The comparative effects of isoprinosine, levamisole, muramyl dipeptide and SM1213 on lymphocyte and macrophage proliferation and activation in vitro

Int. J. Immunopharmac., Vol. 1. pp. 17-27 Pergamon Press Ltd. 1979. Printed in Greal Britain.

THE COMPARATIVE EFFECTS OF ISOPRINOSINE, LEVAMISOLE, MURAMYL DIPEPTIDE AND SM1213 ON LYMPHOCYTE AND MACROPHAGE PROLIFERATION AND ACTIVATION I N VITRO* JOHN W. HADDEN, ARTHUR ENGLARD, JOHN R. SADLIK and ELBA M. HADDEN Laboratory of Immunopharmacology, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, U.S.A. (Received 22 May 1978) A b s t r a c t - - F o u r immunopotentiators, levamisole, isoprinosine, muramyl dipeptide (MDP) and SM 1213 were analysed in vitro on phytohemagglutinin-induced lymphocyte proliferation and lymphokine-induced macrophage proliferation and activation to kill Lister& rnonocytogenes. Levamisole and isoprinosine augmented all three functions. MDP and SM1213 induced macrophage activation directly. The effects of these agents on these ceils at concentrations relevant to in vivo therapy supports their immunopharmacologic capacity to modify cellular immunity.

The recent emphasis on the development of immunopotentiators as adjunctive agents in cancer therapy has led to the discovery of a number of different chemically-defined compounds having immunomodulatory properties. These compounds have been applied in turn to a variety of disease states in addition to cancer; these include acute and chronic viral and bacterial infections, immunodeficiencies and collagen disorders. We (Hadden, Lopez, O'Reilly & Hadden, 1976a) have characterised this as a " p r o h o s t " form of therapy in which the first objective is to restore a deficient immune response or to enhance a specific type of immune response and the second objective is to treat a particular disease state. In general, the experimental and therapeutic applications of these prohost agents have been in disorders in which the augmentation of the cellular immune response is considered the primary therapeutic objective. The mediators of this form of immunity, the thymus-derived lymphocyte and the monocyte-derived macrophage, are thought to be the principal cellular targets for this type of immunopharmacologic therapy. In the present report we have attempted to clarify and compare the actions of four chemically-defined compounds by analysing their effect in vitro on mitogen-induced T cell-dependent lymphocyte proliferation, lymphokine-induced macrophage proliferation and activation of macrophages to kill Listeria m o n o c y t o g e n e s . The background for analysing the action of the immunopotentiators on

macrophage proliferation and activation in vitro lies in the understanding of the r61e played by activated macrophages in vivo during states of intense expression of delayed hypersensitivity (see Hadden & Englard, 1977, for review). Of importance to immunotherapy is that the macrophage, once activated, has the capacity to recognise transformed cells and to kill them while sparing normal cells. It is presumed that the mechanisms and the cell populations involved in the generation of bactericidal and tumoricidal macrophages are the same. Many of the current efforts at immunotherapy employ bacterial preparations such as Mycobacteria (BCG), Corynebacterium parvurn, mixed bacterial vaccine, P s e u d o m o n a s vaccine, etc., which presumably act in part through these mechanisms. It is important to understand the actions of such agents on more precise molecular terms in order to analyse further how immunopharmacologic therapies might be improved. The following compounds were selected for study: Levamisole (LMS) is a phenylimidothiazole anthelmintic which modifies a variety of T-lymphocyte and macrophage responses both in vitro and in vivo and has found clinical application in cancer, chronic bacterial and viral infections and rheumatoid arthritis (Renoux, 1978). Isoprinosine (Inosiplex) is a complex of p-acetamidobenzoic acid, N,N-dimethylamino-2-propanol and inosine (3:1 molar ratio) (Simon & Glasky, 1978), which has been employed experimentally as an antiviral agent. In vitro studies (Hadden, Hadden &

* This work was supported by grants-in-aid from the National Institutes of Health, Newport Pharmaceuticals International, Strategic Medical Research Corporation, The American Heart Association, and the Alton Jones Foundation. 17

J. W. HADDENet al.

18

Coffey 1976b) indicate that this agent is active on lymphocytes and have led to its evaluation as an immunopotentiator (Simon & Glasky, 1978). Muramyl dipeptide (MDP) is a substituted monosaccharide (N-acetylmuramyl-L-alanyl-D-isoglu-tamine) synthesized by Lefrancier, Choay, Derrien & Lederman (1977) to mimic the immunologic activity of the cell walls of BCG. The immunomodulatory activities of BCG and its use in cancer have been recently reviewed (Hadden, Delmonte & Oettgen, 1977). Muramyl dipeptide and related water soluble analogs have shown potent immunoadjuvant activity both in vitro and in vivo (Chedid & Audibert, 1977). SM 1213 is a synthetic, substituted monosaccharide developed as an antiviral agent and found to have immunomodulatory activity in vivo (Majde & Gordon, 1976). This agent is in the early phases of preclinical development and is of particular interest because of its structural relationship to MDP. These four agents were analysed in vitro for their biologic effects on relevant response parameters of lymphocytes and macrophages. EXPERIMENTAl, PROCEDURES

Materials

Indomethacin, a prostaglandin synthesis inhibitor, was purchased from Sigma (St. Louis): levamisole was provided by Janssen Pharmaceutica (Beerse, Belgium); isoprinosine by Newport Pharmaceuticals International (Newport Beach, CA); muramyl dipeptide and an inactive analog (N-acetyl muramylD-ALA-D-iso-GLM) by E. Lederer (Pasteur Institute, Paris, France); and SM 1213 1,2-O-iso-propylidene-3-O-3' (N', N ' - d i m e t h y l - a m i n o - n - p r o p y l ) D-glucofuranose by Paul Gordon (Strategic Medical Research Corp., Greenwich, CT). Phytohemagglutinin (PHA) (HA-17) was purchased from Burroughs Wellcome (Research Triangle Park, NC). A preparation containing Macrophage Mitogenic Factor (MMF) and Macrophage Activating Factor (MAF) was prepared from antigen-stimulated immune lymph node lymphocytes (guinea pig) as previously described (Hadden, Sadlik & Hadden, 1975a, 1978). Partial purification of this preparation by vacuum dialysis and Sephadex G-100 column chromatography yielded an active fraction in the range of 35-70,000 daltons exhibiting both mitogenic and activating properties. This active fraction was employed in both the proliferation and activation assays. Sephadex fractions were generally optimally active in both assays at a concentration equivalent to a 1:4 dilution of the original lymphokine-rich supernatant. Purified endotoxin (LPS) was provided by Dr. G. Tarnowski (Sloan-Kettering Institute, New York, NY).

Isolation and functional assays o f cells

Ficoll-hypaque purified human peripheral blood lymphocytes were prepared and PHA-induced proliferation was assayed by the incorporation of tritiated thymidine as described (Hadden, Hadden, Coffey, Corrales-Lopez & Sunshine, 1975). Each compound was analysed in the presence of suboptimal, optimal and supraoptimal concentrations of P H A (0.001, 0.01, 0.1 units/ml respectively). Paraffin oil-induced guinea pig peritoneal macrophages were prepared and incubated as a monolayer culture ( > 9 8 % pure macrophages). Lymphokine (MMF)induced proliferation was assayed by the incorporation of tritiated thymidine at 3 and 5 days of culture as described (Hadden et al., 1975a, 1978). Lymphokine (MAF)-induced macrophage activation to kill Listeria m o n o c y t o g e n e s following 5 days of culture in the presence or absence of M A F was performed during a 6-h period as described (Hadden & Englard, 1977). Phagocytosis was quantitated during a 20-min exposure to Listeria rnonocytogenes by counting the number of macrophages containing bacteria and the number of bacteria per phagocytic cell on Gram stained monolayers in Lab tek chambers, lntracellular killing of bacteria was evaluated by counting the number of cells containing bacteria and the number of bacteria/cell 6 h after the initial 20-rain exposure. Parallel experiments in which macrophages were lysed and intracellular bacteria were cultured confirm the validity of bactericidal activity determined by this manner in this system. The four drugs were employed in each of the three systems over serial log concentration range in triplicate in the presence and absence of mitogen or lymphokine. Each type of experiment was performed at least three times.

RESU LTS Phytohemagglutinin-induced

lymphocyte proliferation

(Fig. I). Both levamisole and isoprinosine significantly (p < 0.01 for both drugs; Student's t-test) augmented PHA-induced lymphocyte proliferation; neither had a significant effect in the absence of PHA. The maximal effects of isoprinosine were greater than those of levamisole. MDP marginally depressed proliferation in the presence of P H A and had no effect in the absence of PHA. SM!213 had no significant effect on lymphocyte proliferation in the presence or absence of PHA. It appears then that, in terms of thymidine incorporation, the PHA-stimulated lymphocyte is a particular target for the actions of LMS and isoprinosine.

Effects of Isoprinosine, Levamisole, Muramyl Dipeptide and SMI23

19

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Drug Concentration (ug/ml) Fig. I. Effects of levamisole, isoprinosine, MDP, and SMI213 on PHA-induced h u m a n lymphocyte proliferation in vitro. In the experiments presented ( n > 30) Ficoll-hypaque-purified lymphocytes were incubated for 72 h in the presence of an optimal concentration of P H A and tritiated thymidine incorporation assayed. The effects of the agents are compared to the P H A control as a ratio. Vertical bars indicate standard errors of the mean.

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Drug Concentration (;Jg/ml) Fig. 2. Effects o f levamisole, isoprinosine, M D P , and SM 1213 on MMF-induced guinea pig peritoneal macrophage proliferation. In the experiments presented ( n > 20), n o n - i m m u n e macrophages were incubated for 3 or 5 days in the presence of Sephadex-fractionated M M F (at a concentration equivalent to a 1:4 dilution of the crude antigen-stimulated lymph node cell supernatant) in the presence or absence of indomethacin (10 6M). The effects o f the agents on tritiated thymidine incorporation are assayed and compared to the M M F control and expressed as a ratio•

20

J. W.

HADDEN

MMF-induced macrophage proliferation (Fig. 2) None of the agents had a significant effect on macrophage proliferation in the absence of MMF. In the presence of MMF, isoprinosine either stimulated or inhibited proliferation depending on the MMF preparations used. The isoprinosine A experiment was performed with an MMF-rich Sephadex fraction containing minimal residual macrophage migration inhibitory factor (MIF) and molecules of molecular weight <35,000, while the isoprinosine B experiments (n =3) were performed with a preparation which included a greater contamination with MIF and also contained molecules in the 20-35,000 daltons range, such as interferon. Since endogenous prostaglandin production, particularly in the first day of culture, somewhat limits proliferation in this system (Hadden et al., 1978) and interferon, a possible contaminant in the MMF fraction, has been reported to induce prostaglandin metabolism (Yaron and Yaron, 1977), we performed the isoprinosine B experiments in the presence and absence of indomethacin (10 e'M), a prostaglandin synthesis inhibitor. In the presence of indomethacin, the proliferative effect of MMF is increased approximately 30% and the inhibitory effect of isoprinosine (B) is rev,:rsed so that mild stimulation is observed. SM1213 had no significant effect on MMF-in-

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Activation o f macrophages to kill Listeria monocytogenes MAF derived from antigen-stimulated guinea pig lymph node lymphocytes and fractionated on Sephadex was incubated with macrophages for 5 days of culture. These macrophages were slightly less phagocytic than those of the control following a 20-min exposure to Listeria (Fig. 3, 0 time). In this and the following figures phagocytosis is evaluated by both the percentage of cells on the monolayer containing bacteria (height of column) and the number (figures within column segments) of bacteria per cell. The breakdown of the column for the controls (0 time) indicates that 9°7o of the cells contained 1 2 bacteria; 4%, 3 4 1%, 5 6; and 2%, 7-15. MAF slightly reduced phagocytosis as a result of a lower percent of cells with ingested bacteria. A suboptimal amount of MAF is also depicted here since later experiments with immunopotentiators

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Fig. 3. Effects of optimal and suboptimal preparations of MAF and LPS (0.5 and 0.05 /~g/ml) on macrophage activation to kill Listeria monocytogenes. Non-immune peritoneal macrophages were cultured as a monolayer for 5 days in the presence or absence of Sephadex fraction containing MAF exactly as described for MMF (Fig. 2). The monolayers were then exposed for 20 min to Listeria monocytogenes (ratio 20 bacteria/cell), washed, and the percent of cells bearing bacteria (ordinate) and the number of bacteria/cell (indicated in each column) are determined by counting Gram stained slides. (See text for details.)

Effects of lsoprinosine, Levamisole, Muramyl Dipeptide and SMI23

21

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0 Time 6 Hour Fig. 4. Effects of levamisole on macrophage activation. Macrophage monolayers were incubated for 5 days. The drug was added either during the phagocytosis and killing period of 6 h (A) or at the onset of the 5-day culture in the presence of suboptimal MAF (B). In the experiments depicted, the drugs were added at day 0 with the MAF.

22

J. W . HADDEN et al.

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Fig. 5. Effects of ~soprinosine on macrophage activation. Macrophage monolayers were incubated for 5 days in the presence of suboptimal MAF with and without isoprinosine. employed it. Macrophages, incubated for 5 days with endotoxin (LPS) demonstrated a decrease in phagocytosis at 0.5 but not at 0.05/~g/ml. Following the 6-h incubation the control macrophages showed no significant change in the number of cells with bacteria; however, the number of bacteria per cell increased with 1070 having 3 4 or 5- 6 bacteria per cell; 3°70, 7-15; and 10070, 16 31. Like the control, macrophages stimulated with suboptimal M AF were not able to clear the bacteria or to prevent the increase in bacteria. Macrophages stimulated with optimal MAF or endotoxin (0.5 and 0.05 /~g/m.) cleared themselves of most of their intracellular bacteria, as seen by a reduction in column height, and digested them as adjudged by visualization of fragmented bacteria within them. The residual macrophages containing bacteria (7-16 on average) are calculated to be those which had more than 5 bacteria at time 0. Thus, the macrophage can be activated to kill Listeria by a lymphokine MAF or by LPS, yet the activation process is only expressed if the bacterial load is low ( < 6 bacteria/cell). When levamisole was added during the 20-min pulse exposure to the bacteria over a concentration range of 0.03-300 /ag/ml, it augmented phagocytosis of bacteria by increasing both (for each, p <0.01; Fisher exact test) the percent of cells containing bacteria and the average number of bacteria per cell (Fig. 4A, 0 time). Levamisole, added during the

20-min pulse and the 6-h incubation, induced a moderate degree of killing of Listeria by the macrophages (Fig. 4A, 6-h). If levamisole was added at day 0, little or no effect on phagocytosis and killing was observed on day 5; however, if levamisole was added at day 0 in the presence of a suboptimal concentration of MAF, it potentiated the effect of MAF to induce macrophages to kill intracellular Listeria (Fig. 4B). If MAF was added at day 0 and levamisole at day 5 during pulse exposure with the bacteria, data equivalent to those in Fig. 4A and B, 6 h, resulted. Thus, levamisole added directly or with suboptimal MAF augmented phagocytosis (p <0.01; Fisher exact test) and induced killing ( p < 0.01 ; Fisher exact test) of Listeria. The results observed with levamisole (three experiments) and imidazole (four experiments, date not shown) were nearly identical, indicating that the levamisole effects are principally those of imidazole, and thus probably mediated by an increase in macrophage cyclic GMP and/or a lowering of cyclic AMP (Hadden et al., 1975b). lsoprinosine (four experiments, Fig. 5) had very little effect in the absence of MAF, except that at 1 /~g/ml it slightly enhanced killing when added with the bacteria at day 5 (data not shown), lsoprinosine at concentrations of 1, 25 and 100/zg/ml augmented the effect of suboptimal MAF to induce macrophages to kill Listeria whether it was added at day 0 with the MAF or at day 5 with the bacteria, lsoprinosine mildly increased phagocytosis when added

23

Effects of lsoprinosine, Levamisole, Muramyl Dipeptide and SM123 at day 5 (Fig. 5, time 0). It had a significant (p <0.01; Fisher exact test) effect in augmenting the effect of MAF (suboptimal) to induce macrophage killing (Fig. 5, 6 h). Muramyl dipeptide significantly enhanced both phagocytosis and killing (p~<0.01; Fisher exact test) when added at day 0 of culture or at the onset of the exposure to the bacteria. As seen in Fig. 6A, at time 0, MDP increased the number of macrophages which ingested bacteria and the number of bacteria ingested

per cell. During the 6-h incubation following the pulse exposure to the bacteria, nearly all of the MDP treated macrophages completely killed their intracellular bacteria. The optimal effects of MDP in four similar experiments on both phagocytosis and killing of intracellular bacteria were observed between 1.2 and 12 Fg/ml. In each experiment, 0.12 # g / m l was active. SM1213 at concentrations of 12 /~g/ml and below slightly enchanced phagocytosis and significantly

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Fig. 6. Effects of MDP (A) and SM1213 (B) on macrophage activation in the absence of MAF. Macrophage monolayers were incubated for 5 days in the absence of MAF. The drugs were added either at the onset of the 5-day incubation or during the phagocytosis and killing period.

24

J. W. HADDENet al.

(17 <0.01; Fisher exact test) increased killing of intracellular bacteria (Fig. 6B). SM1213 was active whether added at day 0 or at the onset of the exposure to the bacteria. In these nearly identical experiments, the optimal effect of SM1213 on phagocytosis was observed at 1.2 12,F.g/ml and the optimal effect on killing at 0.12/zg/ml. Since their effects were quite significant alone, neither M D P nor SM1213 was analysed in the presence of M A F . None of the four c o m p o u n d s at the concentrations tested had any direct bactericidal or bacteriostatic activity, nor affected the viability of the macrophage monolayer. I)ISCUSSION

1975, 1976b), suggesting that either two cell populations with differing sensitivities to these compounds are involved in the response or that they have two different types of actions depending on their concentrations. Further experiments are needed to resolve the question. It is clear that PHA-induced lymphocyte proliferation involves both T and B cell proliferation, the latter being dependent on recruitment by T cells (Lohrman, Novikovs & Graw, 1974). It may also involve accessory cells for the initiation event; however, their role is controversial (Hadden & Talmage, 1977), so that multiple cellular targets for these drugs remain a distinct possibility. The positive effects of levamisole and isoprinosine under these circumstances do offer some relevant confirmation The following table summarizes the effects of the compounds studied on in vitro immunologic paraof the cellular targets of their actions for interpreting meters. their effects on immune responses in vivo. Table 1. Eftects on in vitro immune parameters

Drug

PHA-induced lymphocyte proliferation

MMF-induced macrophage proliferation

Macrophage activation

MAF-induced macrophage activation

Levamisole

t

±t

Slight increase

t

Isoprinosine

tt

t~*

Slight increase

t

t

not tested

t

not tested

Muramyl dipeptide

0

SMl213

0

0

* Effects of isoprinosine were variable depending on the MMF preparation. Inhibition was apparently mediated by prostaglandin(s). It is clear from these and other data (Hadden et al., 1976a, 1976b, 1977; Renoux, 1978) that both levamisole and isoprinosine can modulate lymphocyte function. The action of levamisole on lymphocyte proliferation in vitro is mimicked by its imidazole component (Hadden et al., 1975b) but not by sulfur-containing compounds like dithiosemicarbamate or thiabendazole (Renoux, 1978). The effects of both imidazole and levamisole on proliferation in vitro are closely paralleled by their capacity to increase lymphocyte levels of cyclic G M P (Hadden et al., 1975b). The action of levamisole in vivo, however, appears to involve the effects of both imidazole and the sulfur moiety (Renoux, 1978). lsoprinosine's mechanism of action does not appear to involve early changes in lymphocyte cyclic nucleotide levels, and actions linked to R N A metabolism seem likely (Gordon, Ronsen & Brown, 1974). Both levamisole and isoprinosine show double-peaked dose response curves on P H A induced lymphocyte proliferation (Hadden et al.,

The lack of significant effects of M D P and SM1213 do not rule out the lymphocyte as a target for their actions; the data merely indicate that their action is not apparent in a proliferation assay, it will be necessary to perform further examination of mitogen-induced proliferation in mouse spleen cells since M D P has been reported to stimulate lymphocyte proliferation in this type of assay (Specter, Friedman & Chedid, 1977). Whether the response of mouse cells indicates a differential effect of M D P based on species or the action of accessory cells such as macrophages needs to be determined. It also seems relevant to examine cyclic nucleotide and R N A metabolism of lymphocytes as well as other functions before concluding that M D P and SM 1213 do not act on lymphocytes. In the assays of macrophage function we examined the effects of the drugs on macrophage proliferation induced by a newly described lymphokine M M F (Hadden et al., 1975a, 1978). This lymphokine can be tentatively dissociated from MIF, colony stimulating

Effects of isoprinosine, Levamisole, Muramyl Dipeptide and SM 123 factor and interferon, based on Sephadex chromatography, isoelectric focusing, stability to vacuum dialysis and serologic criteria (Hadden et al., 1975a, 1978 & unpublished results). Since macrophage production of prostaglandins constitutes a negative regulatory influence to their own proliferation (Hadden et al., 1978; Kurland, Hadden & Moore, 1977; Kurland, Bockman, Broxmeyer & Moore, 1978) all of the agents used in this study were analysed in the presence and absence of indomethacin. A factor which is so far indistinguishable from MMF in our hands is MAF, which activates macrophages in vitro to kill both Listeria monocytogenes and tumor cells (Piessens, Churchill & David, 1975; Churchill, Piessens, Sulis & David, 1975). We employed MAF in the present studies with the presumption that MMF and MAF are the same molecule; however, final biochemical proof of this assumption has not yet been achieved. We can show that the activating factor is not MIF or interferon by exclusion chromatography and isoelectric focusing (unpublished observations). We do not mean to imply that either MIF or interferon is not also an activator of macrophages as has been reported (Piessens et al., 1975; Churchill et al., 1975; Schwartz, Papamatheakis & Chirigos, 1977), since there seems to be a great variety of agents in addition to those presented here which will activate macrophages to become either bactericidal or tumoricidal. The activation process can be achieved within 24 h with agents such as LPS, Listeria factor, ascorbate (Englard, Johnson & Hadden, unpublished) and by a soluble mediator (Ruco & Meltzer, 1978) while 3-5 days is required for activation with MAF (Hadden & Englard, 1977; Piessens et al., 1975, Churchill et al., 1975). Thus in the system employed in the present study, both immediate and delayed activation were analysed and the agents were tested alone, and, if ineffective alone, in combination with a suboptimal preparation of MAF. Levamisole had marginal effects on both basal and MMF-induced macrophage proliferation; however, the marginal effects on proliferation were consistent enough to convince us that levamisole was active on macrophage proliferation. J. P. Giroud (personal communication) has shown that pretreatmem of macrophages with levamisole will augment their subsequent proliferative response to a mitogenic factor. The effects of levamisole were mimicked by imidazole and were unaffected by indomethacin. Levamisole alone augmented phagocytosis, produced a modest increase in bactericidal capacity and potentiated the effect of suboptimal MAF to induce bactericidal capacity. Imidazole simulated each of these actions so that the mechanism presumably involves

25

cyclic GMP, as in the lymphocyte (Hadden et al., 1975b). Others have demonstrated the effects of levamisole to increase phagocytosis and chemotaxis and related these effects to increases in levels of cyclic GMP in phagocytic cells (see lgnarro, 1977, for review). These observations seem particularly relevant to those of in vivo studies in which increased chemotaxis, inflammatory infiltration, or bone marrow recovery in patients or animals treated with levamisole have been shown (Renoux, 1978). Isoprinosine's effect on macrophage proliferation was variable depending on the preparation of MMF. The positive effects of isoprinosine on MMF-induced macrophage proliferation paralleled those on PHAinduced lymphocyte proliferation and presumably result from a similar mechanism. The negative effects of isoprinosine on MMF-induced proliferation result apparently from an action of isoprinosine to promote a factor which induces prostaglandin production. The action of isoprinosine to promote an antiproliferative influence mediated by prostaglandin makes it seem likely that interferon is involved since interferon is both antiproliferative and implicated in prostaglandin metabolism (Yaron & Yaron, 1977). The observation that isoprinosine has an action in relation to prostaglandin metabolism, even if indirect, may be relevant to its application as an immunopotentiator in cancer where prostaglandins have been implicated in the suppression of the immune response, lsoprinOsine may have to be employed with PG inhibitors in order to show beneficial effects. On the other hand, when isoprinosine is to be used to potentiate an antitumor effect of interferon, PG inhibitors would not be indicated. lsoprinosine also appeared to augment phagocytosis and bactericidal capacity, particularly in the presence of suboptimal MAF. The latter effect may have involved either an interaction with MAF or trace interferon since this preparation of MAF was the same used in the proliferation experiments. These in vitro observations with isoprinosine may relate to the in vivo immunoadjuvant actions of isoprinosine seen in mice inoculated with L1210 leukemic cells (Spreafico, 1978), influenza virus and sheep erythrocytes (Simon & Glasky, 1978). It is notable that isoprinosine has been observed to potentiate interferon's effects in vivo (Chany, 1978). Further in vivo analyses with this agent seem warranted, particularly in combination with interferon and in those circumstances in which levamisole is active. Muramyl dipeptide potently inhibited MMFinduced macrophage proliferation with an IDs0 of 0.01/~g/ml. This inhibition was only slightly modified by indomethacin (10 6M). Since the drug only

26

J. W. HADDENet al.

slightly inhibited lymphocyte proliferation, a general antiproliferative action can be ruled out. The bases for a selective antiproliferative action on the macrophage remain to be determined. M D P was very active in increasing phagocytosis and bactericidal activity of the macrophage, both early and late. The differential action of M D P to inhibit macrophage proliferation yet p r o m o t e activation implies that M D P is a complex agent with different mechanisms of action in separate macrophage subpopulations. In preliminary experiments (Hadden, 1978, unpublished results), we have observed that M D P increases both cyclic A M P and cyclic G M P in macrophages; however, we have not as yet analysed subpopulations to determine whether these increases are in the same cells or different subpopulations. The in vivo relevance of these in vitro observations seem to lie in the potent i m m u n o a d j u v a n t activity of M D P and its active analogs in circumstances where macrophages are known to play a critical r61e (Chedid & Audibert, 1977; Juy & Chedid, 1975). SM1213, a c o m p o u n d very similar in structure to muramyl dipeptide, was selective in its action since it activated macrophages and increased their phagocytosis but showed neither pro- nor anti-proliferative activity. Preliminary work indicates that SM1213 increases macrophage levels of cyclic G M P but has no effect on cyclic A M P levels. This suggests that SM1213 may share M D P ' s positive cyclic G M P -

related action without possessing its negative, cyclic AMP-related effect on proliferation. Both M D P and SM1213 are substituted monosaccharides and their activity may derive from the effects of low concentrations of sugars to act as hormonal agonists to induce the so-called "glucose effect". In this regard, Goldberg (1975) has previously speculated that cyclic G M P is the intracellular mediator of this effect. Further experiments with this agent are required, both in vitro and in vivo, to determine its therapeutic potential. The in vitro analyses o f these four agents have provided a spectrum of observations which, in addition to indicating two cells which are targets of their action, suggest to us a basis for predicting their possible applications in vivo. The two agents principally active in facilitating mitogen and lymphokine action, levamisole and isoprinosine, have both demonstrated in vivo effects in facilitating on-going immune responses initiated by specific antigenic challenge. The two agents principally active on the macrophage, M D P and SM1213, have been particularly effective when challenged in vivo with infectious agents, the resistance to which are highly macrophage dependent. Both types of agents appear to be effective in adjuvant immunization protocols and therefore future work on their combined use will be regarded with interest.

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