lmmunopharmacology, 13 (1987) 159-171

Elsevier IMO 00368

159

The immunosuppressive activities of two abortifacient proteins isolated from the seeds of bitter melon (Momordica charantia)
S.O. Leung, H.W. Yeung and K.N. Leung
Department of Biochemistry and Chinese Medicinal Material Research Centre, Chinese University of Hong Kong, Hong Kong
(Received 1 April 1986; accepted 27 January 1987)

Abstract:

Two abortifacient proteins, ct- and fl-momorcharin, have been purified from the seeds of the bitter melon (Momordica charantia). It was found that non-cytotoxic concentrations of these plant proteins can significantly inhibit the mitogenic responses of

mouse splenocytes to concanavalin A, phytohaemagglutinin and lipopolysaccharide in a dose-dependent manner. In addition, the alloantigen-induced lymphoproliferation and the in vitro generation of a primary cytotoxic lymphocyte response were severely suppressed in the presence of these proteins. In contrast, the cytolytic activity of cytotoxic lymphocytes and natural killer cells was unimpaired by in vitro exposure to momorcharin. On the other hand, a clear decrease in the functional capacity of macrophages, such as the cytostatic and phagocytic activities, was observed under similar conditions. In vivo studies have shown that single injections of nontoxic microgram amounts of momorcharin into mice resulted in a significant depression of the delayed-type hypersensitivity response as well as the humoral antibody formation to sheep red blood cells. Similarly, the thioglycollate-induced in vivo migration of macrophages was also suppressed. Interestingly, the in vivo activation of natural killer cells was not appreciably affected. Our data suggests that the observed potent immunosuppressive effect of ct- and fl-momorcharin is unlikely to be due to direct lymphocytotoxicity or due to a shift in the kinetic parameter of the immune response. Key words: Abortifacient protein; Bitter melon; Immunosuppression; Murlne immune response

Introduction
The bitter melon (Momordica charantia) of the family Cucurbitaceae is a plant widely cultivated in many tropical and subtropical regions and is frequently used in the Orient as a foodstuff or tonic.
Correspondence: Dr. K.N. Leung, Department of Biochemistry, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong. Abbreviations: Con A, concanavalin A; CTL, cytotoxic T lymphocytes; DTH, delayed type hypersensitivity; FCS, foetal calf serum; 3H-TdR, [3H]thymidine; LPS, lipopolysaccharide; MCI, Momordica charantia inhibitor; MLR, mixed lymphocyte reaction; NK, natural killer; PBS, phosphate-buffered saline; PEC, peritoneal exudate cells; PFC, plaque-forming cells; PHA, phytohaemagglutinin; SRBC, sheep red blood cells.

Extracts from various components of this plant have been reported to have hypoglycaemic activity (Akhtar et al., 1981; Meir and Yaniv, 1985), protein synthesis inhibitory activity (Lin et al., 1978; Barbieri et al., 1979), guanylate cyclase inhibitory activity (Vesely et al., 1977; Takemoto et al., 1980), antitumor properties (Claflin et al., 1978; Jilka et al., 1983), antifertility effects (Dixit et al., 1978) and abortifacient activity (Morton, 1967). Of particular interest is that a number of bioactive proteins have recently been isolated and characterized from the seeds of the bitter melon. Thus, Lin et al. (1978) have purified two galactose-binding lectins from M. charantia seeds and one of them (known as momordin) exhibits moderate inhibitory activity on protein synthesis of Ehrlich ascites tumor cells. Bar-

0162-3109/87/$03.50 1987 Elsevier Science Publishers B.V. (Biomedical Division)

160 bieri et al. (1980), on the other hand, have purified two ribosome-inactivating proteins from the seeds of M. charantia. One of these is a haemagglutinating protein known as the M. charantia lectin, while the other is a non-haemagglutinating protein called the M. charantia inhibitor (MCI). MCI was subsequently shown to possess immunomodulatory effects both in vivo and in vitro (Spreafico et al., 1983). More recently, we have isolated and purified two basic glycoproteins, ~- and /~-momorcharin, from the seeds of M. charantia (Yeung et al., 1985). Although these two proteins have similar physicochemical properties, they are immunologically distinct (Yeung et al., 1985). Both proteins were shown to be effective in inducing early and mid-term (post-implantation) abortion in the mouse (Law et al., 1983; Chan et al., 1984). In vitro studies have demonstrated that these abortifacient proteins adversely affected the compaction of blastomeres and disrupted the formation of the blastocyst (Tam et al., 1985). In view of the potential application of this class of abortifacient proteins for clinical use and the fact that many plant proteins can affect immune reactivity (Edwards et al., 1981; Spreafico et al., 1983; Descotes et al., 1985), it is of interest to study the immunomodulatory activities of these two abortifacient proteins. In this paper, we report that ~- and /~-momorcharin are not directly cytotoxic to mouse lymphocytes in vitro, yet they display potent immunosuppressive effects for a variety of cell-mediated and humoral immune responses.
Materials and Methods

seeds had been described in detail previously (Yeung et al., 1985). Briefly, the proteins were prepared by subjecting an extract of the decorticated seeds of M. charantia to acetone fractionation followed by purification on CM Sepharose CL6B and Sephadex G100 columns (Pharmacia, Sweden). The final preparations were shown to be homogeneous by SDS-polyacrylamide gel electrophoresis and immunoelectrophoresis (Yeung et al., 1985). The abortifacient activity of ~- and/3-momorcharin was assayed by injecting 20-50 /~g of the protein into pregnant mice on day 12 of gestation. This resulted in the death of over 90% of the fetuses and abortion in all the pregnant mice (Chan et al., 1984). The proteins were dissolved in phosphate-buffered saline (PBS, pH 7.3) and sterilized by millipore filtration before use. Protein concentrations were determined by the Folin-Lowry procedure (Lowry et al., 1951). Mitogen-induced lymphocyte transformation Mouse spleen cells were cultured in flat-bottomed 96-well microtiter plates (Flow Laboratories, U.K.) at 5 105 cells/well in a final volume of 0.2 ml RPMI-1640 medium (Gibco, U.S.A.) supplemented with 10% foetal calf serum (FCS) (Gibco) and containing 100 units/ml of penicillin G, 100 pg/ml of streptomycin sulfate and 3 #g/ml of fungizone (Gibco). Mitogen and/or various concentrations of abortifacient protein were added and the cultures were kept in a humidified atmosphere containing 10% C O / i n air. Mitogens (Sigma, St. Louis, MO) used include concanavalin A (Con A, 3/~g/ml), phytohaemagglutinin (PHA, 10 #g/ml) and lipopolysaccharide (LPS, 30/~g/ml). After 48 h of incubation at 37C, 0.5 /~Ci [3H]thymidine (3H-TdR) (2 Ci/mM; Amersham, U.K.) was added to each well and incubation was continued for 6 h. Cells were harvested with a Titertek multiharvester (Flow Laboratories) and radioactivity was counted in a liquid scintillation counter (LS 1801; Beckman, U.S.A.). Incubations were usually done in quadruplicates and DNA synthesis, as measured by 3H-TdR incorporation, was expressed as counts per minute (CPM).

Mice Inbred BALB/c (H-2 d) and C57BL/6J (H-2 b) mice were bred at the University Animal House, Chinese University of Hong Kong. Mice of the same age (6-10 weeks old) and same sex were used in each experiment. Abortifacient proteins The procedure used for the isolation and purification of ~- and /3-momorcharin from bitter melon

161

Mixed lymphocyte culture

Mixed lymphocyte reaction (MLR) was carried out as described by Bradley (1980), Briefly, 5 x 105 BALB/c splenocytes were treated with mitomycin C (Sigma; 50 #g/ml for 30 min at 37C) and cocultured with an equal number of C57BL/6J splenocytes in the presence or absence of momorcharin. After incubation for 72 h in a humidified atmosphere containing 10% CO2 in air at 37C, the cells were given a 20-h pulse with 0.5 pCi 3H-TdR and radioactivity incorporated was determined.

spontaneous release counts (cs) and the maximum releasable counts (Cmax) using the equation: LCt - Cma x --

Cs
Cs

Measurement (DTH)

of

delayed-type

hypersensitivity

Generation of alloreactive cytotoxic T lymphocytes in vitro and assay of cell~mediated lympholysis

The procedure described by Engers et al. (1976) was essentially followed. In brief, 2 105 mitomycin C-treated BALB/c splenocytes were co-cultured with 105 C57BL/6J splenocytes in each well of the round-bottomed 96-well microtiter plate (Costar, Cambridge, MA). Various concentrations of the abortifacient proteins were added to a final volume of 0.2 ml/well and the culture was incubated at 37C under a gas phase of 10% CO2 in air. Each well was replenished on day 2 and day 5 with 50 #1 RPMI medium supplemented with 10% heat-inactivated FCS. Cytotoxicity of the effector cells generated was measured on day 6, using the standard 51Cr-release assay. For the measurement of cellmediated lympholysis, 5 106 P815 targets (H-2 d) were labelled with 200 ktCi SlCr (NaSlCrO4, 350 600 mCi/mg chromium; Amersham) for 1 h at 37C. The cells were then washed twice with RPMI medium and the cell concentration adjusted to 105/ml. On day 6, 100 #1 supernatant was removed from each well of the primary culture and 104 5~Cr-labelled P8 t 5 cells in 0.1 ml volume were added. After a further incubation period of 8 h, 100 #1 supernatant was carefully sucked up from each well and radioactivity counted in a gamma counter. For spontaneous lysis, 0.1 ml RPMI medium was added to 0.1 ml labelled targets. For maximum releasable 51Cr, 0.1 ml 10% Triton-X100 was added to 0.1 ml labelled target cells. The percentage specific lysis (L) was calculated from the test culture counts (ct), the

Mice in groups of four were given an intraperitoneal (i.p.) injection of ~- or fl-momorcharin (4 mg/kg body weight) either two days before, on the same day as, or two days after intravenous sensitization with 106 sheep red blood cells (SRBC). Control mice were injected with an equal volume of PBS on the same day as antigen sensitization. Four days after antigen sensitization, each mouse was challenged with 108 SRBC in 50 #1 PBS, injected subcutaneously into the right hind footpad. The same volume of PBS was injected into the left hind footpad as a control. Footpad thickness was measured 24-72 h after antigen challenge and results were expressed as the percentage mean increase in footpad thickness as described in detail previously (Leung et al., 1980).

Haemolytic plaque assay

The method of Cunningham and Szenberg (1968) was principally followed with minor modifications. Groups of C57BL/6J mice were pre-treated with ~- or fl-momorcharin (4 mg/kg body weight i.p.) or PBS two days before i.p. immunization with 4 x 108 SRBC. Spleen cells obtained 4 days later were mixed with 10% guinea pig complement and SRBC to form a monolayer in the microchamber. The number of direct (IgM) plaque-forming cells (PFC) were enumerated after incubation for 1 h at 37C. Incubations were usually done in triplicates and results were expressed as the number of PFC per 106 spleen cells.

Haemagglutination assay
C57BL/6J mice were injected i.p. with the abortifacient proteins (4 mg/kg) or an equal volume of

162 PBS. Two days later, all mice were immunized with 4 x 108 SRBC given i.p. The sera of mice collected on days 7, 14 and 21 after antigen immunization were heat-inactivated at 56C for 30 min. Serial twofold dilutions of the immune sera were made with PBS in 96-well round-bottomed microtiter plates in a final volume of 50 /d. 50 /d of 0.5% SRBC was then added to each well, the plates were allowed to stand at room temperature for 2 h and the haemagglutination endpoints were recorded. The titre of the serum was expressed as the reciprocal of the highest dilution of the serum which gave a positive result of haemagglutination.

Phagocytosis assay

Natural killer cell assay

Natural killer (NK) cell activity was assessed by the ability of Corynebacterium parvum~activated mouse spleen cells to lyse the NK-sensitive YAC-1 target cells in a 4-h s~Cr-release assay as described in detail elsewhere (Ojo et al., 1978). In essence, mice were injected intravenously with 350/~g formalinkilled C. parvum (Wellcome Research Laboratories, U.K.) and splenic effector cells were assayed for N K activity 4 days later. The effect of ~- and pmomorcharin was determined by either pre-treatment of mice with the proteins (4 mg/kg i.p. 2 days before injection of C. parvum) or by adding the proteins (100 #g/ml) directly to the mixture of effector and target cells in the assay.

The latex uptake method, as described by Kohl et al. (1977), was followed with minor modification. C57BL/6J mice were injected i.p. with 1 ml 10% proteose peptone (Difco). The PEC harvested 3 days later were suspended in RPMI medium + 10% FCS at 2 x 106/ml. The in vitro effect of momorcharin on the phagocytic activity of proteose peptone-elicited PEC was examined by pre-incubating 0.5 ml PEC with a final concentration of 100 /~g/ml protein for 6 h at 37C in a siliconized borosilicate test-tube. 25/A of 1% latex particles (0.8 /~m; Sigma) was then added and the mixture was further incubated for 1 h in a shaking water bath at 37C. The cell suspension was then layered onto 2 ml heat-inactivated calf serum and centrifuged at 300 g for 5 min to remove excess latex. The cell pellet was resuspended in RPMI medium + 10% FCS and the percentage phagocytosis was calculated by cell counting using a haemocytometer. Cells containing three or more latex particles as assessed by phase-contrast microscopy were considered as phagocytic.

Assay of macrophage-mediated cytostasis

In vivo migration of macrophages

The method described by Gervais et al. (1984) was adopted. In brief, C57BL/6J mice pre-treated either with momorcharin (4 mg/kg i.p. on day - 2 and day 0) or PBS were injected with 1 ml 10% Brewers' thioglycollate medium (Difco, Detroit, MI). Three days later, the elicited plastic-adherent peritoneal exudate cells (PEC) were evaluated as described in detail previously (Mak et al., 1982).

This is carried out as described by Ruffmann et al. (1984). Briefly, 2.5 mg picolinic acid (Sigma) dissolved in PBS was injected into C57BL/6J mice. Three days later, the PEC were harvested and resuspended in RPMI medium + 10% FCS at 2 x 106/ml. 0.1 ml PEC suspension was added onto each well of a flat-bottomed 96-well microtiter plate together with 0.1 ml momorcharin or control medium. After 6 h of incubation at 37C, non-adherent cells and momorcharin were removed by three washes with warm medium. 104 MBL-2 cells (a Moloney virus-induced T cell lymphoma of C57BL/6J mice) supplemented with LPS (2 ng/ml) were added to each well in a final volume of 0.2 ml. After incubating for 48 h at 37C in a humidified

163 atmosphere containing 10% CO2 in air, the cells in each well were pulsed with 0.5 #Ci 3H-TdR and then harvested 5 h later using a Titertek multiharvester. The percentage growth inhibition of MBL-2 cells induced by macrophages was calculated from the 3H-TdR incorporation of MBL-2 cells in the presence (I+m) or absence ( l - m ) of macrophages using the equation: Results

Effect of momorcharin on mitogen-induced and aL loantigen-induced lymphoproliferation

The in vitro effect of :- and fl-momorcharin on DNA synthesis in Con A-stimulated lymphocyte transformation is shown in Table I. Both proteins brought about a decrease in D N A synthesis in a dose-dependent manner from 0.1 100 #g/ml to nearly the same extent. Similar results were obtained using PHA and LPS as the mitogens (Table I). It is clear that both proteins were more inhibitory fo the PHA response than to the Con A and LPS responses. In all cases, almost complete suppression was seen at protein concentrations of 50-100 /~g/ml. This is also true for the allogeneic stimulation in the one-way mixed lymphocyte reaction (Table II), using splenocytes from C57BL/6J

% Cytostasis = 1 - - I

I+m
m

x 100

Stat&tical analysis
All results are expressed as the arithmetic mean =1= standard error. Student's t-test was used to determine the confidence limits in group comparison.

TABLE I Effect of in vitro exposure to ~- and fl-momorcharin on the response of murine splenocytes to mitogens a

Protein added to culture

Protein concentration (#g/ml)

3H-TdR incorporation (CPM S.E. x 10 -3) Con A (3 #g/ml) 105.4 111.0 95.4 N Dc 66.4 N D 11.0 100.3 104.0 N D 72.6 N D 29.4 + 6.5 6.0 5.5 -5 10 37* 90* 5 1 31" 72* Suppressionb LPS (%) (30 #g/ml) 40.9 2.5 38.4 34.3 N D 16.9 N D 3.2 36.6 28.1 N D 19.5 N D 4.2 2.7 2.5 1.7 0.2 5:1.3 2.8 1.2 0.3 6 16 59* 92* 11 31" 52* 90* Suppressionb PHA (%) (10 ,ug/ml) 4.1 0.1 2.7 1.5 1.1 0.7 0.2 N D 2.8 + 2.0 + 0.9 + 0.7 0.3 + N D 0.2 0.1 0.02 0.02 0.02 34* 63* 73* 83* 95* Suppressionb (%)

-~-Momorcharin 0.1 1 5 10 50 100 0.1 1 5 10 50 I00

11.5 4- 2.0 + 7.0 4.0 9.0 3.5

fl-Momorcharin

0.2 0.1 0.03 0.03 0.02

32* 51" 78* 83* 93*

a Mouse spleen cells (5 x 10S/well) were stimulated with a predetermined optimal concentration of mitogen in the presence or absence of momorcharin. Cells cultured for 48 h were given a 6-h pulse with 3H-TdR (0.5 /~Ci/well) and radioactivity incorporated was measured. b Percent suppression was obtained by comparison with the corresponding control culture in which no momorcharin was added. c Not done. * Significantly different from control, p < 0.05.

164 TABLE II Effect of in vitro exposure to ~- and/~-momorcharin on mixed lymphocyte reaction a Protein added to culture -~-Momorcharin Protein 3H-TdR concentration incorporation (#g/ml) (CPM S.E.) -0.1 1 5 10 50 0.1 1 5 10 50 1804 97 1504 1045 514 343 [92 1294 761 447 214 95 77 142 42 37 12 17 42* 72* 81" 89* 28 58* 75* 88* 95* Suppression b (%)

Effect of momorcharin on the primary alloreactive cytotoxic T lymphocyte response in vitro

D a t a in T a b l e II1 showed that there is a m a r k e d inhibition o f c y t o t o x i c T l y m p h o c y t e ( C T L ) gene r a t i o n at m o m o r c h a r i n c o n c e n t r a t i o n s as low as 0.1 /~g/ml. In c o n t r a s t , the lytic activity o f the alloreactive C T L was not a p p r e c i a b l y affected in the presence o f p r o t e i n over a wide range o f c o n c e n t r a tions (0.1 50/~g/ml) (Table IIl).

Suppression of a DTH response by momorcharin

Mice are k n o w n to develop a D T H response following i n t r a v e n o u s injection o f S R B C , a n d the m a x i m u m response is detected 4 d a y s after antigen sensitization ( M i t s u o k a et al., 1978). E x p e r i m e n t s were d o n e to e x a m i n e whether a d m i n i s t r a t i o n o f c~- a n d / L m o m o r c h a r i n into mice can affect their ability to develop a D T H response in vivo. G r o s s o b s e r v a t i o n s have shown t h a t p r e - t r e a t m e n t o f C57BL/6J mice with m o m o r c h a r i n alone (4 m g / k g b o d y weight) h a d no general toxicity a n d no significant cellular d e p l e t i o n in the l y m p h o i d o r g a n s was detected. The results in Fig. 1 show that ~- a n d / % m o m o r c h a r i n b o t h significantly inhibited the in vivo i n d u c t i o n o f a D T H response to S R B C when a d m i n i s t e r e d 2 d a y s p r i o r to, on the same d a y as, or 2 days after antigen injection, indicating that these two p r o t e i n s m a y interfere with the activation a n d / o r differentiation o f D T H T cells in vivo. The o b s e r v e d suppression is a p p a r e n t l y not due to a shift in the kinetic p a r a m e t e r o f the i m m u n e response, as the kinetics o f the D T H response in the c o n t r o l mice a n d p r o t e i n - t r e a t e d mice were similar a n d m a r k e d suppression o f the D T H response was seen b o t h 24 a n d 48 h after challenge (Fig. 1).

fl-Momorcharin

216 156 41 9 8

a 5 x 105 BALB/c splenocytes were treated with mitomycin C (50/~g/ml for 30 min at 37C) and co-cultured with an equal number of C57BL/6] splenocytes in the presence or absence of momorcharin. After incubation for 72 h at 37C, the cells were pulsed with 0.5 ~tCi 3H-TdR for 20 h and radioactivity incorporated was measured. b Percent suppression was obtained by comparison with the control culture in which no momorcharin was added. * Significantly different from control, p < 0.05.

mice as r e s p o n d e r s a n d m i t o m y c i n C - t r e a t e d B A L B / c splenocytes as stimulators. The o b s e r v e d suppressive effect o f c~- a n d / L m o m o r c h a r i n on mitogen- a n d a l l o a n t i g e n - i n d u c e d l y m p h o p r o l i f e r a tion does n o t seem to result from a direct c y t o t o x i c effect o f these p r o t e i n s on m o u s e l y m p h o c y t e s in vitro since no significant decrease in the viability o f m o u s e splenocytes was o b s e r v e d after i n c u b a t i n g these cells with a high c o n c e n t r a t i o n (100 #g/ml) o f p r o t e i n for up to two d a y s ( d a t a n o t shown), as a s s a y e d by the t r y p a n blue dye exclusion m e t h o d (Philip, 1973).

Humoral immune response of momorcharin-treated mice

C57BL/6J mice were either treated with m o m o r charin (4 m g / k g b o d y weight i.p.) or PBS 2 days

165 TABLE Ill Effect of momorcharin on the primary alloreactive CTL response in vitro" Protein added to culture Protein concentration (#g/ml) Specific s'Cr release (%; mean 3: S.E.)

Induction phase b 80.7 4- 2.4 e-Momorcharin 0. 1

1

Effector phase c 80.7 4- 2.4 72.7 76.8 77.4 74.3 71.3 82.8 75.7 79.0 76.3 71.3 4- 2.9 4- 1.6 3:7.8 3:6.8 3:5.5 3:1.4 4- 4.6 3:3.0 3:3.0 3:5.0

5 10 50 /~-Momorcharin 0.1 1 5 10 50

31.4 4- 6.7* 16.7 4.5* 3.6 3: 1.5" 0* 0* 49.8 32.0 9.2 3.6 1.3 3: 9.8* 3: 0.9* 4- 3.0* 3: 0.9* 3: 0.9*

" Primary alloreactive CTL were generated by co-culturing l0 s C57BL/6J (H-2 b) splenocytes with 2 x 105 BALB/c (H-2d) splenocytes for 6 days at 37C. Cytotoxicity was measured on P815 (H-2d) targets using a 8-h 51Cr-release assay. b Various concentrations of momorcharin were added at the beginning of the mixed lymphocyte culture. c Various concentrations of momorcharin were added together with l04 51Cr-labelled P815 target cells to each well containing the 6 day primary effector cells. Momorcharin alone was found to have no effect on the spontaneous release of the target cells over the 8-h assay period. * Significantly different from control, p < 0.05. before i m m u n i z a t i o n with S R B C (4 x 108 i.p.). T h e splenic P F C c o u n t was e n u m e r a t e d 4 d a y s after antigen sensitization whereas the circulating h a e m a g g l u t i n a t i n g a n t i b o d y titre was d e t e r m i n e d at d a y 7, 14 a n d 21 p o s t - i m m u n i z a t i o n . It was f o u n d t h a t p r e - t r e a t m e n t o f mice with e- o r / ? - m o m o r c h a r i n caused a d r a s t i c r e d u c t i o n (70-110-fold) in the n u m b e r o f a n t i b o d y - p r o d u c i n g cells in the spleens o f i m m u n i z e d mice ( T a b l e IV). In a d d i t i o n , the levels o f circulating a n t i b o d y to S R B C , m e a s u r e d up to 3 weeks after antigen injection, were signific a n t l y depressed at all times as c o m p a r e d to those o f c o n t r o l mice ( T a b l e IV). c o n t r o l mice receiving C. parvum only. As seen in T a b l e V, the cytolytic activity o f splenocytes f r o m p r o t e i n - t r e a t e d mice t o w a r d s N K - s e n s i t i v e Y A C - 1 target cells was c o m p a r a b l e to t h a t o f the c o n t r o l mice. In a n o t h e r set o f experiments, the co-culturing o f C. parvum-activated splenocytes with ~- or /~-momorcharin (100 p g / m l ) in vitro h a d no effect on their cytolytic activity (Table V), i n d i c a t i n g that the lytic function o f a c t i v a t e d N K cells was n o t imp a i r e d in the presence o f m o m o r c h a r i n , u n d e r the p r e s c r i b e d e x p e r i m e n t a l conditions. Similar results were o b t a i n e d by p r i o r e x p o s u r e o f the effector cells to m o m o r c h a r i n for 6 h before a d d i t i o n o f the target cells ( d a t a n o t shown).

Effect of momorcharin on the induction and effector function of N K cells

In the first set o f experiments, mice were p r e - t r e a t e d with ~- o r / ? - m o m o r c h a r i n (4 m g / k g i.p.) 2 d a y s before i n t r a v e n o u s injection o f C. parvum a n d the gene r a t i o n o f splenic N K activity was c o m p a r e d to

Effect of momorcharin on macrophage migration in vivo

It has been k n o w n t h a t i.p. injection o f i r r i t a n t substances such as t h i o g l y c o l l a t e a n d p r o t e o s e p e p t o n e can increase the yield o f m a c r o p h a g e s in the peri-

166 induce the a c c u m u l a t i o n o f m a c r o p h a g e s in vivo. It was f o u n d that t r e a t m e n t with m o m o r c h a r i n alone did n o t induce a n influx o f m a c r o p h a g e s into the peritoneal cavity of mice (data n o t shown). The effect of e- a n d /~-momorcharin on the influx of m a c r o p h a g e s in response to locally administered sterile thioglycollate in vivo was then examined. The results in Table VI showed that mice treated with m o m o r c h a r i n two days before a n d on the same day as thioglycollate injection had a m u c h lower P E C n u m b e r as c o m p a r e d to mice treated with thioglycollate alone. In addition, the a c c u m u l a t i o n of m a c r o p h a g e s in the peritoneal cavity of m o m o r charin-treated mice was also significantly reduced
24 48 72 Time a f t e r footpad challenge (hours)

30
v

,~ 2o

~ lO

Fig. I. Suppression of DTH response by momorcharin. Mice in groups of four were injected with ~- or/~-momorcharin (4 mg/kg i.p.) either two days before (~1,/~1), on the same day as (e2,/~2), or two days after (e3, /~3) intravenous sensitization with 100 SRBC. Control mice (C) were injected with an equal volume of PBS on the same day as SRBC sensitization. Four days after antigen sensitization, each mouse was challenged with 10s SRBC into the right hind footpad. The DTH response, as determined by the percent specific increase in footpad thickness, was measured 24-72 h after antigen challenge. Vertical bars represent one standard error. toneal cavity of mice ( O g m u n d s d o t t i r a n d Weir, 1980). Initial experiments were p e r f o r m e d to determ i n e whether injection of m o m o r c h a r i n per se can TABLE IV

(Table VI). Thus, o u r results show that the two abortifacient proteins are p o t e n t inhibitors of cell m i g r a t i o n in vivo.

Effect of momorcharin on macrophage function in vitro

The effects o f e- a n d / % m o m o r c h a r i n o n the in vitro effector functions of the elicited a n d activated m a c r o p h a g e s were investigated. The results in T a b l e VII show that p r e - i n c u b a t i o n of the proteose peptone-elicited m a c r o p h a g e s with m o m o r c h a r i n (100/~g/ml) for 6 h at 37C significantly inhibited the phagocytic activity of the macrophages. Similarly, in vitro exposure of picolinic acid-activated

Humoral antibody response to SRBC in mice treated with ~- and #-momorcharin Pre-treatment of mice with" Number of PFC/106 splenocytesb Serum haemagglutinating antibody titerc Day 7 PBS :~-Momorcharin #-Momorcharin 644 4- 25 9 4- 5* 6 + 3* 320 0 70 + 10" 30 4- 6* Day 14 300 4- 20 24 4- 6* 33 + 18" Day 21 200 :t: 23 28 :t: 8* 33 :k 18"

a C57BL/6J mice in groups of four were pre-treated with PBS or momorcharin (4 mg/kg i.p.) 2 days before i.p. injection with 8 108 SRBC. b The number of PFCs in spleens of mice were enumerated on day 4 after SRBC administration using the modified haemolytic plaque assay (Cunningham and Szenberg, 1968). c The haemagglutinating antibody titer was expressed as the reciprocal of the highest dilution of the serum which gave a positive result of haemagglutination. * Significantlydifferent from control, p < 0.05.

167 TABLE V Momorcharin has no effect on the induction and effector function of mouse natural killer activity Pre-treatment of mice with? Co-culture of C. parvum activated spleen cells with: b Specific slCr release (%) at E/T: 50:1 PBS c~-Momorcharin fl-Momorcharin -----~-Momorcharin fl-Momorcharin 25.3 25.3 31.8 28.5 24.1 25.7 444444l.l 0.9 0.4 1.6 0.9 0.8 100:1 41.5 41.4 45.6 40.7 37.4 37.5 4444441.0 0.4 1.2 0.9 1.3 1.0

" Mice in groups of four were pre-treated with PBS or momorcharin (4 mg/kg i.p.) 2 days before intravenous injection of C. parvum (350 #g/mouse). Splenocytes were assayed for NK activity 4 days later, using YAC-1 cells as the target. b Day-4 splenocytes from C. parvum-injected mice were co-cultured with momorcharin (100 #g/ml) and assayed for NK activity on YAC-1 target cells. Momorcharin alone was found to have no effect on the spontaneous release of the target cells over the 4-h assay period. TABLE VI Effect of momorcharin on the in vivo migration of macrophages elicited by thioglycollate Mice treated with? Total PEC Adherent PEC b recovered per mouse recovered per ( 10 -5 ) mouse( 10 -5 ) 429.0 4- 11.8 141.2 4- 4.2* 162.0 4- 25.2* 290.0 4- 8.0 45.6 4- 1.4" 69.8 4- 11.0" TABLE VII Effect of momorcharin on macrophage effector functions in vitro Pre-incubation of PEC with:" Phagocytosisb (%) Cytostatic activityc (%)

PBS ~-Momorcharin fl-Momorcharin

Control medium 52.8 + 1.9 c~-Momorcharin 25.9 4- 1.9" /LMomorcharin 28.1 4- 1.3"

65.8 4- 6.4 3.4 4- 2.3* 16.9 4- 3.0*

a C57BL/6J mice in groups of four were pre-treated with PBS or momorcharin (4 mg/kg i.p. on day - 2 and day 0) and then injected with I ml 10% Brewers' thioglycollate medium i.p. Three days later, the elicited PEC were harvested and counted. b PEC that remain attached to the plastic surface after incubation at 37C for 3 h and were not removed by several washing with warm medium. The adherent PEC consisted mainly of macrophages as judged by the non-specific esterase staining method. * Significantly different from control, p < 0.05. macrophages to momorcharin tostatic activity (Table VII). for 6 h also caused

a PEC were pre-incubated with control medium (RPMI) or momorcharin (100 #g/ml) for 6 h before assay for the macrophage effector functions. b Phagocytic activity of proteose peptone-elicited PEC was assayed by the latex uptake method (Kohl et al., 1977). c Cytostatic activity of picolinic acid-activated PEC was measured by their ability to inhibit the growth of MBL-2 cells, as determined from the 3H-TdR incorporation of the MBL-2 cells grown in the presence or absence of the peritoneal macrophages. * Significantly different from control, p < 0.05. l a t e d f r o m t h e s e e d s o f t h e b i t t e r m e l o n (Momordica

a d r a s t i c r e d u c t i o n in t h e m a c r o p h a g e - m e d i a t e d cy-

charantia), c a n e f f e c t i v e l y i n d u c e e a r l y a n d m i d t e r m a b o r t i o n i n t h e m o u s e ( L a w e t al., 1983; C h a n et al., 1984). I n t h i s p a p e r , w e s h o w t h a t t h e s e t w o abortifacient proteins are extremely potent modulators o f a variety o f cell-mediated a n d h u m o r a l immune r e s p o n s e s , b o t h in v i v o a n d in v i t r o . A l -

Discussion
P r e v i o u s w o r k in t h i s l a b o r a t o r y h a s d e m o n s t r a t e d t h a t ~- a n d f l - m o m o r c h a r i n , t w o g l y c o p r o t e i n s

iso-

t h o u g h ~- a n d f l - m o m o r c h a r i n d i f f e r b o t h in m o -

168 lecular weight (c~-form, 31 000;//-form, 29 000) and immunological property (Yeung et al., 1985), their immunosuppressive activities are very much alike and both proteins appear to have the same degree of potency and similar activity on the murine immune system. However, it is noteworthy that these two proteins are not totally non-selective in their suppressive effects. Thus, in vitro studies have shown that momorcharin present at non-cytotoxic concentrations (50-100 Ftg/ml) can give rise to maximum suppression of mitogen-induced and alloantigen-induced lymphoproliferation but did not affect the cytolytic activity of N K cells or CTL activity. On the other hand, a clear decrease in the functional capacity of macrophages, such as the cytostatic and phagocytic activities, was observed under similar conditions. Moreover, the in vivo activation of N K cells was not significantly affected by treatment of mice with momorcharin whereas similar treatment of mice resulted in a significant depression of the D T H response as well as humoral antibody formation to SRBC. The precise cellular target(s) for the immunosuppressive effect of ~- and fi-momorcharin has not yet been identified. Since both Con A and PHA are T cell mitogens, suppression of their stimulation for lymphocyte transformation by momorcharin implied an effect of these two proteins on the T cell population. This was further substantiated by the fact that the in vitro specific proliferation of T cells in M L R and the in vivo generation of effector T cells mediating the D T H reaction were markedly suppressed by treatment with momorcharin. In addition, our preliminary results have shown that momorcharin administered in vivo or present in vitro can inhibit the production of interleukin-2 from Con A-activated mouse splenocytes (K.N. Leung, unpublished observations). Since interleukin-2 is a T cell-derived lymphokine (Robb, 1984), our results indicate that momorcharin is not only able to suppress T cell proliferation but can also reduce the capacity of T cells, either directly or indirectly, to liberate lymphokines in vitro. Since T cells are heterogenous in nature, whether momorcharin might have a differential effect on certain subpopulations of T cells is as yet unclear. It is known that T cell subpopulations with distinct reactivities to Con A and PHA are present in the spleen of the mouse (Stobo et al., 1972). It is therefore possible that momorcharin may have a greater effect on certain subpopulations of T cells, as the proteins were found to be more inhibitory to the PHA response than to Con A response under similar conditions. Obviously, more experiments are needed to clarify this point. Moreover, our data also showed that momorcharin can cause the suppression of LPS-induced B cell proliferation in vitro and the production of specific antibodies in vivo. Thus momorcharin appears to modulate both T cell and B cell responses of the murine immune system. However, it is still unclear whether it exerts its effects directly on the T and B cells or indirectly on accessory cells such as macrophages which are known to play an essential role in the induction of specific immune responses. In fact, we have found that momorcharin can inhibit macrophage functions both in vitro and in vivo. In vitro exposure of macrophages to m o m o r c h a r i n not only reduces their phagocytic activity but also diminishes their cytostatic activity. Although single injections of momorcharin per se did not appear to affect the number of resident peritoneal macrophages, nevertheless, migration of monocytes into the peritoneal cavity as a result of thioglycollate-elicited acute inflammation was markedly suppressed by in vivo treatment of mice with the proteins. The mechanism(s) by which the proteins might exert a suppressive effect on macrophage migration in vivo has not yet been resolved. It may be a result of reduced secretion of soluble mediators or due to the reduced chemotactic response of macrophages. While the primary target(s) for momorcharin remains to be established, the available evidence so far suggests that the primary effect is unlikely to be at the NK cell level as neither the in vivo activation nor the. in vitro effector function of this cell type were appreciably affected in the presence of momorcharin under the prescribed experimental conditions. Our present finding, that proteins with immunosuppressive activities can be isolated from the seeds of the bitter melon plant, is compatible with those of others who have reported that immuno-

169 suppressive factors could be isolated from the fruits (Vesely et al., 1977; Takemoto et al., 1980) and seeds (Spreafico et al., 1983) of the bitter melon plant. Thus, Vesely et al. (1977) have shown that a guanylate cyclase inhibitor from the ripe fruit of M. charantia could block [3H]thymidine incorporation in Con A-stimulated rat splenocytes. Likewise, Takemoto et al. (1980) have demonstrated that a crude bitter melon factor derived from the aqueous extract of the fruit could cause growth inhibition of human lymphocytes and lymphoblastoid cell lines. Perhaps our results are more analogous to those of Spreafico et al. (1983), who have isolated an immunomodulatory protein from the seeds of the bitter melon. The protein, known as the M . charantia inhibitor (MCI), is a potent inhibitor of protein synthesis in cell-free systems. It is clear from our studies that ~- and fl-momocharin exhibited some immunodulatory activities similar to those observed for MCI. These include suppression of the mitogen-induced lymphocyte transformation and inhibition of the humoral antibody response to SRBC. Since ~- and fl-momorcharin and MCI were all isolated from the seeds of M'. eharantia and they possess similar molecular weights (Spreafico et al., 1983; Yeung et al., 1985) and immunomodulatory activities, it is quite possible that they are very similar, if not identical, molecules. However, a number of differences have been noted. Firstly, no neutral sugar was detected in MCI (Barbieri et al., 1980)~ whereas both ~- and fi-momorcharin were found to be glycoproteins (Yeung et al., 1985). Secondly, MCI was reported to be more inhibitory to Con A and PHA responses than to LPS responses and thus appears to be a primary inhibitor of T cells (Spreafico et al., 1983). We, on the other hand, have found that both the Con A and LPS responses were inhibited to almost the same extent. Finally, MCI could enhance spontaneous macrophage-mediated cytotoxicity, both in vivo and in vitro (Spreafico et al., 1983). In contrast, we have found that the phagocytic and cytostatic activities of the macrophages were depressed by prior exposure of the cells to the proteins in vitro. In addition, thioglycollate-induced macrophage accumulation in vivo was significantly inhibited by administration of momorcharin into mice. Hence, the identity and the relationship between momorcharin and MCI remain to be determined. The mechanism(s) whereby ~- and fl-momorcharin exert their immunosuppressive activities has not yet been resolved. Data in this paper clearly demonstrated that immunosuppression is not a direct result of lymphocytotoxicity as protein concentrations up to 100 pg/ml did not cause a significant loss of cell viability over a period of 48 h whereas such dosages almost completely abrogated a variety of immune responses. In addition, the observed suppression is apparently not due to a time-shift in the kinetic parameter of an immune response as the kinetics of the D T H and humoral antibody responses were not appreciably affected by the proteins and significant suppression was observed both at the optimum and suboptima of an immune response. Since rapid proliferation of immunocompetent cells is known to be required for the expression of certain immune reactivity, it is quite possible that momorcharin might exert its immunosuppressive effect through its selective cytostatic action on the immune cells. Alternatively, immunosuppression might simply result from a direct inhibition of protein synthesis of the immune cells, as had been suggested by Barbieri et al. (1980) for the immunomodulatory activity of MCI. Experiments testing these hypotheses are currently in progress and results will shortly be reported. In conclusion, we have shown that two glycoproteins isolated from the seeds of bitter melon can display both abortifacient and immunomodulatory activities. It would be of great interest to determine whether there is any relationship between these two types of activities. Finally, the possibility of induction of abortion through immunologic responses or other molecular mechanism(s) has yet to be elucidated.

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