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Facilitation of adenylate cyclase stimulation in macrophages by lectins
CELLULAR
45, 415-427 (1979)
IMMUNOLOGY
Facilitation
ANNA Section
of Immunology,
of Adenylate in Macrophages
Cyclase Stimulation by Lectinsl
GRUNSPAN-SWIRSKY Department Tel-Aviv
AND
of Human University
Received
EDGAR
Microbiology, Sackler Tel-Aviv, Israel
October
PICKY School
of Medicine,
2. 1978
Preincubation of guinea pig peritoneal macrophages with concanavalin A (Con A) markedly enhanced the accumulation of 3’,5’-cyclic-adenosine monophosphate (CAMP) in response to the adenylate cyclase (AC) stimulators prostaglandin E, (PGEJ and isoproterenol (IP). Basal CAMP levels were not altered. Maximal enhancement of CAMP accumulation was induced by preincubation with 50-100 pg/ml Con A for 10 min at 37°C. Con A-induced facilitation of macrophage responsiveness was prevented by cu-methyl-omannoside (@MM). No facilitation was induced by the divalent derivative, succinyl-Con A or by Con A immobilized on Sepharose beads. Con A-induced facilitation developed normally in macrophages treated with the microfilament blocking agent, cytochalasin B. The responsiveness of macrophages to PGE, and IP was also augmented by phytohemagglutinin (PHA) but wheat germ agglutinin (WGA), soy bean agglutinin (SBA), pokeweed mitogen (PWM), and Lotus tetragonolobus lectin (LL) showed no enhancing effect. The effect of Con A on CAMP levels was the result of augmented CAMP synthesis and not of reduced degradation or a block in CAMP egress from the cells. Lectin-induced facilitation of AC stimulation could be mediated via one of the following mechanisms: (i) induction of receptor clustering; (ii) causing a conformational change in the receptors; (iii) inhibition of negative cooperativity; (iv) causing an increase in membrane fluidity; (v) disruption of microtubules by acting as a CaZ+ lonophore; or (vi) inactivation of a sugar-containing inhibitor of AC.
INTRODUCTION Macrophages of several animal species were found to respond to incubation with P-adrenergic agents, prostaglandins E, and E, (PGE,, PGE2),” and cholera toxin by an elevation in the cellular level of cyclic 3’,5’-adenosine monophosphate (CAMP) (l-6). There is also direct evidence for the presence of /3-adrenergic receptors on the membrane of peritoneal macrophages (7). The cellular concentra’ Supported by National Institutes ofHealth Grant AI-l 1194, Bethesda, Maryland and by U.S.-Israel Binational Science Foundation Grants 480 and 1505. * Address reprint requests to Edgar Pick. ’ Abbreviations used in this paper: PGE,, prostaglandin E,; CAMP, 3’,5’-cyclic-adenosine monophosphate; MIF, migration inhibitory factor; Con A, concanavalin A; PHA, phytohemagglutinin; PWM, pokeweed mitogen; LL, Lotus tetragonolobus lectin; WGA, wheat germ agglutinin: SBA, soybean agglutinin; IP, isoproterenol; PDE, phosphodiesterase; DMSO, dimethylsulfoxide; aMM, a-methyl-o-mannoside; PEC, peritoneal exudate cells; MEM, Eagle’s minimum essential medium; FCS, fetal calf serum; AC, adenylate cyclase. 415 0008-&X749/79/080415-13$02.00/O Copyright 0 1979 by Academic Press, All
rights
of reproduction
in any
form
Inc. reserved.
416
GRUNSPAN-SWIRSKY
AND
PICK
tion of CAMP has a profound influence on several macrophage functions, such as phagocytosis (8, 9), spontaneous motility (lo), responsiveness to chemotactic stimuli (11) and to migration inhibitory factor (MIF) (12, 13), and enzyme release (14). Adenylate cyclase (AC) is exclusively a membrane-bound enzyme (15). The currently accepted model visualizes it as being composed of two distinct components, a stimulant-specific receptor facing outwards and a catalytic unit localized deeper in the membrane. The receptors and, perhaps, catalytic units too, are seen as freely moving in the plane of the membrane (16, 17). These properties suggested that AC might serve as a sensitive functional marker for membrane perturbations induced by exogenous agents interacting directly with the membrane or by intracellular events reflected on the membrane. A number of lectins including concanavalin A (Con A), phytohemagglutinin (PHA), pokeweed mitogen (PWM), and Lotus tetragonofobus lectin (LL) were found to bind to macrophage membrane receptors (18-20). Most studies were performed with Con A, which was also found to influence macrophage physiology, as illustrated by stimulation of endocytosis (21), formation of cytoplasmic vesicles (21, 22), and induction of plasminogen activator (23). It was therefore of interest to study the possible effect of lectins able to react with the macrophage membrane on the activity of AC. Additional reasons for initiating this study were the reports that PHA (24), Con A (25), and wheat germ agglutinin (WGA) (26) elevate CAMP levels in lymphocytes and the findings that Con A and WGA demonstrate both stimulatory and inhibitory effects on the AC of a variety of nonlymphoid cell types (27-30). In the present paper we report that Con A and PHA markedly enhance CAMP generation by guinea pig peritoneal macrophages stimulated by PGE, or IP but have no effect as basal CAMP levels. These findings indicate that agents reacting with membrane glycoproteins can modulate the responsiveness of macrophages to hormonal stimuli acting via the CAMP messenger. Part of this work was published in abstract (31). While this work was in progress, Gemsa et al. (32) reported that Con A moderately enhances CAMP production in rat macrophages stimulated with PGE1, IP, and cholera toxin. MATERIALS AND METHODS Lectins. Con A (3 x crystallized, lyophylized, desalinated powder) was obtained from Miles-Yeda and a stock solution of 1 mg/ml was prepared in 0.01 M phosphate buffer, pH 7.2, containing 1 M NaCl. PHA-P was obtained from Difco Laboratories and a stock solution of 17.5 mg/ml was prepared in distilled water. WGA, SBA, and LL were all obtained from Sigma in the form of lyophilized powder and dissolved in 0.15 M NaCl to 0.5 mg/ml. PWM was obtained from Gibco and dissolved in 0.15 M NaCl to a concentration of 0.5 mg/ml. All mitogen stock solutions were kept frozen at -70°C. Succinyl-Con A was prepared from Con A by twice derivatization with succinic anbydride (Sigma), as described by Gunther et a/. (33). Con A-Sepharose (Con A covalently bound to Sepharose 4B by the cyanogen bromide method) was a commercial preparation obtained from Pharmacia Fine Chemicals, containing 8 mg Con A per milliliter of gel. Just before use, the gel was washed five times with an alternation of 0.1 M acetate
LECTINS
AFFECT
MACROPHAGE
ADENYLATE
CYCLASE
417
buffer, ph 4, and 0.1 M borate buffer, pH 8.6, both containing 1 M NaCl, followed by five washes with 0.15 M NaCl. The washed gel was suspended in 0.15 M NaCl containing 1O-3 M MnCl,, MgC&, and CaCl,. Before use, each preparation of Con A-Sepharose was tested for the presence of reactive Con A by incubation with a 1% suspension of guinea pig red cells for 15 min at 37°C in a shaking water bath. Adherence of numerous erythrocytes to the surface of Con A-Sepharose beads was taken as an indicator that the preparation is suitable for use. Drugs and chemicals. PGE1, a kind gift of Dr. John E. Pike, The Upjohn Company, was dissolved in 95% ethanol to a concentration of 10 mg/ml and stored at -70°C. L-Isoproterenol-D-bitartrate was obtained from Sigma and solutions were prepared just prior to use. The CAMP phosphodiesterase (PDE) inhibitor d, 1-4-(3-butoxy-4-methoxybenzyl)-2-imidazolinone (Ro 20-1724) was a kind gift of F. Hoffmann-LaRoche and Company Ltd., Basel, Switzerland. A stock solution of 0.2 M was prepared in dimethyl sulfoxide (BMSO) and stored at 4°C. Preparation of macrophage monolayers. Male guinea pigs, Hartley strain, weighing 300-500 g, were injected intraperitoneally with 20 ml light paraffin oil (British Drug Houses) and peritoneal exudate cells (PEC) were harvested 3-4 days subsequent to injection. PEC were washed three times in Earle’s balanced salt solution and suspended at a concentration of 5- 10 X 10” cells/ml in Eagle’s minimum essential medium (MEM), supplemented with nonessential amino acids and sodium pyruvate, and containing 15% heat-inactivated fetal calf serum (FCS, Gibco). The suspension was divided among 9-cm-diameter plastic petri dishes (tissue culture grade, Nunc), 5 ml/dish, and incubated for 1 hr at 37°C in 90% air- 10% CO, to allow for preferential adherence of macrophages to the dish surface. Unattached cells were then removed by rinsing the dishes 3 times with 5 ml warm MEM. Viability of macrophages in monolayers (assessed by trypan blue exclusion) was close to 100% and was not affected by any of the lectins, drugs. or stimulants used. Determination of lectin effect on macrophage CAMP levels. Macrophage monolayers were covered with 5 ml MEM containing the lectin to be tested and incubated for predetermined time intervals. This stage was termed the preincubation period. Following preincubation, AC stimulators were added together with the PDE inhibitor Ro 20-1724 (2 x 10e4 M) and incubation continued for 15 min more. The reaction was terminated by placing the dishes on ice-cooled metal trays. The medium was rapidly removed by vacuum suction and 1.5 ml of ice-cold 0.05 M Tris-HCI buffer, pH 7.5, containing 0.004 M EDTA, were added per dish. The cells were removed by means of a rubber policeman and transferred into 100 x 16-mm conical glass tubes, kept in ice water. A sample of 0.1 ml was removed for protein determination by the method of Lowry et al. (34). The conical tubes were then immersed for 5 min in boiling water to precipitate protein, centrifuged for 10 min at 2OOOg, and 1 ml of supernatant was lyophilized. It was then redissolved in 0.2-0.5 ml of distilled water and CAMP was measured in duplicate 50-~1 samples by the competitive protein binding assay of Brown et al. (35), as modified by Tovey et al. (36), using a kit commercially manufactured by the Radiochemical Center, Amersham, England (Code No. TRK 432). Results were expressed as picomoles of CAMP per milligram of macrophage protein, representing the average derived from two identically-treated petri dishes. De-
418
GRUNSPAN-SWIRSKY
AND PICK
tailed CAMP extraction and determination procedures have been described previously (4). Measurement of CAMP release from macrophages. Macrophage monolayers were covered with 1.5 ml MEM containing the lectin and incubated for 10 min at 37°C. Following this, AC stimulators were added together with Ro 20-1724 (2 x lop4 M) and incubation continued for further 15 min. The experiment was stopped by placing the dishes on ice-cooled metal trays and rapidly transferring the supernatant into conical glass tubes maintained in ice water and containing 0.075 ml of 1 M Tris-HCl buffer, pH. 7.5, and 0.08 M EDTA. The content of the tubes was rapidly mixed on a Vortex apparatus and centrifuged for 5 min at 2OOOg, at 4”C, to sediment cells which may have become detached from the dish surface. The supematants were transferred to new tubes which were boiled for 5 min and centrifuged for 10 min at 2000g. Supematant, 1 ml amounts, were lyophilized and redissolved in 0.2 ml of distilled water, and duplicate 50 ~1 samples of this were used in the CAMP assay. Cellular CAMP levels were determined in the same dishes by extracting the macrophage monolayer, after removal of the supematant , as described above. RESULTS Enhancement
by Con A of Macrophage
Responsiveness
to AC Stimulators
We have previously demonstrated that CAMP levels in purified guinea pig peritoneal macrophages are elevated in a highly reproducible manner by incubation with PGEl (4). CAMP generation in response to IP is more erratic and occasionally absent (37). Preincubation of macrophages in monolayer with Con A resulted in a marked increase in CAMP accumulation in response to both PGE, and IP (Table 1). TABLE
1
Effect of Con A on Responsiveness of Macrophages to Adenylate Cyclase Stimulators
Macrophages preincubated with:
Concentration Cwh-4
Medium Con A
-
Medium Con A Medium Con A D Cells were 2 x lo+ M. P pretreated with t test for paired
Preincubation time (min)
CAMP level (pmol per mg macrophage protein k SEM)” No. of expt
Basal
+PGE, (lO-4 M)
+Isoproterenol ( 1O-3 M)
10
9
22.8 c 5.1 21.7 2 3.4 (P > 0.50)
139 rf: 13.5 462 f 56.0 (P < 0.001)
43.4 Ii 9.1 168 2 45.7 (P < 0.02)
100
10
4
28.8 2 10.9 18.2 k 6.9 (P < 0.50)
152 k 20.5 372 -c 12.5 (P < 0.001)
33.8 f 13.1 115 5 20.0 (P c 0.10)
100
60
8
19.3 ” 3.6 14.7 + 2.8 (P < 0.10)
170 2 14.3 323 r 30.8 (P ==c0.002)
18.5 f 2.8 57.4 + 7.7 (P c 0.001)
50
stimulated with PGE, or isoproterenol for 15 min, in the presence of Ro 20-1724, values express the significance of difference between CAMP levels in macrophages medium compared to macrophages pretreated with Con A, calculated by Student’s data.
LECTINS
AFFECT
MACROPHAGE TABLE
Prevention of Con A-Induced
Macrophages preincubated with: Medium Con A (YMM Con A + cvMM Con A + aMM
Concentration 50 &ml 5 x 10-z M 50 &ml
ADENYLATE
419
CYCLASE
2
Enhancement of Macrophage Adenylate Cyclase Responsiveness by a-Methyl-o-Mannoside (aMM)” Preincubation time (min)
IO
CAMP level (pmol per mg macrophage protein) Basal
+PGE, (lo-’ M)
+lsoproterenol (IO 2 M)
19.1 28.4 13.8 18.1
190 469 185 224
33.4 136 27.7 38. I
16.5
167
28.5
IO-'M 50 &ml 5 x 10-z M
’ Cells were preincubated with Con A in the presence or absence of aMM or with cvMM alone. They were then stimulated with PGE, or isoproterenol for 15 min in the presence of Ro 20-1724. 2 x lo-’ M. Data represent means of values from duplicate dishes.
The enhancement of macrophage responsiveness was even more striking when the stimulant was IP since cells totally unresponsive to IP could be induced to respond briskly by mere preexposure to Con A. Con A had no effect on basal CAMP levels. This phenomenon was termed “lectin-induced AC facilitation.” Facilitation developed both in the presence and in the absence of a PDE inhibitor but such inhibitor (Ro 20-1724, 2 x 1O-4 M) was regularly present in our experiments in order to enhance and stabilize CAMP accumulation. Dose response studies indicated that facilitation is induced by concentrations of Con A from 10 to 400 j&ml with a peak being reached at 50- 100 &ml. Facilitation was seen after incubation of macrophages with Con A for 10 to 120 min; maximal enhancement was obtained after exposure to Con A for 10 min. Preliminary results indicate that preincubation of macrophages with Con A (50 &ml) for 10 min does not enhance CAMP accumulation in response to a maximally stimulatory concentration of cholera toxin (10 @ml). Very moderate enhancement (123%) was found when macrophages were stimulated with 50 or 1000 &ml cholera toxin. The effect of Con A is exerted via its binding to specific sugar-containing macrophage membrane structures. This was demonstrated by the total elimination of facilitation, when incubation with Con A was in the presence of the competitive inhibitor aMM (1-5 x lo-* M) (Table 2). Inability
of Succinylated Con A and Insolubilized Con A to Induce Facilitation
Succinylation of native Con A converts the lectin from a tetramer to a dimer (33). Succinyl-Con A possesses identical sugar-binding properties to the native molecule but has a lesser capacity to agglutinate red cells and does not induce cap formation in lymphocytes. Macrophages were preincubated with succinyl-Con A for intervals of 10 or 60 min after which time they were stimulated with PGE, or IP. As apparent from
420
GRUNSPAN-SWIRSKY
AND PICK
TABLE
3
Inability of Succinyl-Con A to Enhance the Responsiveness of Macrophages to Adenylate Cyclase Stimulators
Macrophages preincubated with:
Concentration (dmU
Medium Con A Succinyl-Con A Con A Succinyl-Con A
10 10 50 50
Medium Con A Succinyl-Con A Con A Succinyl-Con A
10 10 50 50
CAMP level (pmol per mg macrophage protein)
Preincubation time (min)
No. of expt
10
60
Basal
+PGE, (1O-4 M)
+Isoproterenol (1O-3 M)
2
42.9 49.2 51.2 49.7 48.3
354 467 246 624 246
106 172 106 289 122
2
27 28 32 23 35
174 354 224 389 210
23.6 49.3 27.2 45.8 28.0
” Cells were stimulated with PGEl or isoproterenol for 15 min, in the presence of Ro 20-1724, 2 x 10e4 M. Data represent means of two experiments for every preincubation interval. For each experiment data were derived from duplicate dishes.
Table 3, succinyl-Con A, at concentrations of 10 and 50 &ml, was not capable of inducing AC facilitation comparable to that induced by identical concentrations of native Con A. We next examined the ability of Con A immobilized on Sepharose beads, to cause facilitation. Immobilized Con A was shown to be an effective lymphocyte mitogen (38), although the contrary opinion was also expressed (39). Macrophage monolayers were incubated with a suspension of Con A-Sepharose beads, equivalent to SO or 200 pg/ml of soluble Con A, on a rocking platform for 15 min at TABLE
4
Inability of Insolubilized Con A (Con A-Sepharose) to Enhance the Responsiveness of Macrophages to Adenylate Cyclase Stimulators’
Macrophages preincubated with:
Concentration of Con A (&ml)
Medium Con A Con A-Sepharose Con A-Sepharose
50 50 200
Preincubation time (min)
15
CAMP level (pmol per mg macrophage protein) Basal
+PGE, (1O-4 M)
6.9 9.1 4.5 3.8
81.6 259 81 148
+Isoproterenol (1O-3 M) 8.2 64.8 8.0 14.2
(’ Cells were preincubated with soluble Con A or Con A-Sepharose beads for 15 min at 37°C on a rocking platform, to ensure optimal contact between the beads and the macrophage monolayers. They were then stimulated with PGE, or isoproterenol for 15 min, in the presence of Ro 20-1724, 2 x 10e4 M. Data represent means of values from duplicate dishes.
LECTINS
AFFECT
MACROPHAGE
ADENYLATE
421
CYCLASE
37°C. Attachment of beads to the upper surface of cells in monolayer was clearly visible by examination of the dishes on an inverted microscope. The cells were subsequently exposed to PGE, or IP for 15 min at 37”C, without rocking. As seen in Table 4, preincubation with 50 pg/ml Con A-Sepharose did not result in facilitation of AC stimulation. Con A-Sepharose, 200 &ml, induced some facilitation but this could be accounted for by the presence in the medium covering the macrophages of soluble Con A, which had leaked from the beads during the incubation on the rocking platform. Leakage of Con A was detected by testing the macrophage supernatants for the ability to agglutinate guinea pig red cells. No free Con A was found in supernatants derived from macrophages incubated with 50&ml Con A-Sepharose. It was, however, possible that the lack of facilitation by insoluble Con A was due to the fact that 15 min was not sufficient time for an optimal interaction between Sepharose beads and the cells. Experiments were therefore performed in which macrophages were incubated with Con ASepharose for 30 min or 1 hr but no facilitation was obtained under these conditions, either. The Influence
of the Cytoskeleton
on Con A-Induced
Facilitation
Macrophages possess an extensive array of microfilaments and microtubules (40, 41). There is a large body of evidence indicating cytoskeletal control of membrane receptors in general and Con-A receptors in particular [reviewed in (42)]. We have recently shown that guinea pig peritoneal macrophages do not exhibit spontaneous capping of Con-A receptors but capping is made possible by treating the cells with microtubule disrupting drugs such as colchicine, vinblastine, or podophyllotoxin (20). We have also shown that such capping requires the integrity of microfilaments. The possibility was therefore considered that Con A exerted its action on AC with the participation of microfilaments and/or microtubules associated with the cell membrane. Preincubation of macrophage monolayers for 1 hr with 10 pg/ml TABLE
5
Lack of Effect of Colchicine on Con A-Induced Enhancement of Macrophage Adenylate Cyclase Responsiveness” CAMP level (pmol per mg macrophage protein) Macrophages preincubated with:
Concentration
Basal
+PGE, (1O-4 M)
Medium Con A Colchicine Colchicine + Con A
50 pgirnl lo-” M 10-6M 50 &ml
15.0 14.0 21.9 23.1
161 761 966 843
+Isoproterenol (IO-:’ M) 37.8 189 243 271
n Cells were preincubated in medium with or without colchicine for 60 min. Following this, Con A or an equivalent volume of saline solution was added and the dishes reincubated for further 10 min. The cells were then exposed for 15 min to PGE, or isoproterenol in the presence of Ro 20-1724, 2 x 10s4 M. Data represent means of values from duplicate dishes.
422
GRUNSPAN-SWIRSKY
AND PICK
cytochalasin B, a drug interfering with microfilament function, did not alter the enhancement of responsiveness to PGE, and IP induced by a subsequent exposure to Con A (50 pg/ml, 10 min). Pretreatment of macrophages with 10m6M colchicine for 1 hr resulted in a marked enhancement of the PGE,- and IP-induced CAMP accumulation, in accordance with our earlier findings (37). Preincubation of macrophages with lop6 M colchicine for 1 hr followed by a IO-min exposure to 50 pg/ml Con A did not result in either summation or synergism of facilitating effects (Table 5). The enhancement induced by the combination of colchicine and Con A was close to that induced by colchicine alone. Release of CAMP from Con A-Treated Macrophages
Release of CAMP into the surrounding medium has been described in rat and guinea pig macrophages stimulated with prostaglandins and IP (2,3). One possible explanation for the enhanced CAMP accumulation in Con A-treated macrophages could be a reduction in the release of CAMP from the cells. However, preexposure to 50 &ml Con A for 10 min resulted in an increased liberation of CAMP from PGE,- and IP-stimulated cells into the medium, which paralleled the elevated intracellular concentrations (Table 6). Therefore, a block in CAMP egress from macrophages cannot be the explanation for Con A-induced facilitation. Effect of other Lectins on Responsiveness of Macrophages to AC Stimulators
The question which immediately arose in the light of the marked effect of Con A on macrophage AC was whether other lectins can exert a similar influence. We therefore examined the effects of PHA, WGA, SBA, PWM, and LL. The ability of PHA, WGA, and SBA to bind to guinea pig peritoneal macrophages is shown by their ability to agglutinate and inhibit the migration of these cells in the conTABLE
6
Effect of Con A on CAMP Release from Macrophages” CAMP level (pmol per mg macrophage protein) Macrophages preincubated with:
Concentration Wml)
Basal
+PGE, (lo-4 M)
+Isoproterenol ( 1O-3 M)
In cell extract Medium Con A
50
31.5 22.5
215 685
53.1 261
In cell-free supernatant Medium Con A a The experimental 10 min and stimulated CAMP concentrations phage monolayers. b Concentration of
50
00 Ob
9.3 15.5
3.7 7.5
procedure was as described in the text. Cells were preincubated with Con A for with PGEi or isoproterenol for 15 min, in the presence of Ro 20-1724,2 x 10m4M. were determined both in the cell extracts and in the medium covering the macroCAMP was below the limit of sensitivity of our assay.
LECTINS
AFFECT
MACROPHAGE TABLE
ADENYLATE
423
CYCLASE
7
Effect of PHA on Responsiveness of Macrophages to Adenylate Cyclase Stimulators
Macrophages preincubated with:
Concentration (wdml)
Medium PHA PHA PHA
10 50 200
Medium PHA PHA PHA
3.5 35 350
Preincubation time (min)
CAMP level (pmol per mg macrophage protein)” Basal
+PGE, (10m4 M)
+Isoproterenol (lO-3 M)
10
7.6 7.1 7.4 6.8
223 379 338 511
32.4 66.7 172 154
60
12.0 7.7 3.8 2.9
192 219 383 424
23.1 25.8 67.8 114.1
(’ Cells were stimulated with PGE, or isoproterenol for 15 min in the presence of Ro 20-1724, 2 x 10m4M. Data represent means of values from duplicate dishes.
centration range of 10 to 40pg/ml (E. Pick, unpublished observation). The capacity of the fucose-binding LL to react with guinea pig macrophages is suggested by the detection ofa-L-fucose in the cell membrane (19, 43). We have no proof for binding of PWM to guinea pig macrophages but this has been demonstrated for mouse peritoneal macrophages (18). Incubation of macrophages with a wide variety of PHA concentrations for 10 or 60 min resulted in a marked enhancement of responsiveness to PGE, and IP. (Table 7). In contrast, the basal CAMP level was not elevated by PHA. It is therefore evident that PHA behaves in a fashion similar to Con. A. On the other hand, preincubation of macrophages with WGA, SBA, PWM, and LL had no significant influence on CAMP accumulation in response to PGE, or IP (Table 8). In all these experiments a positive Con-A control was introduced, serving as proof that facilitation does occur with the appropriate lectin. A limited number of experiments were performed with the above lectins using a preincubation time of 1 hr. Under these conditions, WGA reduced the basal CAMP concentration to 25% of the control level and the CAMP accumulation in response to PGE, and IP, to 50% of the control levels. DISCUSSION We have shown that two lectins, Con A and PHA, which exhibit specificity for sugars present in the macrophage membrane, markedly enhance CAMP generation in guinea pig macrophages stimulated with PGE, and IP. Basal CAMP levels were not modified. Facilitation of AC responsiveness was not induced by any of the four other lectins tested: WGA, SBA, PWM, and LL. Con A-induced facilitation developed as early as 10 min from the exposure of cells to the lectin, and concentrations of Con A from 10 to 400 &ml were effective. The Con-A induced enhancement did not extend to all AC activators; the response to cholera toxin was apparently not augmented. The effect of Con A required its
GRUNSPAN-SWIRSKY TABLE
AND PICK 8
Lack of Effect of Four Different Lectins on Responsiveness of Macrophage Adenylate Cyclase to Stimulators
Macrophages preincubated with:
Concentration (&ml)
Medium WGA WGA
-
Medium SBA SBA
-
Medium PWM PWM
-
Medium LL LL
-
10 50 10 50 10 50 10 50
Preincubation time (min)
CAMP level (pmol per mg macrophage protein ? SEM) No. of expt
Basal
+PGE, ( lO-4 M)
+Isoproterenol ( lO-3 M)
10
3
14.9 + 5.5 5.9 k 1.3 9.0 + 1.0
192 + 25.0 146 2 26.5 226 + 52.5
81.0 k 9.5 73.0 2 6.8 123 k 24.1
10
4
19.7 f 4.9 17.2 2 6.2 11.5 2 3.2
241 k 45.1 217 2 28.3 239 k 15.6
85.3 k 39.3 76.1 k 22.8 72.5 + 22.3
10
3
17.6 f 6.9 13.2 2 3.7 15.5 2 6.5
231 k 30.6 202 2 22.1 219 ? 23.5
52.2 k 6.9 51.6 2 8.7 43.6 2 3.3
10
1
12.9 10.5 10.3
240 247 209
71.0 79.5 70.8
u Cells were stimulated with PGE, or isoproterenol for 15 min in the presence of Ro 20-1724, 2 x low4 M. Data represent means ? SEM. For each experiment, data were derived from duplicate dishes.
attachment to specific sugar-containing receptors on the membrane, as shown by the prevention offacilitation by aMM. For effective facilitation to occur, Con A had to be presented to the macrophage as a soluble tetrameric molecule. The dimeric succinyl-Con A and Sepharose-attached Con A were both inactive. The requirement for tetrameric Con A closely parallels the finding that while native Con A elevates CAMP levels in human lymphocytes, succinyl-Con A does not (44). Sepharose-attached Con A was also found to be less effective than soluble Con A in stimulating CAMP synthesis in lymphocytes (25). Lectins have been shown in the past to be capable of modulating the activity of a number of membrane-bound enzymes, including AC, in a variety of cells or membrane preparations. Con A inhibits basal and stimulated AC in fat cell (27) and brain synaptic membranes (29). WGA also inhibits AC in fat cell (27) and pancreas plasma membranes (30). As opposed to these inhibitory actions, Con A improved ACTH stimulation of AC in adrenocortical plasma membranes (28) and restored activity of an ATPase preparation inactivated by detergent treatment (45). The ectoenzyme 5’-nucleotidase of liver plasma membrane could be stimulated by low concentrations and inhibited by high concentrations of Con A while succinyl-Con A was unable to inhibit (46). The findings most closely related to ours are those of Gemsa et al. (32), who described enhancement of responsiveness of rat macrophages to PGE1, IP, and cholera toxin by preincubation with Con A. Interestingly, these authors found no enhancement with PHA, under conditions in which it was found active by us on guinea pig macrophages. Our findings also differ from those of Gemsa et al. (32) by
LECTINS
AFFECT
MACROPHAGE
ADENYLATE
CYCLASE
425
our failure to see significant enhancement by Con A of macrophage responsiveness to cholera toxin. It therefore appears that binding of Con A or PHA to macrophages is followed by a perturbation of the membrane resulting in an increased sensitivity of the cells to some but not all AC stimulators but having no influence on the basal enzyme activity. Mere binding of the lectin to cell-surface sugars is not a sufficient condition for the induction of facilitation. A requirement seems to exist for multivalent interaction between Con A and membrane receptors, as shown by the inability of the dimeric succinyl-Con A to induce facilitation. A similar requirement was described for the mitogenicity of some but not all lectins (47). The lack of activity of succinyl-Con A is unlikely to be due to its inability to induce capping since macrophages, unlike lymphocytes, do not exhibit spontaneous capping of Con-A receptors (20). However, the possibility must be considered that capping of Con-A receptors might occur in macrophages incubated with AC stimulators, as a consequence of the elevation in the cellular level of CAMP. It has been found that leukocytes of patients with Chediak-Higashi syndrome, which have a markedly increased CAMP concentration, exhibit an abnormally high frequency of Con-A capping (48). This is probably mediated by the detrimental effect of high cellular CAMP levels on microtubules (49); indeed, disruption of microtubules was shown by us to make Con-A capping in macrophages possible (20). The inability of Sepharose-bound Con A to induce facilitation suggests that multivalent attachment per se is not sufficient and that clustering of receptors is obligatory. Such clustering is obviously impossible under conditions in which Con A is attached to a solid matrix. There is recent evidence indicating that, contrary to earlier reports, insolubilization of lectins results in loss of mitogenic activity (39). While receptor movement seems to be essential for the development of facilitation, it does not appear to be effected with the aid of actin microfilaments, as shown by the fact that cytochalasin B-treated macrophages demonstrate undiminished Con A-induced facilitation. The role of microtubules in Con-A enhancement is more difficult to determine because of the marked enhancement of AC activity in colchicine-treated cells (32, 37) and the lack of evidence for either synergism or antagonism between the two agents. The central issue raised by our findings is the mechanism by which lectins influence stimulated but not basal AC activity. Enhancement of CAMP accumulation could be the result of: (a) increased synthesis, (b) reduced degradation, or (c) block of egress from the cells. Reduced degradation can be eliminated since all experiments were performed in the presence of the CAMP-specific PDE inhibitor Ro 20-1724, which was reported to be 5000 times more potent than theophylline (50). Release of CAMP from Con A-treated macrophages was not reduced but actually augmented, a reflection of the elevated intracellular levels. An effect on degradation or release is also incompatible with the fact that Con A and PHA do not affect basal CAMP levels. It can therefore be concluded that the target for lectin enhancement is the AC complex in the membrane. This complex is composed of the stimulant-specific receptor physically separate from the catalytic unit of the enzyme (16, 17). Lectins could act by an effect on the receptors, on the enzyme itself, or on the receptor-enzyme complex in coupled form. In all eventuality, we must assume that the hormone receptors or the Catalytic unit are either glycoproteins, possessing a-D-mannosyl, a-D-glucosyl (as
426
GRUNSPAN-SWIRSKY
AND PICK
shown by the Con-A effect), or N-acetyl-D-galactosamine (as shown by the PHA effect) residues, or are interacting with and influenced by such glycoproteins in their membrane microenvironment. The following mechanisms could account for the lectin-induced enhancement of AC stimulation: (i) induction of receptor clustering, assuming that clustered receptors are more effective in interacting with the enzyme; (ii) causation of a conformational change in the receptor molecule which augments receptor exposure, increases affinity of ligand for receptor, or stabilizes the receptor-catalytic unit coupling; (iii) inhibition of negative cooperativity which results in slow dissociation of ligands at high ligand concentrations; negative cooperativity was described for P-adrenergic stimulants (5 1) and Con A was found to markedly inhibit negative cooperativity of insulin receptors in lymphocytes (52); (iv) increase in membrane fluidity which facilitates receptor movement in the lipid bilayer and augments the chance of collision with the catalytic unit of the enzyme; mitogenic lectins were found to induce an increase in membrane fluidity of lymphocytes within 30 min of incubation (53); (v) induction of a Caz+ influx, causing depolymerization of submembranal microtubules, which is known to induce facilitation of AC stimulation (32, 37); mitogenic lectins produce early increases in Ca*+ uptake in lymphocytes (54) and elevated Ca2+ concentrations were reported to disrupt microtubules (55); and (vi) finally, the possibility exists that lectins bind to and inactivate a sugar-containing inhibitor of AC; there is no experimental evidence in support of this. An important question is raised by our finding which cannot be answered at present: Why do Con A and PHA induce facilitation while WGA, SBA, PWM, and LL are inactive? These differences could depend on the presence and density of membrane receptors or on special properties of the lectins, such as their valency. These results, obtained with model membrane-reactive agents such as lectins, suggest that under physiologic or pathologic conditions macrophage reactivity to hormonal stimulation is determined by the state of the membrane. Obvious examples for such modulation are the enhanced responsiveness to PGE, of macrophages during phagocytosis (56) and the refractoriness of MIF-treated macrophages to several AC stimulators (4, 57). ACKNOWLEDGMENTS We thank Dr. John E. Pike, The Upjohn Company, Kalamazoo, Michigan, for repeated gifts to prostaglandin E,; F. Hoffmann-LaRoche and Company Ltd., Base], Switzerland, for gifts of Ro 20-1724; and Ms. H. Cohen for excellent secretarial assistance.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
Remold-O’Donnell, E., J. Biol. Chem. 249, 3615, 1974. Gemsa, D., Steggeman, L., Menzel, J., and Till, G., J. Immunol. 114, 1422, 1975. Higgins, T. J., and David, J. R., Cell. Immunol. 27, 1, 1976. Pick, E., Cell. Immunol. 32, 329, 1977. Ikegami, K., Biochem. Pharmacol. 26, 1813, 1977. 8, 180, 1978. Welscher, H. D., and Cruchaud, A., Eur. J. Zmmunol. Nowell, G. H., Cruse, J. M., and Lewis, R. E., Am. J. Pathol. 86, 81a, 1977. Seyberth, H. W., Schmidt-Gayk, J., Jacobs, K. H., and Hackenthal, E.,J. Cell. Bid. 57,567,1973. Muschel, P. J., Rosen, N., Rosen, 0. M., and Bloom, B. R., J. fmmunol. 119, 1813, 1977. Pick, E., Nature New Bid. 238, 176, 1972.
LECTINS 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57.
AFFECT
MACROPHAGE
ADENYLATE
CYCLASE
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Genta, G. E., and Hill, H. R., Clin. Res. 24, 180A, 1976. Koopman, W. J., Gillis, M. H., and David, J. R., J. Immunol. 110, 1609, 1973. Pick, E., and Manheimer, S., Cell. Immunol. 11, 30, 1974. Vassalli, J. D., Hamilton, J., and Reich, E., Cell 8, 271, 1976. Perkins, J. P., Adv. Cyclic Nucl. Res. 3, 1, 1973. Cuatrecasas, P., Ann. Rev. Biochem. 43, 169, 1974. De Haen, P., J. Theor. Biol. 58, 383, 1976. Loor, F., and Roelants, G. E., Eur. J. Immunol. 4, 649, 1974. Newman, W., Bloom, B. R., and Satoh, P., Cell. Immunol. 27, 343, 1976. Pick, E., and Wilner, I., Exp. Cell Res. 118, 151, 1979. Edelson, P. J., and Cohn, Z. A., J. Exp. Med. 140, 1364, 1974. Goldman, R., and Raz, A., Exp. Cell Res. 96, 393, 1975. Vassalli, J. D., Hamilton, J., and Reich, E., Cell 11, 695, 1977. Smith, J. W., Steiner, A. L., and Parker, C. W., J. C/in. Invest. 50, 442, 1971. Lyle, L. R., and Parker, C. W., Biochemistry 13, 5415, 1974. Greene, W. C., Parker, C. M., and Parker, C. W., Fed. Proc. 34, 1029, 1975. Cuatrecasas, P., and Tell, G. P. E., Proc. Nat. Acad. Sci. U.S.A. 70, 485, 1973. Schlegel, W., FEBS Lett. 69, 67, 1976. Michaelis, E. K., and Michaelis, M. L., Life Sci. 18, 1021, 1976. Deschodt-Lanckman, M., Robberecht, P., Camus, J. C., and Cristophe, J., J. Cycl. NW/. Res. 3, 177, 1977. Grunspan-Swirsky, A., and Pick, E., fsr. J. Med. Sci., in press. Gemsa, D., Steggemann, L., Till, G., and Resch, K., J. Immunol. 119, 524, 1977. Gunther, G. R., Wang, J. L., Yahara, I., Cunningham, B. A., and Edelman, G. M., Proc. Nut. Acad. Sci. U.S.A. 70, 1012, 1973. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J., J. Biol. Chem. 193,265, 1951. Brown, B. L., Albano, J. D. M., Ekins, R. P., and Sgherzi, A. M., Biochem. J. 121, 561, 1971. Tovey, K. E., Oldham, K. G., and Wheelan, J. A. M., Clin. Chim. Acta 56, 221, 1974. Grunspan-Swirsky, A., and Pick, E., Immunopharmacology, 1, 71, 1978. 2, 233, 1972. Anderson, J., Edelman, G. M., Moller, G., and Sjoberg, 0.. Eur. J. Immunol. Avrameas, S., Ternynck, T., Macieira-Coelho, A., and Garcia-Giralt, E., Biochem. Biophys. Rrs. Commun. 72, 790, 1976. Reaven, E. P., and Axline, S. G., J. Cell. Biol. 59, 12, 1973. Pick, E., Honig, S., and Griffel, B., Int. Arch. Allergy Appl. Immunol., 58, 149, 1979. Nicolson, G. L., Biochim. Biophys. Acta 457, 57, 1976. Remold, H. G., J. Exp. Med. 138, 1065, 1973. Coffey, R. G., Hadden, E. M., and Hadden, J. W., J. Immunol. 119, 1387, 1977. Ronquist, G., and Hedstrom, M., Biochim. Biophys. Acta 483, 483, 1977. Slavik, M., Kartner, N., and Riordan, J. R., Biochrm. Biophys. Res. Commun. 75, 342, 1977. Schechter, B., Lis, H., Lotan, R., Nowogrodsky, A., and Sharon, N., Eur. J. Immunol. 6, 145, 1976. Boxer, L. A., Watanabe, A. W., Rister, M., Besch, H. R., Allen, J., and Baehner, R. L.. New EngI. J. Med. 295, 1041, 1976. Weissmann, G., Goldstein, I., Hoffstein, S., and Tsung, P. K.,Ann. N. Y. Acad. Sri. 253,750, 1975. Sheppard, H., and W&an, G., Molec. Pharmacol. 7, 111, 1971. Limbird, L. E., De Meyts, P., and Lefkowitz, R. J., Biochem. Biophys. Res. Commun. 64, I 160. 1975. De Meyts, P., J. Supramolec. Struct. 4, 241, 1976. Toyoshima, S., and Osawa, T., J. Biol. Chem. 250, 1655, 1975. Parker, C. W., Biochem. Biophys. Res. Commun. 61, 1180, 1974. Fuller, G. M., and Brinkley, B. R., J. Supramolec. Struct. 5, 497, 1976. Gemsa, D., Seitz, M., Kramer, W., Till, G., and Resch, K., J. fmmunol. 120, 1187, 1978. Pick, E., and Grunspan-Swirsky, A., Cell. Immunol. 32, 340, 1977.
45, 415-427 (1979)
IMMUNOLOGY
Facilitation
ANNA Section
of Immunology,
of Adenylate in Macrophages
Cyclase Stimulation by Lectinsl
GRUNSPAN-SWIRSKY Department Tel-Aviv
AND
of Human University
Received
EDGAR
Microbiology, Sackler Tel-Aviv, Israel
October
PICKY School
of Medicine,
2. 1978
Preincubation of guinea pig peritoneal macrophages with concanavalin A (Con A) markedly enhanced the accumulation of 3’,5’-cyclic-adenosine monophosphate (CAMP) in response to the adenylate cyclase (AC) stimulators prostaglandin E, (PGEJ and isoproterenol (IP). Basal CAMP levels were not altered. Maximal enhancement of CAMP accumulation was induced by preincubation with 50-100 pg/ml Con A for 10 min at 37°C. Con A-induced facilitation of macrophage responsiveness was prevented by cu-methyl-omannoside (@MM). No facilitation was induced by the divalent derivative, succinyl-Con A or by Con A immobilized on Sepharose beads. Con A-induced facilitation developed normally in macrophages treated with the microfilament blocking agent, cytochalasin B. The responsiveness of macrophages to PGE, and IP was also augmented by phytohemagglutinin (PHA) but wheat germ agglutinin (WGA), soy bean agglutinin (SBA), pokeweed mitogen (PWM), and Lotus tetragonolobus lectin (LL) showed no enhancing effect. The effect of Con A on CAMP levels was the result of augmented CAMP synthesis and not of reduced degradation or a block in CAMP egress from the cells. Lectin-induced facilitation of AC stimulation could be mediated via one of the following mechanisms: (i) induction of receptor clustering; (ii) causing a conformational change in the receptors; (iii) inhibition of negative cooperativity; (iv) causing an increase in membrane fluidity; (v) disruption of microtubules by acting as a CaZ+ lonophore; or (vi) inactivation of a sugar-containing inhibitor of AC.
INTRODUCTION Macrophages of several animal species were found to respond to incubation with P-adrenergic agents, prostaglandins E, and E, (PGE,, PGE2),” and cholera toxin by an elevation in the cellular level of cyclic 3’,5’-adenosine monophosphate (CAMP) (l-6). There is also direct evidence for the presence of /3-adrenergic receptors on the membrane of peritoneal macrophages (7). The cellular concentra’ Supported by National Institutes ofHealth Grant AI-l 1194, Bethesda, Maryland and by U.S.-Israel Binational Science Foundation Grants 480 and 1505. * Address reprint requests to Edgar Pick. ’ Abbreviations used in this paper: PGE,, prostaglandin E,; CAMP, 3’,5’-cyclic-adenosine monophosphate; MIF, migration inhibitory factor; Con A, concanavalin A; PHA, phytohemagglutinin; PWM, pokeweed mitogen; LL, Lotus tetragonolobus lectin; WGA, wheat germ agglutinin: SBA, soybean agglutinin; IP, isoproterenol; PDE, phosphodiesterase; DMSO, dimethylsulfoxide; aMM, a-methyl-o-mannoside; PEC, peritoneal exudate cells; MEM, Eagle’s minimum essential medium; FCS, fetal calf serum; AC, adenylate cyclase. 415 0008-&X749/79/080415-13$02.00/O Copyright 0 1979 by Academic Press, All
rights
of reproduction
in any
form
Inc. reserved.
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GRUNSPAN-SWIRSKY
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PICK
tion of CAMP has a profound influence on several macrophage functions, such as phagocytosis (8, 9), spontaneous motility (lo), responsiveness to chemotactic stimuli (11) and to migration inhibitory factor (MIF) (12, 13), and enzyme release (14). Adenylate cyclase (AC) is exclusively a membrane-bound enzyme (15). The currently accepted model visualizes it as being composed of two distinct components, a stimulant-specific receptor facing outwards and a catalytic unit localized deeper in the membrane. The receptors and, perhaps, catalytic units too, are seen as freely moving in the plane of the membrane (16, 17). These properties suggested that AC might serve as a sensitive functional marker for membrane perturbations induced by exogenous agents interacting directly with the membrane or by intracellular events reflected on the membrane. A number of lectins including concanavalin A (Con A), phytohemagglutinin (PHA), pokeweed mitogen (PWM), and Lotus tetragonofobus lectin (LL) were found to bind to macrophage membrane receptors (18-20). Most studies were performed with Con A, which was also found to influence macrophage physiology, as illustrated by stimulation of endocytosis (21), formation of cytoplasmic vesicles (21, 22), and induction of plasminogen activator (23). It was therefore of interest to study the possible effect of lectins able to react with the macrophage membrane on the activity of AC. Additional reasons for initiating this study were the reports that PHA (24), Con A (25), and wheat germ agglutinin (WGA) (26) elevate CAMP levels in lymphocytes and the findings that Con A and WGA demonstrate both stimulatory and inhibitory effects on the AC of a variety of nonlymphoid cell types (27-30). In the present paper we report that Con A and PHA markedly enhance CAMP generation by guinea pig peritoneal macrophages stimulated by PGE, or IP but have no effect as basal CAMP levels. These findings indicate that agents reacting with membrane glycoproteins can modulate the responsiveness of macrophages to hormonal stimuli acting via the CAMP messenger. Part of this work was published in abstract (31). While this work was in progress, Gemsa et al. (32) reported that Con A moderately enhances CAMP production in rat macrophages stimulated with PGE1, IP, and cholera toxin. MATERIALS AND METHODS Lectins. Con A (3 x crystallized, lyophylized, desalinated powder) was obtained from Miles-Yeda and a stock solution of 1 mg/ml was prepared in 0.01 M phosphate buffer, pH 7.2, containing 1 M NaCl. PHA-P was obtained from Difco Laboratories and a stock solution of 17.5 mg/ml was prepared in distilled water. WGA, SBA, and LL were all obtained from Sigma in the form of lyophilized powder and dissolved in 0.15 M NaCl to 0.5 mg/ml. PWM was obtained from Gibco and dissolved in 0.15 M NaCl to a concentration of 0.5 mg/ml. All mitogen stock solutions were kept frozen at -70°C. Succinyl-Con A was prepared from Con A by twice derivatization with succinic anbydride (Sigma), as described by Gunther et a/. (33). Con A-Sepharose (Con A covalently bound to Sepharose 4B by the cyanogen bromide method) was a commercial preparation obtained from Pharmacia Fine Chemicals, containing 8 mg Con A per milliliter of gel. Just before use, the gel was washed five times with an alternation of 0.1 M acetate
LECTINS
AFFECT
MACROPHAGE
ADENYLATE
CYCLASE
417
buffer, ph 4, and 0.1 M borate buffer, pH 8.6, both containing 1 M NaCl, followed by five washes with 0.15 M NaCl. The washed gel was suspended in 0.15 M NaCl containing 1O-3 M MnCl,, MgC&, and CaCl,. Before use, each preparation of Con A-Sepharose was tested for the presence of reactive Con A by incubation with a 1% suspension of guinea pig red cells for 15 min at 37°C in a shaking water bath. Adherence of numerous erythrocytes to the surface of Con A-Sepharose beads was taken as an indicator that the preparation is suitable for use. Drugs and chemicals. PGE1, a kind gift of Dr. John E. Pike, The Upjohn Company, was dissolved in 95% ethanol to a concentration of 10 mg/ml and stored at -70°C. L-Isoproterenol-D-bitartrate was obtained from Sigma and solutions were prepared just prior to use. The CAMP phosphodiesterase (PDE) inhibitor d, 1-4-(3-butoxy-4-methoxybenzyl)-2-imidazolinone (Ro 20-1724) was a kind gift of F. Hoffmann-LaRoche and Company Ltd., Basel, Switzerland. A stock solution of 0.2 M was prepared in dimethyl sulfoxide (BMSO) and stored at 4°C. Preparation of macrophage monolayers. Male guinea pigs, Hartley strain, weighing 300-500 g, were injected intraperitoneally with 20 ml light paraffin oil (British Drug Houses) and peritoneal exudate cells (PEC) were harvested 3-4 days subsequent to injection. PEC were washed three times in Earle’s balanced salt solution and suspended at a concentration of 5- 10 X 10” cells/ml in Eagle’s minimum essential medium (MEM), supplemented with nonessential amino acids and sodium pyruvate, and containing 15% heat-inactivated fetal calf serum (FCS, Gibco). The suspension was divided among 9-cm-diameter plastic petri dishes (tissue culture grade, Nunc), 5 ml/dish, and incubated for 1 hr at 37°C in 90% air- 10% CO, to allow for preferential adherence of macrophages to the dish surface. Unattached cells were then removed by rinsing the dishes 3 times with 5 ml warm MEM. Viability of macrophages in monolayers (assessed by trypan blue exclusion) was close to 100% and was not affected by any of the lectins, drugs. or stimulants used. Determination of lectin effect on macrophage CAMP levels. Macrophage monolayers were covered with 5 ml MEM containing the lectin to be tested and incubated for predetermined time intervals. This stage was termed the preincubation period. Following preincubation, AC stimulators were added together with the PDE inhibitor Ro 20-1724 (2 x 10e4 M) and incubation continued for 15 min more. The reaction was terminated by placing the dishes on ice-cooled metal trays. The medium was rapidly removed by vacuum suction and 1.5 ml of ice-cold 0.05 M Tris-HCI buffer, pH 7.5, containing 0.004 M EDTA, were added per dish. The cells were removed by means of a rubber policeman and transferred into 100 x 16-mm conical glass tubes, kept in ice water. A sample of 0.1 ml was removed for protein determination by the method of Lowry et al. (34). The conical tubes were then immersed for 5 min in boiling water to precipitate protein, centrifuged for 10 min at 2OOOg, and 1 ml of supernatant was lyophilized. It was then redissolved in 0.2-0.5 ml of distilled water and CAMP was measured in duplicate 50-~1 samples by the competitive protein binding assay of Brown et al. (35), as modified by Tovey et al. (36), using a kit commercially manufactured by the Radiochemical Center, Amersham, England (Code No. TRK 432). Results were expressed as picomoles of CAMP per milligram of macrophage protein, representing the average derived from two identically-treated petri dishes. De-
418
GRUNSPAN-SWIRSKY
AND PICK
tailed CAMP extraction and determination procedures have been described previously (4). Measurement of CAMP release from macrophages. Macrophage monolayers were covered with 1.5 ml MEM containing the lectin and incubated for 10 min at 37°C. Following this, AC stimulators were added together with Ro 20-1724 (2 x lop4 M) and incubation continued for further 15 min. The experiment was stopped by placing the dishes on ice-cooled metal trays and rapidly transferring the supernatant into conical glass tubes maintained in ice water and containing 0.075 ml of 1 M Tris-HCl buffer, pH. 7.5, and 0.08 M EDTA. The content of the tubes was rapidly mixed on a Vortex apparatus and centrifuged for 5 min at 2OOOg, at 4”C, to sediment cells which may have become detached from the dish surface. The supematants were transferred to new tubes which were boiled for 5 min and centrifuged for 10 min at 2000g. Supematant, 1 ml amounts, were lyophilized and redissolved in 0.2 ml of distilled water, and duplicate 50 ~1 samples of this were used in the CAMP assay. Cellular CAMP levels were determined in the same dishes by extracting the macrophage monolayer, after removal of the supematant , as described above. RESULTS Enhancement
by Con A of Macrophage
Responsiveness
to AC Stimulators
We have previously demonstrated that CAMP levels in purified guinea pig peritoneal macrophages are elevated in a highly reproducible manner by incubation with PGEl (4). CAMP generation in response to IP is more erratic and occasionally absent (37). Preincubation of macrophages in monolayer with Con A resulted in a marked increase in CAMP accumulation in response to both PGE, and IP (Table 1). TABLE
1
Effect of Con A on Responsiveness of Macrophages to Adenylate Cyclase Stimulators
Macrophages preincubated with:
Concentration Cwh-4
Medium Con A
-
Medium Con A Medium Con A D Cells were 2 x lo+ M. P pretreated with t test for paired
Preincubation time (min)
CAMP level (pmol per mg macrophage protein k SEM)” No. of expt
Basal
+PGE, (lO-4 M)
+Isoproterenol ( 1O-3 M)
10
9
22.8 c 5.1 21.7 2 3.4 (P > 0.50)
139 rf: 13.5 462 f 56.0 (P < 0.001)
43.4 Ii 9.1 168 2 45.7 (P < 0.02)
100
10
4
28.8 2 10.9 18.2 k 6.9 (P < 0.50)
152 k 20.5 372 -c 12.5 (P < 0.001)
33.8 f 13.1 115 5 20.0 (P c 0.10)
100
60
8
19.3 ” 3.6 14.7 + 2.8 (P < 0.10)
170 2 14.3 323 r 30.8 (P ==c0.002)
18.5 f 2.8 57.4 + 7.7 (P c 0.001)
50
stimulated with PGE, or isoproterenol for 15 min, in the presence of Ro 20-1724, values express the significance of difference between CAMP levels in macrophages medium compared to macrophages pretreated with Con A, calculated by Student’s data.
LECTINS
AFFECT
MACROPHAGE TABLE
Prevention of Con A-Induced
Macrophages preincubated with: Medium Con A (YMM Con A + cvMM Con A + aMM
Concentration 50 &ml 5 x 10-z M 50 &ml
ADENYLATE
419
CYCLASE
2
Enhancement of Macrophage Adenylate Cyclase Responsiveness by a-Methyl-o-Mannoside (aMM)” Preincubation time (min)
IO
CAMP level (pmol per mg macrophage protein) Basal
+PGE, (lo-’ M)
+lsoproterenol (IO 2 M)
19.1 28.4 13.8 18.1
190 469 185 224
33.4 136 27.7 38. I
16.5
167
28.5
IO-'M 50 &ml 5 x 10-z M
’ Cells were preincubated with Con A in the presence or absence of aMM or with cvMM alone. They were then stimulated with PGE, or isoproterenol for 15 min in the presence of Ro 20-1724. 2 x lo-’ M. Data represent means of values from duplicate dishes.
The enhancement of macrophage responsiveness was even more striking when the stimulant was IP since cells totally unresponsive to IP could be induced to respond briskly by mere preexposure to Con A. Con A had no effect on basal CAMP levels. This phenomenon was termed “lectin-induced AC facilitation.” Facilitation developed both in the presence and in the absence of a PDE inhibitor but such inhibitor (Ro 20-1724, 2 x 1O-4 M) was regularly present in our experiments in order to enhance and stabilize CAMP accumulation. Dose response studies indicated that facilitation is induced by concentrations of Con A from 10 to 400 j&ml with a peak being reached at 50- 100 &ml. Facilitation was seen after incubation of macrophages with Con A for 10 to 120 min; maximal enhancement was obtained after exposure to Con A for 10 min. Preliminary results indicate that preincubation of macrophages with Con A (50 &ml) for 10 min does not enhance CAMP accumulation in response to a maximally stimulatory concentration of cholera toxin (10 @ml). Very moderate enhancement (123%) was found when macrophages were stimulated with 50 or 1000 &ml cholera toxin. The effect of Con A is exerted via its binding to specific sugar-containing macrophage membrane structures. This was demonstrated by the total elimination of facilitation, when incubation with Con A was in the presence of the competitive inhibitor aMM (1-5 x lo-* M) (Table 2). Inability
of Succinylated Con A and Insolubilized Con A to Induce Facilitation
Succinylation of native Con A converts the lectin from a tetramer to a dimer (33). Succinyl-Con A possesses identical sugar-binding properties to the native molecule but has a lesser capacity to agglutinate red cells and does not induce cap formation in lymphocytes. Macrophages were preincubated with succinyl-Con A for intervals of 10 or 60 min after which time they were stimulated with PGE, or IP. As apparent from
420
GRUNSPAN-SWIRSKY
AND PICK
TABLE
3
Inability of Succinyl-Con A to Enhance the Responsiveness of Macrophages to Adenylate Cyclase Stimulators
Macrophages preincubated with:
Concentration (dmU
Medium Con A Succinyl-Con A Con A Succinyl-Con A
10 10 50 50
Medium Con A Succinyl-Con A Con A Succinyl-Con A
10 10 50 50
CAMP level (pmol per mg macrophage protein)
Preincubation time (min)
No. of expt
10
60
Basal
+PGE, (1O-4 M)
+Isoproterenol (1O-3 M)
2
42.9 49.2 51.2 49.7 48.3
354 467 246 624 246
106 172 106 289 122
2
27 28 32 23 35
174 354 224 389 210
23.6 49.3 27.2 45.8 28.0
” Cells were stimulated with PGEl or isoproterenol for 15 min, in the presence of Ro 20-1724, 2 x 10e4 M. Data represent means of two experiments for every preincubation interval. For each experiment data were derived from duplicate dishes.
Table 3, succinyl-Con A, at concentrations of 10 and 50 &ml, was not capable of inducing AC facilitation comparable to that induced by identical concentrations of native Con A. We next examined the ability of Con A immobilized on Sepharose beads, to cause facilitation. Immobilized Con A was shown to be an effective lymphocyte mitogen (38), although the contrary opinion was also expressed (39). Macrophage monolayers were incubated with a suspension of Con A-Sepharose beads, equivalent to SO or 200 pg/ml of soluble Con A, on a rocking platform for 15 min at TABLE
4
Inability of Insolubilized Con A (Con A-Sepharose) to Enhance the Responsiveness of Macrophages to Adenylate Cyclase Stimulators’
Macrophages preincubated with:
Concentration of Con A (&ml)
Medium Con A Con A-Sepharose Con A-Sepharose
50 50 200
Preincubation time (min)
15
CAMP level (pmol per mg macrophage protein) Basal
+PGE, (1O-4 M)
6.9 9.1 4.5 3.8
81.6 259 81 148
+Isoproterenol (1O-3 M) 8.2 64.8 8.0 14.2
(’ Cells were preincubated with soluble Con A or Con A-Sepharose beads for 15 min at 37°C on a rocking platform, to ensure optimal contact between the beads and the macrophage monolayers. They were then stimulated with PGE, or isoproterenol for 15 min, in the presence of Ro 20-1724, 2 x 10e4 M. Data represent means of values from duplicate dishes.
LECTINS
AFFECT
MACROPHAGE
ADENYLATE
421
CYCLASE
37°C. Attachment of beads to the upper surface of cells in monolayer was clearly visible by examination of the dishes on an inverted microscope. The cells were subsequently exposed to PGE, or IP for 15 min at 37”C, without rocking. As seen in Table 4, preincubation with 50 pg/ml Con A-Sepharose did not result in facilitation of AC stimulation. Con A-Sepharose, 200 &ml, induced some facilitation but this could be accounted for by the presence in the medium covering the macrophages of soluble Con A, which had leaked from the beads during the incubation on the rocking platform. Leakage of Con A was detected by testing the macrophage supernatants for the ability to agglutinate guinea pig red cells. No free Con A was found in supernatants derived from macrophages incubated with 50&ml Con A-Sepharose. It was, however, possible that the lack of facilitation by insoluble Con A was due to the fact that 15 min was not sufficient time for an optimal interaction between Sepharose beads and the cells. Experiments were therefore performed in which macrophages were incubated with Con ASepharose for 30 min or 1 hr but no facilitation was obtained under these conditions, either. The Influence
of the Cytoskeleton
on Con A-Induced
Facilitation
Macrophages possess an extensive array of microfilaments and microtubules (40, 41). There is a large body of evidence indicating cytoskeletal control of membrane receptors in general and Con-A receptors in particular [reviewed in (42)]. We have recently shown that guinea pig peritoneal macrophages do not exhibit spontaneous capping of Con-A receptors but capping is made possible by treating the cells with microtubule disrupting drugs such as colchicine, vinblastine, or podophyllotoxin (20). We have also shown that such capping requires the integrity of microfilaments. The possibility was therefore considered that Con A exerted its action on AC with the participation of microfilaments and/or microtubules associated with the cell membrane. Preincubation of macrophage monolayers for 1 hr with 10 pg/ml TABLE
5
Lack of Effect of Colchicine on Con A-Induced Enhancement of Macrophage Adenylate Cyclase Responsiveness” CAMP level (pmol per mg macrophage protein) Macrophages preincubated with:
Concentration
Basal
+PGE, (1O-4 M)
Medium Con A Colchicine Colchicine + Con A
50 pgirnl lo-” M 10-6M 50 &ml
15.0 14.0 21.9 23.1
161 761 966 843
+Isoproterenol (IO-:’ M) 37.8 189 243 271
n Cells were preincubated in medium with or without colchicine for 60 min. Following this, Con A or an equivalent volume of saline solution was added and the dishes reincubated for further 10 min. The cells were then exposed for 15 min to PGE, or isoproterenol in the presence of Ro 20-1724, 2 x 10s4 M. Data represent means of values from duplicate dishes.
422
GRUNSPAN-SWIRSKY
AND PICK
cytochalasin B, a drug interfering with microfilament function, did not alter the enhancement of responsiveness to PGE, and IP induced by a subsequent exposure to Con A (50 pg/ml, 10 min). Pretreatment of macrophages with 10m6M colchicine for 1 hr resulted in a marked enhancement of the PGE,- and IP-induced CAMP accumulation, in accordance with our earlier findings (37). Preincubation of macrophages with lop6 M colchicine for 1 hr followed by a IO-min exposure to 50 pg/ml Con A did not result in either summation or synergism of facilitating effects (Table 5). The enhancement induced by the combination of colchicine and Con A was close to that induced by colchicine alone. Release of CAMP from Con A-Treated Macrophages
Release of CAMP into the surrounding medium has been described in rat and guinea pig macrophages stimulated with prostaglandins and IP (2,3). One possible explanation for the enhanced CAMP accumulation in Con A-treated macrophages could be a reduction in the release of CAMP from the cells. However, preexposure to 50 &ml Con A for 10 min resulted in an increased liberation of CAMP from PGE,- and IP-stimulated cells into the medium, which paralleled the elevated intracellular concentrations (Table 6). Therefore, a block in CAMP egress from macrophages cannot be the explanation for Con A-induced facilitation. Effect of other Lectins on Responsiveness of Macrophages to AC Stimulators
The question which immediately arose in the light of the marked effect of Con A on macrophage AC was whether other lectins can exert a similar influence. We therefore examined the effects of PHA, WGA, SBA, PWM, and LL. The ability of PHA, WGA, and SBA to bind to guinea pig peritoneal macrophages is shown by their ability to agglutinate and inhibit the migration of these cells in the conTABLE
6
Effect of Con A on CAMP Release from Macrophages” CAMP level (pmol per mg macrophage protein) Macrophages preincubated with:
Concentration Wml)
Basal
+PGE, (lo-4 M)
+Isoproterenol ( 1O-3 M)
In cell extract Medium Con A
50
31.5 22.5
215 685
53.1 261
In cell-free supernatant Medium Con A a The experimental 10 min and stimulated CAMP concentrations phage monolayers. b Concentration of
50
00 Ob
9.3 15.5
3.7 7.5
procedure was as described in the text. Cells were preincubated with Con A for with PGEi or isoproterenol for 15 min, in the presence of Ro 20-1724,2 x 10m4M. were determined both in the cell extracts and in the medium covering the macroCAMP was below the limit of sensitivity of our assay.
LECTINS
AFFECT
MACROPHAGE TABLE
ADENYLATE
423
CYCLASE
7
Effect of PHA on Responsiveness of Macrophages to Adenylate Cyclase Stimulators
Macrophages preincubated with:
Concentration (wdml)
Medium PHA PHA PHA
10 50 200
Medium PHA PHA PHA
3.5 35 350
Preincubation time (min)
CAMP level (pmol per mg macrophage protein)” Basal
+PGE, (10m4 M)
+Isoproterenol (lO-3 M)
10
7.6 7.1 7.4 6.8
223 379 338 511
32.4 66.7 172 154
60
12.0 7.7 3.8 2.9
192 219 383 424
23.1 25.8 67.8 114.1
(’ Cells were stimulated with PGE, or isoproterenol for 15 min in the presence of Ro 20-1724, 2 x 10m4M. Data represent means of values from duplicate dishes.
centration range of 10 to 40pg/ml (E. Pick, unpublished observation). The capacity of the fucose-binding LL to react with guinea pig macrophages is suggested by the detection ofa-L-fucose in the cell membrane (19, 43). We have no proof for binding of PWM to guinea pig macrophages but this has been demonstrated for mouse peritoneal macrophages (18). Incubation of macrophages with a wide variety of PHA concentrations for 10 or 60 min resulted in a marked enhancement of responsiveness to PGE, and IP. (Table 7). In contrast, the basal CAMP level was not elevated by PHA. It is therefore evident that PHA behaves in a fashion similar to Con. A. On the other hand, preincubation of macrophages with WGA, SBA, PWM, and LL had no significant influence on CAMP accumulation in response to PGE, or IP (Table 8). In all these experiments a positive Con-A control was introduced, serving as proof that facilitation does occur with the appropriate lectin. A limited number of experiments were performed with the above lectins using a preincubation time of 1 hr. Under these conditions, WGA reduced the basal CAMP concentration to 25% of the control level and the CAMP accumulation in response to PGE, and IP, to 50% of the control levels. DISCUSSION We have shown that two lectins, Con A and PHA, which exhibit specificity for sugars present in the macrophage membrane, markedly enhance CAMP generation in guinea pig macrophages stimulated with PGE, and IP. Basal CAMP levels were not modified. Facilitation of AC responsiveness was not induced by any of the four other lectins tested: WGA, SBA, PWM, and LL. Con A-induced facilitation developed as early as 10 min from the exposure of cells to the lectin, and concentrations of Con A from 10 to 400 &ml were effective. The Con-A induced enhancement did not extend to all AC activators; the response to cholera toxin was apparently not augmented. The effect of Con A required its
GRUNSPAN-SWIRSKY TABLE
AND PICK 8
Lack of Effect of Four Different Lectins on Responsiveness of Macrophage Adenylate Cyclase to Stimulators
Macrophages preincubated with:
Concentration (&ml)
Medium WGA WGA
-
Medium SBA SBA
-
Medium PWM PWM
-
Medium LL LL
-
10 50 10 50 10 50 10 50
Preincubation time (min)
CAMP level (pmol per mg macrophage protein ? SEM) No. of expt
Basal
+PGE, ( lO-4 M)
+Isoproterenol ( lO-3 M)
10
3
14.9 + 5.5 5.9 k 1.3 9.0 + 1.0
192 + 25.0 146 2 26.5 226 + 52.5
81.0 k 9.5 73.0 2 6.8 123 k 24.1
10
4
19.7 f 4.9 17.2 2 6.2 11.5 2 3.2
241 k 45.1 217 2 28.3 239 k 15.6
85.3 k 39.3 76.1 k 22.8 72.5 + 22.3
10
3
17.6 f 6.9 13.2 2 3.7 15.5 2 6.5
231 k 30.6 202 2 22.1 219 ? 23.5
52.2 k 6.9 51.6 2 8.7 43.6 2 3.3
10
1
12.9 10.5 10.3
240 247 209
71.0 79.5 70.8
u Cells were stimulated with PGE, or isoproterenol for 15 min in the presence of Ro 20-1724, 2 x low4 M. Data represent means ? SEM. For each experiment, data were derived from duplicate dishes.
attachment to specific sugar-containing receptors on the membrane, as shown by the prevention offacilitation by aMM. For effective facilitation to occur, Con A had to be presented to the macrophage as a soluble tetrameric molecule. The dimeric succinyl-Con A and Sepharose-attached Con A were both inactive. The requirement for tetrameric Con A closely parallels the finding that while native Con A elevates CAMP levels in human lymphocytes, succinyl-Con A does not (44). Sepharose-attached Con A was also found to be less effective than soluble Con A in stimulating CAMP synthesis in lymphocytes (25). Lectins have been shown in the past to be capable of modulating the activity of a number of membrane-bound enzymes, including AC, in a variety of cells or membrane preparations. Con A inhibits basal and stimulated AC in fat cell (27) and brain synaptic membranes (29). WGA also inhibits AC in fat cell (27) and pancreas plasma membranes (30). As opposed to these inhibitory actions, Con A improved ACTH stimulation of AC in adrenocortical plasma membranes (28) and restored activity of an ATPase preparation inactivated by detergent treatment (45). The ectoenzyme 5’-nucleotidase of liver plasma membrane could be stimulated by low concentrations and inhibited by high concentrations of Con A while succinyl-Con A was unable to inhibit (46). The findings most closely related to ours are those of Gemsa et al. (32), who described enhancement of responsiveness of rat macrophages to PGE1, IP, and cholera toxin by preincubation with Con A. Interestingly, these authors found no enhancement with PHA, under conditions in which it was found active by us on guinea pig macrophages. Our findings also differ from those of Gemsa et al. (32) by
LECTINS
AFFECT
MACROPHAGE
ADENYLATE
CYCLASE
425
our failure to see significant enhancement by Con A of macrophage responsiveness to cholera toxin. It therefore appears that binding of Con A or PHA to macrophages is followed by a perturbation of the membrane resulting in an increased sensitivity of the cells to some but not all AC stimulators but having no influence on the basal enzyme activity. Mere binding of the lectin to cell-surface sugars is not a sufficient condition for the induction of facilitation. A requirement seems to exist for multivalent interaction between Con A and membrane receptors, as shown by the inability of the dimeric succinyl-Con A to induce facilitation. A similar requirement was described for the mitogenicity of some but not all lectins (47). The lack of activity of succinyl-Con A is unlikely to be due to its inability to induce capping since macrophages, unlike lymphocytes, do not exhibit spontaneous capping of Con-A receptors (20). However, the possibility must be considered that capping of Con-A receptors might occur in macrophages incubated with AC stimulators, as a consequence of the elevation in the cellular level of CAMP. It has been found that leukocytes of patients with Chediak-Higashi syndrome, which have a markedly increased CAMP concentration, exhibit an abnormally high frequency of Con-A capping (48). This is probably mediated by the detrimental effect of high cellular CAMP levels on microtubules (49); indeed, disruption of microtubules was shown by us to make Con-A capping in macrophages possible (20). The inability of Sepharose-bound Con A to induce facilitation suggests that multivalent attachment per se is not sufficient and that clustering of receptors is obligatory. Such clustering is obviously impossible under conditions in which Con A is attached to a solid matrix. There is recent evidence indicating that, contrary to earlier reports, insolubilization of lectins results in loss of mitogenic activity (39). While receptor movement seems to be essential for the development of facilitation, it does not appear to be effected with the aid of actin microfilaments, as shown by the fact that cytochalasin B-treated macrophages demonstrate undiminished Con A-induced facilitation. The role of microtubules in Con-A enhancement is more difficult to determine because of the marked enhancement of AC activity in colchicine-treated cells (32, 37) and the lack of evidence for either synergism or antagonism between the two agents. The central issue raised by our findings is the mechanism by which lectins influence stimulated but not basal AC activity. Enhancement of CAMP accumulation could be the result of: (a) increased synthesis, (b) reduced degradation, or (c) block of egress from the cells. Reduced degradation can be eliminated since all experiments were performed in the presence of the CAMP-specific PDE inhibitor Ro 20-1724, which was reported to be 5000 times more potent than theophylline (50). Release of CAMP from Con A-treated macrophages was not reduced but actually augmented, a reflection of the elevated intracellular levels. An effect on degradation or release is also incompatible with the fact that Con A and PHA do not affect basal CAMP levels. It can therefore be concluded that the target for lectin enhancement is the AC complex in the membrane. This complex is composed of the stimulant-specific receptor physically separate from the catalytic unit of the enzyme (16, 17). Lectins could act by an effect on the receptors, on the enzyme itself, or on the receptor-enzyme complex in coupled form. In all eventuality, we must assume that the hormone receptors or the Catalytic unit are either glycoproteins, possessing a-D-mannosyl, a-D-glucosyl (as
426
GRUNSPAN-SWIRSKY
AND PICK
shown by the Con-A effect), or N-acetyl-D-galactosamine (as shown by the PHA effect) residues, or are interacting with and influenced by such glycoproteins in their membrane microenvironment. The following mechanisms could account for the lectin-induced enhancement of AC stimulation: (i) induction of receptor clustering, assuming that clustered receptors are more effective in interacting with the enzyme; (ii) causation of a conformational change in the receptor molecule which augments receptor exposure, increases affinity of ligand for receptor, or stabilizes the receptor-catalytic unit coupling; (iii) inhibition of negative cooperativity which results in slow dissociation of ligands at high ligand concentrations; negative cooperativity was described for P-adrenergic stimulants (5 1) and Con A was found to markedly inhibit negative cooperativity of insulin receptors in lymphocytes (52); (iv) increase in membrane fluidity which facilitates receptor movement in the lipid bilayer and augments the chance of collision with the catalytic unit of the enzyme; mitogenic lectins were found to induce an increase in membrane fluidity of lymphocytes within 30 min of incubation (53); (v) induction of a Caz+ influx, causing depolymerization of submembranal microtubules, which is known to induce facilitation of AC stimulation (32, 37); mitogenic lectins produce early increases in Ca*+ uptake in lymphocytes (54) and elevated Ca2+ concentrations were reported to disrupt microtubules (55); and (vi) finally, the possibility exists that lectins bind to and inactivate a sugar-containing inhibitor of AC; there is no experimental evidence in support of this. An important question is raised by our finding which cannot be answered at present: Why do Con A and PHA induce facilitation while WGA, SBA, PWM, and LL are inactive? These differences could depend on the presence and density of membrane receptors or on special properties of the lectins, such as their valency. These results, obtained with model membrane-reactive agents such as lectins, suggest that under physiologic or pathologic conditions macrophage reactivity to hormonal stimulation is determined by the state of the membrane. Obvious examples for such modulation are the enhanced responsiveness to PGE, of macrophages during phagocytosis (56) and the refractoriness of MIF-treated macrophages to several AC stimulators (4, 57). ACKNOWLEDGMENTS We thank Dr. John E. Pike, The Upjohn Company, Kalamazoo, Michigan, for repeated gifts to prostaglandin E,; F. Hoffmann-LaRoche and Company Ltd., Base], Switzerland, for gifts of Ro 20-1724; and Ms. H. Cohen for excellent secretarial assistance.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
Remold-O’Donnell, E., J. Biol. Chem. 249, 3615, 1974. Gemsa, D., Steggeman, L., Menzel, J., and Till, G., J. Immunol. 114, 1422, 1975. Higgins, T. J., and David, J. R., Cell. Immunol. 27, 1, 1976. Pick, E., Cell. Immunol. 32, 329, 1977. Ikegami, K., Biochem. Pharmacol. 26, 1813, 1977. 8, 180, 1978. Welscher, H. D., and Cruchaud, A., Eur. J. Zmmunol. Nowell, G. H., Cruse, J. M., and Lewis, R. E., Am. J. Pathol. 86, 81a, 1977. Seyberth, H. W., Schmidt-Gayk, J., Jacobs, K. H., and Hackenthal, E.,J. Cell. Bid. 57,567,1973. Muschel, P. J., Rosen, N., Rosen, 0. M., and Bloom, B. R., J. fmmunol. 119, 1813, 1977. Pick, E., Nature New Bid. 238, 176, 1972.
LECTINS 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57.
AFFECT
MACROPHAGE
ADENYLATE
CYCLASE
427
Genta, G. E., and Hill, H. R., Clin. Res. 24, 180A, 1976. Koopman, W. J., Gillis, M. H., and David, J. R., J. Immunol. 110, 1609, 1973. Pick, E., and Manheimer, S., Cell. Immunol. 11, 30, 1974. Vassalli, J. D., Hamilton, J., and Reich, E., Cell 8, 271, 1976. Perkins, J. P., Adv. Cyclic Nucl. Res. 3, 1, 1973. Cuatrecasas, P., Ann. Rev. Biochem. 43, 169, 1974. De Haen, P., J. Theor. Biol. 58, 383, 1976. Loor, F., and Roelants, G. E., Eur. J. Immunol. 4, 649, 1974. Newman, W., Bloom, B. R., and Satoh, P., Cell. Immunol. 27, 343, 1976. Pick, E., and Wilner, I., Exp. Cell Res. 118, 151, 1979. Edelson, P. J., and Cohn, Z. A., J. Exp. Med. 140, 1364, 1974. Goldman, R., and Raz, A., Exp. Cell Res. 96, 393, 1975. Vassalli, J. D., Hamilton, J., and Reich, E., Cell 11, 695, 1977. Smith, J. W., Steiner, A. L., and Parker, C. W., J. C/in. Invest. 50, 442, 1971. Lyle, L. R., and Parker, C. W., Biochemistry 13, 5415, 1974. Greene, W. C., Parker, C. M., and Parker, C. W., Fed. Proc. 34, 1029, 1975. Cuatrecasas, P., and Tell, G. P. E., Proc. Nat. Acad. Sci. U.S.A. 70, 485, 1973. Schlegel, W., FEBS Lett. 69, 67, 1976. Michaelis, E. K., and Michaelis, M. L., Life Sci. 18, 1021, 1976. Deschodt-Lanckman, M., Robberecht, P., Camus, J. C., and Cristophe, J., J. Cycl. NW/. Res. 3, 177, 1977. Grunspan-Swirsky, A., and Pick, E., fsr. J. Med. Sci., in press. Gemsa, D., Steggemann, L., Till, G., and Resch, K., J. Immunol. 119, 524, 1977. Gunther, G. R., Wang, J. L., Yahara, I., Cunningham, B. A., and Edelman, G. M., Proc. Nut. Acad. Sci. U.S.A. 70, 1012, 1973. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J., J. Biol. Chem. 193,265, 1951. Brown, B. L., Albano, J. D. M., Ekins, R. P., and Sgherzi, A. M., Biochem. J. 121, 561, 1971. Tovey, K. E., Oldham, K. G., and Wheelan, J. A. M., Clin. Chim. Acta 56, 221, 1974. Grunspan-Swirsky, A., and Pick, E., Immunopharmacology, 1, 71, 1978. 2, 233, 1972. Anderson, J., Edelman, G. M., Moller, G., and Sjoberg, 0.. Eur. J. Immunol. Avrameas, S., Ternynck, T., Macieira-Coelho, A., and Garcia-Giralt, E., Biochem. Biophys. Rrs. Commun. 72, 790, 1976. Reaven, E. P., and Axline, S. G., J. Cell. Biol. 59, 12, 1973. Pick, E., Honig, S., and Griffel, B., Int. Arch. Allergy Appl. Immunol., 58, 149, 1979. Nicolson, G. L., Biochim. Biophys. Acta 457, 57, 1976. Remold, H. G., J. Exp. Med. 138, 1065, 1973. Coffey, R. G., Hadden, E. M., and Hadden, J. W., J. Immunol. 119, 1387, 1977. Ronquist, G., and Hedstrom, M., Biochim. Biophys. Acta 483, 483, 1977. Slavik, M., Kartner, N., and Riordan, J. R., Biochrm. Biophys. Res. Commun. 75, 342, 1977. Schechter, B., Lis, H., Lotan, R., Nowogrodsky, A., and Sharon, N., Eur. J. Immunol. 6, 145, 1976. Boxer, L. A., Watanabe, A. W., Rister, M., Besch, H. R., Allen, J., and Baehner, R. L.. New EngI. J. Med. 295, 1041, 1976. Weissmann, G., Goldstein, I., Hoffstein, S., and Tsung, P. K.,Ann. N. Y. Acad. Sri. 253,750, 1975. Sheppard, H., and W&an, G., Molec. Pharmacol. 7, 111, 1971. Limbird, L. E., De Meyts, P., and Lefkowitz, R. J., Biochem. Biophys. Res. Commun. 64, I 160. 1975. De Meyts, P., J. Supramolec. Struct. 4, 241, 1976. Toyoshima, S., and Osawa, T., J. Biol. Chem. 250, 1655, 1975. Parker, C. W., Biochem. Biophys. Res. Commun. 61, 1180, 1974. Fuller, G. M., and Brinkley, B. R., J. Supramolec. Struct. 5, 497, 1976. Gemsa, D., Seitz, M., Kramer, W., Till, G., and Resch, K., J. fmmunol. 120, 1187, 1978. Pick, E., and Grunspan-Swirsky, A., Cell. Immunol. 32, 340, 1977.