production by rat peritoneal macrophages - Springer Link

Agents and Actions, vol. 15, 3/4 (1984)

0065-4299/84/030419-0552.50 1984 Birkh~iuser Vedag, Basel

Leetins modulate prostaglandin E 2 production by rat peritoneal macrophages KAZUO OHUCH1.1, MASAKO WATANABE*, ERIKO TAKAHASHI*, SUSUMU TSURUFUJI*, KEN-ICHI IMANISHIt, |KUO SUZUKIt a n d LAWRENCE LEVINE:~ *Department of Biochemistry, Faculty of Pharmaceutical Sciences, Tohoku University, Aoba, Aramaki, Sendal 980, Japan, ~'The Koriyama Institute of Medical Immunology, Koriyama 936, Japan and ~Department of Biochemistry, Brandeis University, Waltham, MA 02154, USA

Abstract The effect of Aloctln A (Alo A), a leetin having anti-inflammatory activities, on prostaglandln (PG) E2 production by activated rat peritoneal macrophages was compared wlth that of concanavalin A (Con A), wheat germ aRintinin (WGA), plaum safl~m aulutlnin (PSA) and soybean aggintinin (SBA). Alo A, WGA, Con A and PSA at 10 ~g per ml Inhibited PG E 2 production. But SBA, even at a dose of 1 #g per mi, stimulated PG E z production. The Inhibition by AIo A treatment of the release of radioactivity from (3H)arachldonlc acid-inl~lcd macrophagee and the stimulation of this release by SBA treatment wcrr observed. The apUtkg of (SICr)-labelcd sheep red blood cells by the macrophagc was Inhibited by Alo A, Con A, and PSA, all at I0/~g per ml and SBA at I #g per ml, however, WGA at I0 /ag per ml stimulated the uptake of the sheep red blood cells. The mccinmlsm of the anti-lnflammatory properties of Alo A was discussed. Introduction

Aloctin A (Alo A), a lectin isolated from leaves of Aloe arborescens Mill [1], has many biological activities, e.g. mitogenic activity for lymphocytes [1], cytoagglutination [2], growth inhibition of methylcholanthrene-induced fibrosarcoma in vivo [3], and anti-inflammatory activities [4]. Indeed, Aloe arborescens Mill has been used as an anti-inflammatory agent as folklore medicine. However, the mechanism of its anti-inflammatory activities has not been studied. The present investigation was designed to see if Alo A, like the non-steroidal anti-inflammatory drugs, indomethacin or aspirin, inhibits PG E2 biosynthesis. We report here that Alo A does inhibit PG E 2 biosynthesis but unlike indomethacin and aspirin the expression of its inhibitory activity is dependent on incubation To whom correspondence should be addressed.

time. The anti-inflammatory properties of AM A may depend on activities other than that of inhibition of PG E 2 production since several lectins also inhibit PG E 2 production but are not anti-inflammatory. And one of them, Con A, is pro-inflammatory, it induces edema in rat hind paw [5]. The effect of Alo A on the uptake of the foreign particles, sheep red blood cells, by the macrophages was also examined. The inhibitory effect on the uptake of sheep red blood cells was not specific to Alo A. Consequently, the antiinflammatory activities of Alo A cannot be attributed to inhibition of PG E 2 synthesis or to inhibition of phagocytosis of foreign bodies. Materials and methods Preparation of activated peritoneal maerophages Male rats of Sprague-Dawley strain, specific pathogen free (Charles giver Japan, Inc., Kanagawa, Japan), weighing 300-350 g, were used. The activated peritoneal macrophages were prepared according to the procedure already described [6, 7]. In brief, a solution of soluble starch (Wako Pure Chemical, Tokyo, Japan) and bacto peptone (Difco Lab., Detroit, MI, U.S.A.), 5% each, was injected into the rats intraperitoneally at a dose of 5 mi per 100 g body weight in order to stimulate maerophage migration. The stimulant solution was autoclaved at 120~ for 15 rain and cooled to room temperature before injection. Four days after the injection, the rats were kiUed by cutting the carotid artery and the peritoneal cells were harvested as described previously [6, 7]. MacrophaBe culture The peritoneal cells were suspended in Eagle's minimal essential medium (Nissui, Inc., Tokyo, Japan) containing 2 mM 1-glutamate (Wako Pure Chemical, Tokyo, Japan) and supplemented with 10% (v/v) calf serum (Grand Island Biochemical Co., Grand Island, NY, USA) penicillin and streptomycin (Meiji Seika Kaisha, Tokyo, Japan). The macrophages were seeded at 6 • 10+ cells/60 mm Falcon

420


tissue culture dish (Div. Becton, Dickinson and Co., Cockeysvilie, MD, USA) in 4 ml of the medium and incubated for 2 h at 37~ After the incubation, the dishes were washed 3 times with the medium to wash out non-adherent cells. The adherent cells were further incubated with 4 ml of the medium with or without lectins. The lectins examined were Aloctin A (Alo A), which was prepared according to the procedure described by SuzuKi et al. [11, concanavalin A (Con A), soybean agglutinin (SBA), pisum sativum agglutinin (PSA) (Vector Lab. Inc., Burlingame, CA, USA) and wheat germ agglutinin (WGA) (Miles-Yeda, Ltd., Israel). These lectins were dissolved in sterile physiologic saline (1 mg/ml), indomethacin, ibuprofen and aspirin were dissolved in ethanol (1 mg/ml) and an aliquot of the solution was added in the incubation medium. Control medium contained the same amount of the vehicle.

Results

Radioimmunoassay of prosta$1andin E 2 After a specified time of incubation of the macrophages with or without lectins, the medium was withdrawn and centrifuged at 2500 x g for 15 min at 4~ An aliquot of the supernatant fluid was used for measurement of PG E2; the serological specificity of the anti-PG E z has been described elsewhere [8]. There was no interference on PG E 2 radioimmunoassay by the lectins examined.

Measurement of the released radioactivity (3H)araehidonle acid-labeled macrophases

acid insoluble fraction [9] with a slight modification [6]. The activated peritoneal macrophages (6 x 106 cells) were incubated for 26 h in 4 ml of the medium (10% calf serum) containing varying doses of AIo A. Ten/~Ci of (3H)leucine were added to the medium and incubated for another 2 h. After washing the cells 5 times with the medium to remove free (3H)leucine, 1 ml of 1 N perchloric acid was added and the ceils were scraped from the dish. The scraped ceils were washed 3 times with 1 N perchloric acid solution. After centrifugation at 2500 x g for 10 rain, the sedimented cells were transferred into the scintillation vial for quantification of radioactivity. The results have been expressed as mean +_ SEM in the text and figures. For statistical analysis, Student's t-test was applied.

from

To label the cellular lipids, 6 x 106 macrophages were incubated with 1 pCi of (3H)arachidonic acid (61 Ci/mmol, New England Nuclear, Boston, MA, USA) for 20 h in 4 mol of the serum-supplemented medium [6, 7]. To remove free (aH)arachidonic acid, the cells were washed 5 times with 2 ml of this medium. The cells then were incubated in 4 ml of the medium with or without Alo A (10/tg/ml) or SBA (1 ag/ml). At 3, 6 and 9 h, i00 #1 of the medium was withdrawn and counted for the release of radioactivity.

PG E 2 production by the macrophages was not inhibited by Alo A at 1.0 to 10 pg/ml when measured after a 6 h incubation (Fig. 1). In contrast, the non-steroidal anfi-infammatory drugs, indomethacin, ibuprofen and aspirin at similar doses did inhibit PG E 2 production. After 20 h of incubation with Alo A (10 ag/ml), however, PG E 2 production was inhibited 34% (Table 1). After washing these cells that had been incubated for 20 h, additional 6 h incubation with Alo A inhibited PG E2 production more effectively; the levels were inhibited 21 and 57% with 1.0 and 10 #g/ml, respectively (Table 1). Protein

15

Uptake of (stCr)-IaMled sheep red blood cells One ml of packed cell volume of sheep red blood cells (Nippon Bio-Test Lab., Inc., Tokyo, Japan) was suspended in 6 ml of acid-citrate-dextrose solution (pH 5.0). One hundred/JCi of Naz(StCr)O4 (670 mCi/mg Cr, 1 mCi/ml, New England Nuclear, Boston, MA, USA) was added and the suspension was incubated for 1 h at room temperature with occasional mixing. To this solution 6 ml sterile saline containing 60 mg ascorbic acid was added. After 10 rain, the sheep red blood cells were collected by centrifugation (1000 • g for 5 rain at 4~ The sedimented cells, washed 5 times, were suspended in 6 ml of the medium. Two hundred ~1 of this washed suspension (3.5 #Ci) was added into the dish in which 6 • 106 macrophages had already incubated with or without lectins for 26 h in 4 ml of the medium. These cells were incubated for another 1 h, at which time the dishes were washed 4 times with 2 ml of the medium to remove free red blood cells. Then, the macrophages were solubilized in 1 ml of 0.1 N NaOH solution and transferred into the counting tube. The radioactivity of 52Cr was measured in an automatic well type scintillation counter Aloka JDC-751 (Nihon Musen Co., Ltd., Tokyo, Japan).

Protein synthesis Protein synthesis was determined by measuring the incorporation of (JH)leucine (L-(4,5-3H(N))-leueine, 55.9 Ci/mmol, New England Nuclear, Boston, MA, USA) into

-. ~ 10 ~'~ 0. 5

0

0.1

1

Dose

[ pglmt

10

)

Figure 1 The effects of Alo A, indomethacin, ibuprofen and aspirin on PG E z production by activated peritoneal macrophages of rats. Six x 106 macrophages were incubated for 6 h in 4 ml of the medium in the presence of the indicated doses of drugs. The media were removed and assayed for PG E 2. The values are the mean of 2 dishes. O - - - - - O , Alo A; O 0, Aspirin; --~ !1, Ibuprofen; 9 A, Indomethacin.


421

Table 1 The effect of Alo A on PG E2 production by activated peritoneal macrophages.

Table 2 The effects of lectins on PG E2 production by activated peritoneal macrophages.

Incubation period

Alo A (/Jg/ml)

PG E 2 (rig/6 x 106 cells/h)

Lectins

20 h

0 I 10 0 1 10

13.07 + 0.58 11.67 + 0.24 8.67 _+0.47* 3.25 + 0.08 2.57 + 0.02* 1.40 + 0.08*

Additional 6 h

Values are the mean +_ SEM of 3 dishes. Six x 106 macrophages were incubated for 20 h containing indicated doses of Alo A. Then the medium was changed with the fresh medium containing corresponding doses of Alo A and incubated for an additional 6 h. Statistical significance: *p < 0.01.

None WGA Con A PSA SBA "Alo A

Dose (/zg/ml)

10 10 10 1 10

PG E2 (ng/6 • 106 cells/h) 20 h

Additional 6 h

9.21 + 0.85 5.46 + 0.45** 7.08 _+0.18" 10.65 + 1.71 28.80 + 3.18"** 6.15 + 0.63**

2.93 • 0.20 0.77 • 0.02*** 1.17 +_0.05*** 2.13 • 0.08* 7.29 + 0.14"** 1.19 • 0.09**

Values are the mean _+ SEM of 4 dishes. Six x 106 macrophages were incubated for 20 h containing indicated dose of the lectin. Then the medium was changed with the fresh medium containing a corresponding dose of the lectin and incubated for an additional 6 h. Statistical significance: *p < 0.025, **p < 0.01, ***p < 0.001.

'o x

8

K "o E

o= s

do

10 Aloctin A

30

(Hg/ml)

Figure 2 The effects of Alo A on the protein synthesis and the uptake of (S*Cr)-labeled sheep red blood cells by the macrophages. No detachment of macrophages from the dish during the incubation period at indicated doses of Alo A was observed. The values in the protein synthesis experiment are the mean + SEM of 4 dishes. There was no significant difference in the protein synthesis by the treatment with Alo A. In the experiment of (~tCr)-labeled sheep red blood cells uptake, the values are the mean +_ SEM of 3 dishes. Statistical significance; 0/zg/ml vs 10/zg/ml and 0/zg/ml vs 30 #g/ml, p < 0.001.

synthesis, as examined b y the uptake o f (3H)leucine into 1 N perchloric acid insoluble fraction, was not affected during the 26 h incubation with A l o A even at a dose o f 30 # g per ml (Fig. 2). But the u p t a k e o f sheep red blood cells b y the m a e r o p h a g e s was strongly inhibited in a dosedependent m a n n e r (Fig. 2). The inhibitory effect o f Alo A on P G E 2 production was c o m p a r e d with that o f other

lectins. After 20 h incubation with A l o A at a dose o f 10/zg/ml, 33% o f P G E2 production was inhibited; W G A and C o n A at a dose o f 10/zg also inhibited P G E 2 production significantly (Table 2). S B A at a dose o f 1 /tg per ml, stimulated P G E 2 p r o d u c t i o n a b o u t 3 fold (SBA at a dose o f 10 # g per ml also stimulated P G E 2 but the m a c r o p h a g e s detached from the dish). Again, after washing the cells and incubating for additional 6 h with lectins, inhibition o f P G E 2 production by Alo A, W G A , C o n A and P S A at a dose o f 10 /zg per ml was even m o r e p r o n o u n c e d (Table 2). S B A at a dose o f I / l g per ml stimulated P G E 2 production. The effects o f A l o A (10/zg/ml) and SBA (1 # g / m l ) on the release o f radioactivity from (3H)arachidonic acid-labeled m a c r o p h a g e s are shown in Fig. 3. Significant inhibition o f the release o f radioactivity by A l o A treatment was observed after 9 h o f incubation. In contrast, even after 3 h o f incubation with SBA, significant increase in the release o f radioactivity was observed. The effects o f lectins on the uptake of sheep red blood cells by the m a c r o p h a g e s are shown in Fig. 4. SBA, which stimulated P G E~ production at a dose of 1 #g per ml, inhibited the uptake as did A l o A , P S A and C o n A (10/zg/ml). W G A , which inhibits P G E z production, stimulated the uptake o f sheep red blood cells almost 2-fold. N o detachment o f the m a c r o p h a g e s from the dish by the incubation with the medium containing the indicated doses o f lectins was observed.

422

0


X

m

iv. i

i

i

3

6

9

Incubotion Time

{ hr )

Figure 3

The effects of Alo A and SBA on the release of radioactivity from (3H)arachidonic acid-labeled macrophages. 0 - - - O, Control; 9 &, Alo A; ~ O, SBA. The values are the mean +_ SEM of 4 dishes. Statistical significance: SBA vs Control, p < 0.001 at 3 and 6 h , p < 0.01 at 9 hr. Alo A vs Control, p < 0.02 at 9 h.

x 20

r

.m E n o

~10 .g 0

ne Z

~

t~

"O

~

U1

Figure 4 The effects of lectins on the uptake of (~;Cr)-labeled sheep

red blood cellsby the macrophages.The valuesare the mean _+SEM of 4 dishes.Statisticalsignificance:Con A,p < 0.01; WGA, PSA, Alo A and SBA,p < 0.001.

Discussion Alo A, a lectin isolated from leaves of Aloe arborescens Mill, has anti-inflammatory activities. Namely, when Alo A is injected intraperitoneally (0.5-10.0 mg/kg) daily for 15 days, starting from 1 day before intradermal injection of liquid paraffin containing heat-killed Mycobacterium butyricum, adjuvant arthritis formation in rat hind paw was markedly inhibited [4]. A single intraperitoneal injection of Alo A (0.5/10.0 mg/kg) also inhibited the swelling of rat

hind paw induced by subcutaneous injectionof carrageenin solution [4]. The present work was aimed to see ifthe anti-inflammatory activitiesof Alo A, like the activitiesof non-steroidal antiinflammatory agents, reflectedinhibitionof P G production [10]. During the first6 h incubation, Alo A did not inhibitP G E 2 production, whereas indomethacin, ibuprofen and aspirindid (Fig. I). However, Alo A did inhibit P G E 2 production afterrelativelylong incubations.As judged by the time of the effect,the mechanism of inhibitionof P G E L production by Alo A seemed to be different from that of acidic non-steroidal antiinflammatory drugs. The anti-inflammatory activitiesof Alo A had been observed 3.6 h aRer a single intraperitoneal injection [4]. In our cell culture system, within 3.5 h of incubation with Alo A, no inhibitionof P G E 2 production by the activated peritoneal macrophages was observed (Fig. I). Inhibitionof P G E 2 production was not specific to Alo A. Other lectins,such as W G A , Con A and PSA also inhibitedP G E L production (Table I), and W G A , Con A and P S A are not anti-inflammatory; in fact Con A has been reported to induce edema in rat hind paw [5].In contrast with those lectins which inhibit PG E L production, SBA stimulated PG E 2 production even at a dose of 1 pg per ml. Alo A inhibited the release of radioactivity from (3H)arachidonic acid-labeled macrophages but only after a long time of incubation (Fig. 3). A significant increase in the release of radioactivity by SBA treatment was observed, after 3 h of incubation, like stimulation of PG E 2 synthesis by SBA. These data suggest that the inhibition by Alo A and the stimulation by SBA treatment of PG E L production reflect the inhibition and the stimulation of phospholipase A 2, respectively. In lymphocytes, mitogenic lectins stimulate phospholipid methylation and degradation of phospholipids [ 11]. Mitogenic lectins also may stimulate PG synthesis in lymphocytes. In macrophages, however, stimulation of PG E 2 synthesis was observed only by incubation with SBA. The other lectins examined inhibited PG E Lsynthesis. It is suggested [12] that the phagocytosis of foreign particles is accompanied by the release of inflammatory proteases to the extracellular space. To examine the possibility if AIo A has specific inhibitory effect on the phagocytosis of foreign bodies, the effect of Alo A on the uptake of sheep red blood cells by the macrophages was exa-


mined. Inhibition of the uptake of sheep red blood cells by the activated rat macrophages was observed after incubation with Alo A, Con A, PSA and SBA. This inhibition did not reflect loss of protein synthesis capacity. WGA, which, like Alo A, Con A and PSA, inhibited PG E 2 production, specifically stimulated the uptake of sheep red blood cells. Specific effect of WGA on polymorphonuclear leukocytes chemotaxis was also reported [13]. Microscopic observations revealed that WGA stimulated the process of the attachment of the sheep red blood cells to the microvilli rather than that of the engulfment. There raised following possibilities on the stimulation of the uptake by WGA: (a) phagocytotic ability of the macrophages was increased by WGA treatment; (b) sheep red blood cells were changed to be more phagocytizable form by WGA treatment; and (c) both (a) and (b). Preliminary experiment revealed the possibility of (c). In case of the inhibition of the uptake of sheep red blood cells by the several lectins, both macrophages and sheep red blood cells seemed to be changed by the treatment with these lectins. Precise data will be described elsewhere. While our studies do not explain the anti-inflammatory activity of Alo A, its capacity to inhibit PG E 2 production and to inhibit phagocytosis of foreign particles do not appear to be the explanation.

Acknowledgments This work was supported by a grant from Life Science Promotion Center, the Science and Technology Agency, Japan. This paper was presented by Masako Watanabe at the 103rd Annual Meeting of Japanese Pharmaceutical Society held in Tokyo, April 4-6, 1983. Lawrence Levine is a Research Professor of Biochemistry of the American Cancer Society (Award PRP-21).

Received 6 June 1983

423 References [1] I. SuzuKI, H. SAITO, S. INOUE, S. MIGITA and T. TAKAHASHI, Purification and characterization of two lectins from Aloe arborescens Mill, J. Biochem. 85, 163-171 (1979). [2] I. SUZUKI, H. SAITO and S. INOUE, A study of cell agglutination and cap formation on various cells with Alo A, Cell Struct. & Funet. 3, 379 (1979). [3] K. IMAr~ISHI,T. ISHIGURO, H. SAITO and I. SUZUKI, Pharmacological studies on a plant lectin, Alo A. L Growth inhibition of mouse methylcholanthreneinduced fibrosarcoma (Meth A) in ascites form by A loctin A, Expedentia 37, 1186-1187 (1981). [4] H. SAITO, T. ISHIGURO, K. IMANISHIand I. SUZUKI, Pharmacological studies on a plant lectin, Aloctin A. H. Inhibitory effect of Aloctin A on experimental models of inflammation in rats, Jap. J. Pharmac. 32, 139-142 (1982). [5] A.J. LEWIS, J. COTTNEY and D.J. NELSON, Mechanisms of phytohaemagglutinin-P-, concanavalin-A- and kaolin-induced oedemas in the rat, Eur. J. Pharmac. 40, 1-8 (1976). [6] K. OHUCHI,Y. KAMADA,L. LEVINEand S. TSURUFUJI, Glycyrrhizin inhibits prostaglandin E 2 production by activated peritoneal macrophages from rats, Prostaglandins & Med. 7, 457--463 (1981). [7] K. OHUCHI, M. WATANABE, T. TANIGUCHI~ S. TSURUFUJI and L. LEVINE, Inhibition by AA861 of prostaglandin E 2 production by activated peritoneal macrophages of rat, Prostaglandins Leukotrienes & Med. 12, 175-177 (1983). [8] I. ALAM, K. OHUCHIand L. LEVINE,Determination of cyclooxygenase products and prostaglandin metabolites using high pressure liquid chromatography and radioimmunoassay, Analyt. Biochem. 93, 339-345 (1979). [9] M.C. PIKE, N.M. KREDICH and R. SNYDERMAN, Requirement of S-adenosyl-L-methionine-mediated methylation for human monocyte chemotaxis, Proc. natn. Acad. Sei. USA 75, 3928-3932 (1978). [10] J.R. VANE, Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs, Nature New Biol. 231, 232-235 (1971). [1 l] S. TOYOSHIMA,F. HIRATA,M. IWATA,J. AXELROD,T. OSAWAand M.J. WAXDAL,Lectin-induced mitosis and phospholipid methylation, Molec. Immunol. 19, 467475 (1982). [12] R.C. PAGE, P. DAVIES, A.C. ALLISON, International Review of Cytology, vol. 52, 119-157 (Ed. G.H. BOURNEand J.F. DANIELL). 1978. [13] H.D. PEREZ, K. KHANNA, R. ONG, n. BANDA and I.M. GOLDSTEIN, Wheat germ agglutinin specifically inhibits polymorphonuclear leukocyte chemotaxis, Fed. Proc. 41, 373p (1982).