Marked enhancement of macrophage activation induced by synthetic muramyl dipeptide (MDP) conjugate using monoclonal anti-MDP antibodies

CELLULAR

IMMUNOLOGY

86, 269-277 (1984)

Marked Enhancement of Macrophage Activation induced by Synthetic Muramyl Dipeptide (MDP) Conjugate Using Monoclonal Anti-MDP Antibodies C. LECLERC, G. M. BAHR, AND L. CHEDID Immunoth&apie

Exptkmentale,

Institut Pasteur, 28, rue du Dr. Roux, 75724 Paris C&dex 15. France

Received December 2, 1983; accepted February 6. 1984 Activation of peritoneal exudate macrophages of mice to inhibit the in vitro proliferation of tumor target cells was achieved with low concentrations of N-acetyl-L-alanyl-rkoghnamine (MDP for muramyl dipeptide) conjugated to a synthetic carrier. Addition to the cultures of monoclonal anti-MDP or anti-carrier antibodies renders a thousandfold-smaller concentration of the conjugate highly effective in activating macrophages. This synergistic effect was observed neither with a control monoclonal antibody of different specificity nor with an F(ab)r fragment of the monoclonal anti-MDP antibody. Other controls, such as addition to the cultures of the carrier alone with its specific monoclonal antibodies, also demonstrated that there exists a re quirement for the presence of MDP in the conjugate. The possible uses of such a system as well as the underlying mechanisms are discussed.

INTRODUCTION N-Acetylmuramyl-L-alanyl-Disoglutamine (MDP for muramyl dipeptide), besides its adjuvant effect, has been shown to be endowed with numerous immunological or pharmacological properties ( l-4). Most of these studies have demonstrated that MDP activities were mediated through the macrophage; for instance, MDP-activated macrophages can inhibit the proliferation of tumor target cells (5,6) and produce interleukin 1 (IL-l) (7, 8). However, if these effects have been well described, little is known of the mechanisms by which MDP activates these cells. We have recently obtained monoclonal antibodies to MDP which recognize the N-acetylmuramic acid (NAc-Mur) linked to the dipeptide but not NAc-Mur or the dipeptide alone (9). The present study was performed to evaluate whether activation of macrophages by MDP could be modulated by these antibodies. In the following experiments, free or conjugated glycopeptide was used since enhancement of certain biological activities of MDP have been demonstrated after conjugation to a multipoly(DL-alanine)-poly(L-lysine) (A--L) carrier (10, 11). None of the three anti-MDP monoclonal antibodies have modified the activity of free MDP. However, when MDP was conjugated to A--L carrier, the same antibodies strongly enhanced the stimulatory activity of this conjugate (MDP-A--L). Our data also showed that this effect was mediated by Fc receptors and required the presence of an adjuvant-active molecule.

GQO8-8749184 $3.00 Copyright 8 1984 by Academic R-as, Inc. All rights of reproduction in any form ~~~rvcd.

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Mice. Male DBA/2 and (DBA/2 X C57B1/6)F1, 6- to lo-week-old mice were purchased from Iffa Credo (L’Arbresle, France). Immunostimulants and antigens. MDP was MDP-Pasteur (Institut Pasteur Production, Paris). A--L [poly(DL-alanyl)-poly(L-lysine)] and MDP-A--L [multipoly(MDP)-poly(DL-alanyl)-poly(L-lysine)] were prepared by P. Lefrancier (Choay Laboratories) as previously described (10). The conjugate, MDP-A--L, contained 20% MDP. Lipopolysaccharide (LPS) prepared from Salmonella enteritidis by the phenol-water extraction procedure was purchased from Difco Laboratories (Detroit, Mich.). Preparation of dinitrophenylated human albumin (DNP-HA). Five milligrams of human albumin (Serva) and 20 mg of t-DNP-L-lysine (Serva) were dissolved in 2 ml of phosphate-buffered saline (PBS) to which was added 100 ~1 of 8% glutaraldehyde (Sigma). After the coupling was allowed to take place at 4°C for 1 hr under constant stirring, the conjugate was dialyzed extensively against PBS to remove uncoupled DNP. The extent of substitution of DNP groups on the human albumin was estimated spectrally assuming a molar absorbancy of eDNP-L-lysine of 17,530 at 360 nm (12). The molar ratio of HA to DNP-lysine was 1: 16. Murine monoclonal antibodies. The preparation and characterization of monoclonal anti-MDP antibodies have been described elsewhere (9). In this study, three monoclonal antibodies pertaining to different IgG subclasses were used. The clones were 2-4-5 (IgG& M-52-l 1 (IgG&, and M-5-5 (IgG&. All three monoclonals were found to bind to MDP but could not recognize either the N-acetylmuramic acid or the dipeptide separately as determined by an inhibition assay of the binding of peroxidase-labeled MDP-Lys to anti-MDP antibodies (9). Control monoclonal antibodies with anti-DNP specificity, namely U-7-27 (IgGr,) and U-12-5 (h&J, or with anti-A--L specificity, namely X-63-160 (IgG& were kindly provided by Dr. Z. Eshhar of the Weizmann Institute (Israel) and were prepared as described elsewhere (13, 14). Ammonium sulfate precipitates (50%) of the ascitic fluid of each clone were used throughout the study unless otherwise mentioned. The IgG content in the different ascitic immunoglobulin preparations was calculated by passing the ascitic Ig over an S-200 column (Pharmacia) and measuring the 160,000Da peak, recognizing the corresponding hapten or antigen, relative to the total protein collected from the column. The percentage concentrations of IgG in the ascitic Ig of 2-4-5, M-5-5, M-52-l 1, U-7-27, U-12-5, and X-63-160 were 29, 30, 60, 50, 77, and 72, respectively. Preparation of F(ab)z. Ascitic immunoglobulins of clone M-52- 11 were passed over a Sephacryl S-200 column (Pharmacia) and the fractions of the peak corresponding to 160,000 Da and capable of binding to MDP were pooled, concentrated, and dialyzed against 0.1 M sodium acetate buffer, pH 4.5. To 25 mg of IgG in 3 ml, 1 mg of solid pepsin (Sigma) was added and incubated at 37°C for 18 hr. The pH was then adjusted to 8.0 with 1 M Tris and the digested IgG was dialyzed against PBS, pH 7.4. Fc fragments and undigested IgG were removed by passing the preparation twice over a lo-ml Sepharose-protein A column (Pharmacia) and the unbound F(ab)* fragment was collected and tested for its purity by an ELISA technique employing rabbit antimouse subclass-specific antisera (Bionetics). The pure F(ab)? preparation of M-521 1 had an MDP-Lys peroxidase-binding activity identical to that of undigested IgG.

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Cells. Macrophages harvested from peritoneal exudates 4 days after an intraperitoneal injection of 2 ml of thioglycollate medium (Institut Pasteur, Paris) were used as effector cells, The mastocytoma P815 used as target cells were peritoneal ascitic tumor cells recovered from DBA/2 mice. Preparation of macrophage monolayers. After harvesting, macrophages were washed and seeded at a concentration of 3.5 X lo5 cells in 0.2 ml of medium in the wells of microtiter plates (Nunclon, Roskilde, Denmark). The RPM1 1640 culture medium (Flow Laboratories, Rockville, Md.) contained 10% heat-inactivated fetal calf serum, glutamine, and antibiotics. The cultures were incubated at 37”C, with 7.5% CO,. After the cells were allowed to adhere 180 min at 37°C the nonadhering cells were removed by washing the monolayers with 199 medium (Institut Pasteur Production, Paris). Macrophage activation test. Various amounts of the different substances tested were incubated with macrophages during 24 hr, after which they were removed by washing before the target cells were added. P8 15 mastocytoma cells (2 X 104) were then added to the macrophage monolayers and incubated for 24 hr. Four hours before the end of the assay 0.1 &i of [3H]thymidine (1 Ci/nmol, Saclay, France) was added to each well. The labeled cells were collected on glass-fiber disks in a multiple-sample collector (Skatron, Flow Laboratories). Estimation of target cell cytostasis. Results are calculated as percentage growth inhibition = lOO(C - A)/C where C represents radioactivity incorporated by mastocytoma cells cultured on control macrophage monolayers and A represents radioactivity incorporated by mastocytoma cells cultured on activated macrophage monolayers. Each experimental group was tested in triplicate, and data shown are representative of at least two experiments performed under the same conditions. Inhibition of thymidine incorporation was found to be related to a reduction of tumor cell numbers as determinated by dye exclusion.

RESULTS In Vitro Activation of Macrophages by MDP or MDP-A- -L with or without Monoclonal Anti-MDP Antibodies Adherent peritoneal exudate cells (PEC) were incubated for 24 hr with different doses of MDP or MDP-A--L with or without 10 pg/ml of anti-MDP (clone M-52- 11). As can be seen in Fig. 1, a minimum concentration of 0.1 &ml of MDP or of 1 pg/ml MDP-A--L (which represents 0.2 &ml of MDP) was necessary to activate macrophages as measured by inhibition of proliferation of P8 15 tumor cells. M-521 1 antibody had no effect on the cytostatic activity of PEC and did not modify their activation by free MDP. In contrast, the same monoclonal antibody strongly potentiated the activation of these cells by MDP-A- -L: a concentration of 0.00 1 &ml of MDP-A--L was sufficient to activate macrophages. This represents 0.2 ng of MDP/ ml (4 X lo-” miW). A similar synergistic effect was observed between MDP-A--L and anti-MDP with 10 times lower or higher concentrations of monoclonal antibody (data not shown).

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t

0

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FIG. 1. In vitro cytostatic effect of macrophagesafter 24 hr incubation with different doses of MDP (W), or of MDP-A--L alone (0) or in combination with monoclonal anti-MDP, M-52-11 (10 &ml), MDP, and anti-MDP (*), or MDP-A--L and anti-MDP (A).

Activation of Macrophages Anti-A--L, or Anti-DNP

by MDP-AAntibodies

-L in the Presence of Monoclonal

Anti-MDP,

The activation of macrophages by an inactive dose of MDP-A--L (0.1 &ml) has been tested in the presence of different monoclonal antibodies. The enhancing effect of three monoclonal anti-MDP antibodies pertaining to different IgG subclasses, 24-5 (IgG3), M-52-l 1 (IgGh), and M-5-5 (IgG&, have been compared. Their activities have also been compared to a monoclonal antibody recognizing the A--L part of the MDP-A--L conjugate: X-63-160 (IgG3). The specificity of the anti-MDP-enhancing effect has been tested by using two monoclonals having anti-DNP specificity, namely, U-7-27 (IgG& and U-12-5 (IgG&. As can be seen in Fig. 2, in the absence of antibody, again a minimum concentration of 1 &ml MDP-A--L was necessary to activate macrophages. However, as previously observed, the incubation of macrophages with 0.1 &ml MDP-A--L and 10 &ml of M-52- 11 induced a 72% growth inhibition. Significant growth inhibition, although less pronounced, was observed using 2-4-5 and M-5-5 anti-MDP monoclonal antibodies. In the presence of 0.1 &ml of MDP-A--L, the anti-A--L antibody (X-63-160) induced a 70% inhibition of tumor growth. As with anti-MDP, such a synergistic effect could be observed using lower doses of MDP-A--L (from 100 to 0.001 pg of MDP-A--L) (data not shown). The two anti-DNP monoclonal antibodies did not significantly increase the activation of macrophage by MDP-A--L. It was noted that when added alone, none of the tested antibodies had any effect on macrophage activity. It was also noted that

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FIG. 2. In vitro activation of macrophages by MDP-A--L in the presence of monoclonal anti-MDP, antiA--L, or anti-DNP antibodies. Macrophages have been incubated for 24 hr either with different doses of MDP-A--L alone (100, 10, 1, or 0.1 pg) or with 0.1 pg of MDP-A--L in the presence of various monoclonal antibodies. The open area represents the growth inhibition obtained with 0.1 &ml of MDP-A--L in the presence of medium only.

none of these antibodies, with or without MDP-A--L, had any direct effect on P8 15 mastocytoma cells in the absence of macrophages (data not shown). MDP Requirement for Potentiation of Macrophage Activation by Immune Complexes To determine if the observed synergistic effect between MDP-A- -L and antibodies directed against MDP itself or against the carrier was merely due to macrophage activation by antigen-antibody complexes, two types of experiments have been performed: (1) cytostatic activity of macrophages incubated either with MDP-A--L or with A--L in the presence of monoclonal anti-A--L; (2) cytostatic activity of macrophages incubated with DNP-HA in the presence of monoclonal anti-DNP. As can be seen in Fig. 3, incubation of macrophages with A--L (0.1 to 100 &ml) did not induce cytostatic activity. As usual, a high level of cytostatic activity was observed using 0.01 pg/ml of MDP-A--L in the presence of anti-MDP or anti-A--L antibodies. In contrast, the addition of anti-A--L or anti-MDP to A--L (0.1 to 100 &ml) did not modify the PEC activity. Furthermore, stimulation of macrophages with MDP (0.0001 to 100 &ml) in the presence of A--L-anti-A--L complexes did not modify the MDP-induced cytostatic activity (data not shown). Similar results were obtained in the experiment presented in Fig. 4. DNP-HA was ineffective when added alone. Addition of anti-DNP antibodies did not render PEC cytostatic. These experiments clearly demonstrate that the presence of MDP in the immune complexes is required for the potentiation of macrophage activation. Fc Requirement for Potentiation of Macrophage Activation by Immune Complexes F(abh antibodies were prepared from M-52-l 1 as indicated under Materials and Methods. Macrophages were incubated 24 hr with 0.0001 to 100 &ml MDP-A--L

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100

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O.Ol~g/ml

FIG. 3. Lack of activation of macrophages by A--L in the presence of anti-A--L. Macrophages were incubated for 24 hr in the presence of various doses of MDP-A--L or A--L alone (0) or with 10 &ml X-63- 160 (Ed) or M-52- I 1 (0).

either alone or in the presence of 10 @ml M-52-1 1 or of F(ab)2. It can be seen in Fig. 5 that no synergism between MDP-A--L and F(ab)* could be demonstrated, in contrast to what was observed with the intact antibodies. DISCUSSION Muramyl dipeptide (MDP) has been repeatedly shown to activate macrophages, using morphological, biochemical, or functional parameters (3). Therefore, MDP has

\

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10 DNP-

HA

1 pglml

FlG. 4. Lack of activation of macrophages by DNP-HA in the presence of anti-DNP. Macrophages were incubated for 24 hr in the presence of various doses of MDP-A--L or DNF-HA alone (0) or with 10 pg/ ml of U-7-27 (tZ@or M-52-l 1 (II).

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FIG. 5. Macrophages were incubated 24 hr with O.ooOl to 100 &ml the presence of 10 &ml of M-52-I 1 (A) or of M-52-1 1 F(abh (m).

MDP-A--L

either alone (0) or in

been considered as a potential agent for increasing macrophage-mediated immunity and especially antitumor activity ( 15- 17). Several approaches have been successfully used to potentiate the effect of MDP on natural resistance to infection and to tumor. For instance, enhancement of MDP activity has been observed after conjugation to synthetic carrier such as A--L chain (10, 11). Lipophilic derivatives of MDP are also able to strongly stimulate nonspecific immunity against bacterial challenges or tumor grafts (18-20). Another approach has consisted of improving the MDP delivery by using systems such as micropumps (21) or liposomes (22-24). The present study was performed in view of finding whether macrophage activation by MDP could be modified by the use of specific monoclonal antibodies. Our results show that macrophages could be very efficiently activated by using minute amounts of an adjuvant-active hapten conjugated to an inert carrier in the presence of specific monoclonal antibodies. Thus, used alone, at least 1 &ml MDP-A--L was necessary to induce a significant cytostatic activity of murine-elicited peritoneal macrophages. In contrast, in the presence of monoclonal anti-MDP antibodies, as little as 0.001 @ml MDP-A- -L was enough to activate macrophages. This concentration of MDPA--L represents 0.2 ng of MDP/ml (4 X IO-” mM). This synergistic effect with subthreshold amounts of MDP-A--L has been observed with monoclonal anti-MDP antibodies of different isotypes (IgGz,, IgGn,, and IgGJ) and also with a monoclonal antibody directed against the A--L chain. It was noted that this increased MDPA--L activity was observed using a wide range of antigen-to-antibody ratios. In order to demonstrate the potentiating effect of the monoclonal antibddies, three types of requirements have to be ful6lled: (1) MDP has to be conjugated to a carrier since monoclonal antibodies did not modify macrophage activation induced by free MDP. (2) MDP has to be present in the immune complexes. Adequate controls have

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clearly shown that when a hapten devoid of adjuvant activity or a carrier devoid of MDP was mixed with its specific antibodies, no macrophage activation was observed. This lack of activity is not due to a lesser affinity of the control antibodies since the same anti-A--L carrier antibody has produced activation of the macrophages in the presence of MDP-A--L but not in the presence of A--L. (3) The last requirement for the demonstration of synergistic activity between MDP-A- -L and specific antibodies is the Fc part of the immunoglobulin as demonstrated by the inability of the F(abh fragment of anti-MDP antibody to induce such activity. Taken together, these data suggest that the observed phenomenon is mediated through fixation of MDP-antiMDP immune complexes on Fc receptors at the macrophage membrane. Tenu et al. (25) have recently published data which argue in favor of a lack of specific MDP receptors on the macrophage membranes. They proposed that the internalization of muramyl peptides is mediated through a nonspecific fluid-phase pinocytic phenomenon (25). However, it is well established that immune complexes are very efficiently phagocytosed through Fc receptors on the cell surface (26). Thus, the observed synergistic effect could very well be due to an increased MDP internalization via Fc-receptor activation. It has been previously reported that large amounts of antigen-antibody complexes can activate macrophages as measured by production of interleukin 1 (27) or endogenous pyrogen (28, 29) and by increased synthesis of prostaglandins (30). The present study demonstrates a marked potentiation of macrophage activation by a conjugate containing MDP in the presence of specific monoclonal antibodies. Ap propriate controls clearly showed that such an amplification requires the presence of an adjuvant-active hapten and is mediated through Fc receptors. Utilization of such very welldefined agents (monoclonal antibodies, synthetic adjuvant, and carrier) may provide excellent tools for the study of cellular mechanisms or for therapeutic uses. Our preliminary data indicate that potentiation of MDP activity could also be observed in other systems, such as in viva antibody responses to conventional or even synthetic antigens (unpublished observations). ACKNOWLEDGMENTS We thank Dr. P. Lefrancier (Choay Laboratories) for preparing MDP-A--L. We are grateful to E. Deriaud and D. Tello for expert technical assistance and to Ms. de Champs for typing the manuscript.

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Silverstein, S. C., Steinman, R. M., and Cohn, Z. A., Annu. Rev. B&hem. 46, 69, 1911. Blyden, G., and Handschumacher, R. E., J. Immunol. 118, 1631, 1977. 28. Chao, P., Francis, L., and Atkins, A., J. Exp. Med. 145, 1288, 1977. 29. Dinarello, C. A., Fed. Proc. 38, 52, 1979. 30. Rouzer, C. A., Scott, W. A., Kempe, J., and Cohn, Z. A., Proc. Natl. Acad. Sci. USA 77, 4219, 1980. 26. 27.