122.8 _+ 2.8% (NS) 1942 _+ 170
P.I. counts/min
100 636
_+ 11.40/0 _ 17
175.5 + 17.5°70* 1793.7 _ 78.1
100 ___ 7.2070 684.6 _+ 13.5
208.8 _ 8.750/0* 1666 + 163.2
P.I. counts/min
100 _+ 2.7°70 816.5 _+ 26.2
282 3141
100 _+ 2°70 870.4 _+ 21.85
243.4 _+ 9.8O7ot 2607.7 _+ 76.84
P.I.
100
6h
3 days
7 days _+ 4.35% t -4- 120
14 days _+ 3.2O/o
384.7 _+
3.8O7o*
100
+ 0.25o/o
299.6 _+
0.09o/o*
Mean _.+ S.D. of triplicate experiments; */:'<0.05; tp<0.01; *P<0.001. NS = Not significant.
"~
-:
300.
200.
~ ".
o-.--o LL M~
"-
Q
"-
,j
",
I--
~,
0
<
lOO, ---// Controls
l "t---O
0
receive
no T u f t s i n
P.I,=100 *-- S.D. i
7
I/4 DAYS OF CULTURE
Fig. 4. Relationship between age of culture and the phagocytic response to 0.8/aM tuftsin by B T / T T and B L / L L macrophages from eight patients each as measured by the ingestion of ~4C-acetate labelled M. tuberculosis (H37Ra) under serum free assay conditions. The macrophages were pretreated with tuftsin for 10 min prior to interaction for 30 min with bacteria. T u f t s i n treated b a c t e r i a were n o t p h a g o c y t o s e d at significantly greater degrees t h a n u n t r e a t e d bacteria (Table 5). F u r t h e r Mdp p r e t r e a t e d with 0 . 8 / a M tuftsin
for 10 m i n followed by a 30 m i n i n t e r a c t i o n with labelled bacteria exhibited phagocytic s t i m u l a t i o n to the same degree as when tuftsin was present with b a c t e r i a for the entire 30 m i n phagocytic period. F u r t h e r , scanning electron m i c r o g r a p h s clearly d e m o n s t r a t e d two features o f the effect o f tuftsin (Fig. 5): (1) while control m o n o l a y e r s showed the typical fried egg m o r p h o l o g y o f u n s t i m u l a t e d m a c r o p h a g e s with a central ruffled cell b o d y a n d a large spread-out peripheral cytoplasmic area with m i n i m a l ruffling, 0 . 8 / a M tuftsin treated 14-day old n o r m a l a n d B T / T T Mdp cultures showed a d r a m a t i c increase in fllopodia a n d k n o b b e d processes giving the cells sea-urchin or porcupine-like a p p e a r a n c e . T h e peripheral cytoplasm was t h r o w n out as filop o d i a a n d the cell a t t a c h e d to the s u b s t r a t u m by long retraction fibrils. (2) B L / L L Mdp cultures showed m i n i m a l u l t r a s t r u c t u r a l alterations after tuftsin t r e a t m e n t which included a slight increase in filopodia with small retraction fibrils at the cell periphery b u t was otherwise very similar in external m o r p h o l o g y to u n t r e a t e d controls. DISCUSSION Tuftsin is a well d o c u m e n t e d stimulator o f m a c r o phage phagocytic a n d microbicidal functions (Najjar, 1983). O u r results show t h a t 0.66 to 0.8/aM tuftsin is the o p t i m a l range for phagocytic stimulation which is within the observed range (Flemming, 1985). W e have quantitatively assayed phagocytosis by m a c r o p h a g e s using: (a) visual scoring o f ingested
854
R . R . IVE~ et al.
Table 4. The phagocytic response to tuftsin by 7- and 14-day-macrophage cultures from three normal, eight B T / T T and B L / L L subjects assayed by visual counting of the number of macrophages containing >13 M. leprae per 100 macrophages under serum free assay conditions (a) NormalMacrophages Heat killed M. leprae Age of culture
Control (P.I. in %)
7 days 14 days
48.3 _+ 5% 40.4_+8.2%
7 days 14 days
44.1 _+ 1.6% 41.8 _+ 0.9%
Heat killed M. leprae
Live M. leprae
Live M. leprae
Test Control (0.8 taM tuftsin) (P.I. in %) (P.I. in %) 78.2 _+ 3.4%** 45.6 _+ 6.8% 95.6_+ 1.8%** 45 _+ 3.1% (b) B T / T T Macrophages
Test (0.8 taM tuftsin) (P.I. in %) 82.4 _+ 5.6% t 97.2 + 3.01% t
85.6 +_ 2.7%* 40.1 _+ 5.2% 96.7 _+ 1.9%** 36.7 + 3.6% (c) B L / L L Macrophages
78.5 _+ 2%** 94.6 + 2.7%**
Control Tuftsin (0.8 taM) Tuftsin (35 taM) Control Tuftsin (0.8 taM) Tuftsin (35 taM)
7 days (P.I.) 35.8 _+ 8.2% 82.4 _+ 2.1°70** 32.9 _+ 2.6% (NS) 38.1 _+ 5.7% 89.91 _+ 3.6%** 40.2 _+ 4.1 (NS)
14 days (P.I.) 40.8 + 2.3% 38.6 + 4.7% (NS) 85.6 + 5.1%** 44.1 _+ 5.6% 41.3 _+ 6.4% (NS) 92.1 _+ 1.9%**
Mean + S.D. of triplicate experiments; *P<0.05; NS = not significant; tp<0.01. bacteria in stained specimens under the light microscope (Stossel, 1975); (b) measurement of ingested radiolabelled bacteria or particles (Michell, Pancake, Noseworthy & Karnovsky, 1969; Suzuki, Booth & Grecz, 1971); and (c) lysis of macrophages followed by plating on agar. The d o s e - r e s p o n s e profile of phagocytosis of S. aureus and M . tuberculosis (H3vRa)clearly demonstrates two points - - (a) at the optimal tuftsin concentration, 14-day old normal M+ cultures show a greater increase in phagocytosis than 7-day old cultures; and (b) 14-day old B T / T T cultures exhibit a shift to the right in the d o s e - r e s p o n s e curve for S. aureus. In the analysis of r e c e p t o r - l i g a n d binding curves, a shift to the right indicates an increased dissociation of the complex and decreased binding of the ligand to the receptor. The refractoriness of 14-day old B L / L L M+ to the range of tuftsin used (0.8 ~M to 7.0/~M) for assaying the phagocytosis of S. aureus and M . tuberculosis at 0.8 ~M tuftsin respectively, may be explained as an extreme shift to the right of the above d o s e - response curve, raising the possibility that - - (a) B L / L L M+ (14-day) have a decreased number of tuftsin receptors or receptors with altered binding affinities; or (b) the decreased response at higher concentrations may be due to the production of a significant amount of the inhibitory tripeptide, L y s - P r o - A r g due to enzymatic cleavage of tuftsin by macrophage membrane aminopeptidases (Najjar, 1983).
The fact that B L / L L M+ display a differential response at 7 and 14 days of culture to 0.8/aM and 35.0taM tuftsin as measured by ingestion of M . leprae strengthens the earlier inference of altered tuftsin receptor expression (either in number or in affinity for ligand) in these cells with increasing age of culture. The response of 14-day old B L / L L cultures at 35/aM tuftsin may indicate expression of tuftsin receptors with a lowered affinity for its ligand in the older cultures. Multiple receptors with varying affinities for an identical or similar ligand have been documented for Fc r e c e p t o r - I g G interactions in murine and human systems (Diamond & Yelton, 1981; Unkeless, Fleit & Mellman, 1981; Tax, Hermes, Willems, Capel & Koene, 1984; Looney, A b r a h a m & Anderson, 1986). The existence of dual receptors with varying affinity for tuftsin and related peptides was postulated (Bar-Shavit, Terry, Blumberg & Goldman, 1982; Segal, Tzehoval & Feldman, 1983). The expression of these two receptor forms may vary in concentration on different phagocytic cells and stages of differentiation. The isolation of a tuftsin binding protein (Bump, Lee, Wleklik, Reichler & Najjar, 1986) from rabbit peritoneal neutrophil membranes which exists as a dominant 500,000 mol. wt form and a subordinate 250,000 mol. wt form supports the above postulation. In the present study the results point to a decreased ability of B L / L L M+ to exhibit tuftsin-stimulated phagocytosis with increasing age of culture, though
Modulation of Human Lepromatous M o n o c y t e - M ~ Functions by Tuftsin
M~ alone
855
M~ + Tuftsin
Normal
BT/TT
BL/LL
Fig. 5. Morphologic changes induced by 0.8/~M tuftsin treatment of 14-day macrophage cultures derived from normal, B T / T T and B L / L L individuals (three each) as visualized by SEM.
Modulation of Human Lepromatous Monocyte- M~b Functions by Tuftsin
857
Table 5. Assay of opsonic effect in the stimulatory response to tuftsin by 14-day normal macrophage cultures Untreated radiolabelled bacteria S. aureus
Counts/min + S.D. Phagocytic index
1425 + 115.4 100 + 8.09°/0
Tuftsin treated radiolabelled bacteria
H37Ra
S. aureus
H37Ra
2678 _ 33.8 100 _+ 1.3%
1398 _+ 211.8 98.1 _+ 0.0%
3019 _ 412 112.7 + 15.4% (NS)
Mean _+ S.D. of triplicate experiments; NS = not significant.
the basal phagocytic ability o f the u n s t i m u l a t e d M~ cultures was m o r e or less the same in all the three groups. Thus, while B L / L L patients m a y exhibit a d e q u a t e non-specific i m m u n e defences to various b a c t e r i a at a basal level, they a p p e a r u n a b l e to e n h a n c e these f u n c t i o n s o n stimulation. T h e o b s e r v a t i o n t h a t t u f t s i n - t r e a t e d S. a u r e u s a n d M . t u b e r c u l o s i s were p h a g o c y t o s e d to the same extent as u n t r e a t e d b a c t e r i a u n d e r serum free assay c o n d i t i o n s rules o u t the m e c h a n i s m o f non-specific opsonic e f f e c t s / c h a r g e b a s e d p h e n o m e n a for tuftsin o n M~ phagocytic f u n c t i o n . Finally, the S E M p h o t o g r a p h s establish the effect o f tuftsin o n the surface m e m b r a n e o f n o r m a l a n d B T / T T cultures (14-day), while B L / L L cultures lack a similar effect. P h a g o c y t i c activity o f m o n o c y t e / m a c r o p h a g e s in leprosy as a f u n c t i o n of d u r a t i o n o f culture age has
n o t been r e p o r t e d so far. O u r d a t a suggest t h a t the peripheral b l o o d m o n o c y t e s in B L / L L patients exhibit a c h a n g e towards a n end stage t h a t is relatively refractory to activation by agents like tuftsin. O u r results showing a differential response to tuftsin with varying age in culture indicate t h a t the level of d i f f e r e n t i a t i o n o f the M~ p o p u l a t i o n has to be a c c o u n t e d for in studies investigating i m m u n o modulatory approaches. W e are currently studying the possibilities raised in this study by investigating serum tuftsin levels a n d tuftsin receptor characteristics. Acknowledgements - - We thank Prof. I. Nath, Depart-
ment of Biotechnology for providing laboratory facilities and valuable guidance. The assistance of Dr S.N. Upadhya, National Institute of Immunology, New Delhi, during scanning electron microscopy of macrophage cultures is gratefully acknowledged.
REFERENCES
BAR-SHAVIT, Z., TERRY, S., BLUMBERG,S. • GOLDMAN, R. (1982). Neurotensin-macrophage interaction: specific binding and augmentation of phagocytosis. Neuropeptides, 2, 325 - 335. BOYUM, A. (1968). Separation of leukocytes from blood and bone marrow. Scand. J. clin. Lab. Invest., 97, 7 7 - 8 9 . BUMP, N. J., LEE, J., WLEKLIK, M., REICHLER, J. & NAJJAR, V. A. (1986). Isolation and subunit composition of the tuftsin receptor. Proc. natn. Acad. Sci. U.S.A., 83, 7187-7191. DIAMOND, B. & YELTON, D. E. (1981). A new Fc receptor on mouse macrophages binding IgG3. J. exp. Med., 153, 514-519. FLEMMING, K. B. P. (1985). Stimulation and depression of the RES by pharmacological agents. In The Reticuloendothelial System: A Comprehensive Treatise (eds Hadden, J. W. and Szentivanyi, A.) Vol. 8, pp. 247-284. Pharmacology, Plenum Press, New York. GODAL, Z. (1978). Immunological aspects of leprosy - - present status. Prog. Allergy, 25, 211-242. KAPLAN, G. 8z COHN, Z. A. (1984). The immunology of leprosy. Int. Rev. exp. Path., 28, 45 - 7 8 . LEHNERT, B. E. & MORROW, P. E. (1985). The initial lag in phagocytic rate by macrophages in monolayer is related to particle encounters and binding. I m m u n . Invest., 14, 5 1 5 - 521. LOONEY, R. J., ABRAHAM, G. N. 8z ANDERSON, C. L. (1986). Human monocytes and U937 cells bear two distinctive Fc receptors for IgG. J. I m m u n . , 136, 1641 - 1647. MICHELL, R. H., PANCAKE, S. J., NOSEWORTHY, J. & KARNOVSKY, M. (1969). Measurement of rates of phagtocytosis: the use of cellular monolayers. J. cell. Biol., 40, 216-224. NAJJAR, V. A. (1983). Tuftsin, a natural activator of phagocytic cells: an overview. Ann. N . Y . Acad. Sci., 419, I - 11. PRASAD, H. K. & NATH, I. (1981). Incorporation of 3H-thymidine in M. leprae residing in differentiated human macrophages. J. Med. Microbiol., 14, 279-293. RAO, O. N., IYER, R. R., PRASAD, H. K. & NATH, I. (1987). A novel synthetic route to the phagocytosis stimulation tetrapeptide, "tuftsin". Med. Sci. Res., 15, 1199-1201.
858
R . R . IYER et al.
RIDLEY, D. S. & JOPLING, W. H. (1966). Classification of leprosy according to immunity. A five group system. Int. J. Lepr., 34, 2 5 5 - 273. SEGAL, S., TZEHOVAL, E. & FELDMAN, M. (1983). The function of tuftsin and similar sequences in other proteins. Ann. N.Y. Acad. Sci., 419, 168-178. SmLEY, D. L. & KRAHENBUHt, J. L. (1987). Mycobacterium leprae-burdened macrophages are refractory to activation by gamma interferon. Infect. Immun., 55, 446- 450. STOSSEL, T. P. (1975). Phagocytosis: recognition and ingestion. Semin. Hemat., 12, 8 3 - 116. SuzuKh J. B., BOOTH, R. R. & GRECZ, N. (1971). Evaluation of phagocytic activity by ingestion of labelled bacteria. J. Infect. Dis., 123, 9 3 - 96. TAX, W. J. M., HERMES, F. F. M., WILLEMS, R. W., CAPEL, J. A. • KOENE, R. m. P. (1984). Fc receptors for mouse lgGl on human monocytes: polymorphism and role in antibody-induced T cell proliferation. J. Immun., 133, 1185- 1189. UNKELESS, J. C., FLEIT, H. & MELLMAN, I. S. (1981). Structural aspects of the heterogeneity of immunoglobulin Fc receptors. Adv. Immun., 31, 247.