Selective macrophage activation by muramyldipeptide bound to monoclonal antibodies specific for mouse tumor cells

Summary IgM monoclonal antibodies directed against tumor cells which do not mediate antibody-dependent macrophage cytotoxicity (ADMC) even when they are cytotoxic in the presence of complement, have been shown to render macrophages tumoricidal when they carry an immunomodulating agent, i.e., muramyldipeptide (MDP).This statement is based on experiments using two IgM monoclonal antibodies selected for their ability to bind L1210 leukemia cells (F₂-10-23-IgM) and 3LL Lewis lung carcinoma cells (6B6-IgM) specifically, as shown by flow cytofluorometry analysis.The MDP-IgM conjugates, containing 45 MDP molecules per IgM molecule, were prepared by allowing MDP-hydroxy-succinimide ester to react with IgM monoclonal antibodies.The MDP-IgM conjugates are shown to bind to relevant tumor cells and to induce the activation of thioglycolate-elicited peritoneal mouse macrophages leading to 80% growth inhibition of target cells at optimum concentrations of bound MDP. These concentrations of bound MDP were 10 times lower than the concentration of free MDP, giving a maximum activation that is limited to 20% growth inhibition.No macrophage activation was evidenced when tumor cells were incubated in the presence of irrelevant MDP-IgM conjugates and macrophages or when macrophages were preincubated in the presence of MDP-IgM conjugates and then incubated in the presence of relevant or irrelevant tumor cells but in the absence of the MDP-IgM conjugates.The reported results are discussed with reference to the mechanism of activation of macrophage by muramyldipeptide and to the usefulness of such MDP-IgM conjugates as potential antitumor agents in cancer therapy.Abbreviations ADMC antibody dependent macrophage mediated cytotoxicity - F-GAM fluoresceinylthiocarbamyl goat anti mouse antibody - -Man-BSA -mannopyranosyl-phenylthiocarbamyl bovine serum albumin containing some 25 mannose residues (neoglycoprotein) - MDP muramyldipeptide, 2-acetamido-3(2-0-d-lactyl-l-alanyl-d-glutamyl amine) glucopyranose - MDP-F₂-10-23-IgM Murine monoclonal antibody specific of L1210 leukemia cells and substituted with 45 MDP molecules - MDP—6B6-IgM Murine monoclonal antibody specific of 3LL Lewis lung carcinoma cells and substituted with 45 MDP molecules - MEM minimal essential medium - TDM Trehalose-dimycolate     

Abrogation of species specificity for activation of tumoricidal properties in macrophages by recombinant mouse or human interferon-gamma encapsulated in liposomes

Highly purified human blood monocytes, isolated by continuous Percoll density gradients under endotoxin-free conditions, and mouse peritoneal exudate macrophages (PEM) were activated in vitro by the combination of muramyl dipeptide (MDP) and recombinant interferon-gamma (r-IFN-gamma) to become tumoricidal against their respective tumorigenic target cells. The activation of human monocytes or mouse PEM by free unencapsulated r-IFN-gamma and MDP was species specific: human r-IFN-gamma activated human blood monocytes to lyse allogeneic melanoma cells, but did not activate mouse PEM. Mouse r-IFN-gamma activated mouse PEM to lyse syngeneic melanoma cells, but did not activate cytotoxic properties in human monocytes. The encapsulation of either mouse or human r-IFN-gamma with MDP within the same liposome preparation produced synergistic activation of cytotoxic properties in both PEM and monocytes without apparent species specificity. The activation of tumoricidal properties in macrophages by r-IFN-gamma and MDP occurred as a consequence of intracellular interaction. We base this conclusion on the data showing that whereas free r-IFN-gamma and MDP did not activate macrophages pretreated with pronase, liposome-encapsulated r-IFN-gamma and MDP did. Moreover, the i.v. injection of liposomes containing human or mouse r-IFN-gamma and MDP produced in vivo activation of mouse alveolar macrophages. These data suggest that in contrast to activation with free r-IFN-gamma, which requires binding to macrophage surface receptors, the intracellular interaction of r-IFN-gamma, which produces tumoricidal activity in macrophages, is not species specific.     

Production of macrophage-derived cytotoxic factor by N-[3-[(carbamoylmethyl)thio]propionylated] neoglycoproteins

6-Phosphomannosylated bovine serum albumin (Man6P-BSA), a neoglycoprotein endocytosed by macrophages, bearing either 3-(2-pyridyldithio)propionyl or 3-[(carbamoylmethyl)thio]propionyl residues coming from alkylation of thiol residues by iodoacetamide were prepared and tested for their immunomodulator properties. The supernatants of mouse peritoneal macrophages incubated with Man6P-BSA bearing 3-[(carbamoylmethyl)thio]propionyl groups, and by a lesser extent 3-(2-pyridyldithio)propionyl groups, were cytotoxic to L929 cells, suggesting the presence of a tumor necrosis factor like compound. This macrophage-activation process is linked to the capacity of Man6P-BSA to be endocytosed via membrane lectins of macrophages, because the supernatants of macrophages incubated with unglycosylated conjugates were not cytotoxic. The cytotoxic activity induced by 3-[(carbamoylmethyl)thio]propionyl groups bound onto Man6P-BSA was similar to that induced by Man6P-BSA bearing muramyl dipeptide, indicating that endocytosed neoglycoproteins bearing 3-[(carbamoylmethyl)thio]propionyl residues are potent macrophage activators.     

Sugar receptor mediated drug delivery to macrophages in the therapy of experimental visceral leishmaniasis

Methotrexate (MTX) conjugate of a neoglycoprotein, mannosyl bovine serum albumin, containing an average of 30 moles of MTX per mole of neoglycoprotein was taken up efficiently by murine peritoneal macrophages through cell surface mannosyl receptors. The conjugate strongly inhibits the growth of Leishmania donovani inside macrophages, with 50% inhibitory dose of 0.11 micrograms/ml MTX, which makes it 100 times more active than free MTX (50% inhibitory dose of 12.1 micrograms/ml). MTX conjugated to BSA or other non-specific neoglycoproteins like galactose-BSA and glucose-BSA have leishmanicidal effects comparable to free MTX. Moreover, in a murine model of experimental visceral leishmaniasis, the drug conjugate reduced the spleen parasite burden by more than 85% in a 30 day model whereas the same concentration of free drug caused little effect. The results demonstrate that neoglycoproteins may be useful as carriers for receptor mediated drug delivery to treat macrophage associated diseases.     

Neoglycoproteins as Carriers for Receptor-Mediated Drug Targeting in the Treatment of Experimental Visceral Leishmaniasis

ABSTRACT. Methotrexate (MTX) coupled to mannosyl bovine serum albumin (BSA) was taken up efficiently through the mannosyl receptors present on macrophages. Binding experiments indicate that conjugation does not decrease the affinity of the neoglycoprotein for its cell surface receptor. The drug conjugate eliminated intracellular amastigotes of Leishmania donovani in mouse peritoneal macrophages about 100 times more efficiently than free drug on the basis of 50% inhibitory dose. Inhibitory effect of the conjugate was directly proportional to the density of sugar on the neoglycoprotein carrier. Colchicine and monensin, inhibitors of receptor-mediated endocytosis, can prevent the leishmanicidal effect of the conjugate. Antileishmanial effect of the conjugate can be competitively inhibited by mannose-BSA and mannan. In a murine model of experimental visceral leishmaniasis the drug conjugate reduced the spleen parasite burden by more than 85% in a 30-day model whereas the same concentration of free drug caused little effect. These results indicate that MTX-neoglycoprotein conjugate binds specifically to macrophages, and is internalized and degraded in lysosomes releasing the active drug to act on Leishmania parasites. These results also represent the potential for a general approach to intracellular targeting of clinical agents for macrophage-associated disorders.     

Chemical conjugation of muramyl dipeptide and paclitaxel to explore the combination of immunotherapy and chemotherapy for cancer

Paclitaxel (Taxol) conjugated to muramyl dipeptide (MDP) is described. Biological testing showed that the conjugation of MDP at 2'-O-paclitaxel (2'- O -MTC-01) not only has antitumor activity, but also have immunoenhancement capacity. Compared with paclitaxel or MDP alone or with a mixture of paclitaxel + MDP, 2'- O -MTC-01 significantly increases the production and expression of TNF-alpha and IL-12 from mouse peritoneal macrophages, which demonstrates a synergism of MDP and paclitaxel in one conjugated molecule.     

Neoglycoproteins as carriers for receptor-mediated drug targeting in the treatment of experimental visceral leishmaniasis

Methotrexate (MTX) coupled to mannosyl bovine serum albumin (BSA) was taken up efficiently through the mannosyl receptors present on macrophages. Binding experiments indicate that conjugation does not decrease the affinity of the neoglycoprotein for its cell surface receptor. The drug conjugate eliminated intracellular amastigotes of Leishmania donovani in mouse peritoneal macrophages about 100 times more efficiently than free drug on the basis of 50% inhibitory dose. Inhibitory effect of the conjugate was directly proportional to the density of sugar on the neoglycoprotein carrier. Colchicine and monensin, inhibitors of receptor-mediated endocytosis, can prevent the leishmanicidal effect of the conjugate. Antileishmanial effect of the conjugate can be competitively inhibited by mannose-BSA and mannan. In a murine model of experimental visceral leishmaniasis the drug conjugate reduced the spleen parasite burden by more than 85% in a 30-day model whereas the same concentration of free drug caused little effect. These results indicate that MTX-neoglycoprotein conjugate binds specifically to macrophages, and is internalized and degraded in lysosomes releasing the active drug to act on Leishmania parasites. These results also represent the potential for a general approach to intracellular targeting of clinical agents for macrophage-associated disorders.     

Activation of the production of cytotoxic factors by mouse spleen cells exposed to the combined action of lipopolysaccharide and muramyl dipeptide in vitro

Lipopolysaccharide (LPS) and muramyl dipeptide (MDP) stimulated murine splenocytes in vitro to produce cytotoxic factors (CF) that killed target cells L-929. This effect was synergic at LPS dose of 10 ng/ml and MDP dose of 10 micrograms/ml. CF production started 2 hours after spleen cell activation and was maximum in 6 hours. CF were produced by macrophages as well as by lymphocytes stimulated by LPS, MDP or their combination. However, synergic effect of immunomodulators was registered only if nonfractionated spleen cells were stimulated during 24 hours. Lymphocytes depleted on T cells did not lack the ability to generate CF upon activation. In addition, LPS and MDP activated synergically the production of interleukin-I by spleen cells in vitro.     

Effect of albumin on the in vitro conjugation of bilirubin by rat liver microsomes

These studies were carried out to determine whether bovine serum albumin (BSA), which is usually included in the incubation mixture for the in vitro determination of bilirubin-UDP-glucuronyl transferase (GT) activity, affects GT activity. Using bilirubin as substrate, addition of BSA to the enzyme reaction mixture at concentrations varying from 2 to 30 mg/ml resulted in a dose-related inhibition of "native" GT activity of rat liver microsomes. When detergent-activated enzyme was employed, increasing concentrations of BSA also required higher concentrations of deoxycholate, digitonin, or Triton X-100 to produce maximal bilirubin conjugation. Low BSA concentrations (2 mg/ml) prevented enzyme activation by both detergents and UDP-N-acetyl glucosamine. When BSA was omitted and bilirubin dissolved in dimethyl sulfoxide, UDP-N-acetyl glucosamine failed to enhance GT activity, and activation by detergents was only 15-25% of that observed in the presence of optimal concentrations of BSA. When rat albumin was substituted for BSA, a similar dose-related inhibition of in vitro bilirubin conjugation by untreated microsomes was observed, although at any given albumin concentration, GT activity was lower with rat than with bovine albumin. Additionally, both detergents and UDP-N-acetyl glucosamine produced similar GT activation regardless of the rat albumin concentration. Finally, these effects of BSA and rat albumin could not be reproduced when beta-lactoglobulin was employed and/or when p-nitrophenol was the acceptor substrate of GT. These findings indicate that albumin, in particular BSA, profoundly and selectively influences the in vitro activity of microsomal GT toward bilirubin as the acceptor substrate.     

Dendritic cell maturation induced by muramyl dipeptide (MDP) derivatives: monoacylated MDP confers TLR2/TLR4 activation

6-O-acyl-muramyldipeptides (MDP) with various lengths of fatty acid chains were examined for their dendritic cell (DC) maturation activity expressed through TLRs. Judging from anti-TLR mAb/inhibitor-blocking analysis, MDP derivatives with a single octanoyl or stearoyl fatty acid chain were found to activate TLR2 and TLR4 on human DCs, although intact and diacylated MDP expressed no ability to activate TLRs. Human DC activation profiles by the monoacylated MDP were essentially similar to those by Calmette-Guerin (BCG)-cell wall skeleton (CWS) and BCG-peptidoglycan (PGN) based on their ability to up-regulate costimulators, HLA-DR, beta(2)-microglobulin, and allostimulatory MLR. Monoacylated MDP induced cytokines with similar profiles to BCG-CWS or -PGN, although their potency for induction of TNF-alpha, IL-12p40, and IL-6 was less than that of BCG-CWS or -PGN. The MDP derivatives initiated similar activation in normal mouse macrophages, but exhibited no effect on TLR2/4-deficient or MyD88-deficient mouse macrophages. Mutation of d-isoGln to l-isoGln in monoacylated MDP did not result in loss of the DC maturation activity, suggesting marginal participation of nucleotide-binding oligomerization domain 2, if any, in monoacyl MDP-dependent DC maturation. These results define the adjuvant activity of 6-O-acyl MDP compounds at the molecular level. They target TLR2/TLR4 and act through the MyD88-dependent pathway in DCs and macrophages. Hence, the unusual combined activation of TLR2 and TLR4 observed with Mycobacterium tuberculosis is in part reflected in the functional properties of monoacylated MDP compounds. These findings infer that the essential minimal requirement for TLR2/4-mediated adjuvancy of BCG lies within a modified MDP.     

Activation of apoalkaline phosphatase by serum albumin with Zn2+ in rat hepatoma cells.

Reuber rat hepatoma cells (R-Y121B) cultured at 0.5% serum accumulated apoalkaline phosphatase in intact cells. When R-Y121B cells were cultured in the presence of bovine serum albumin, alkaline phosphatase activity increased in the cells, and the associated increase in enzyme activity differed amongst bovine serum albumin preparations. The treatment of bovine serum albumin with activated charcoal not only enhanced the effect of serum albumin on alkaline phosphatase activity, but also cancelled the differences due to different preparations of serum albumin. In contrast, no effect from serum albumin was observed in the increase of alkaline phosphatase activity in R-Y121B cell homogenates incubated at 37 degrees C. The activated-charcoal treatment of bovine serum albumin increased the amount of Zn2+ bound to the protein. When R-Y121B cells were cultured with bovine serum albumin, the concentration of Zn2+ in the cytosol fraction slightly increased. However, the effect of serum albumin on Zn2+ concentration in the cytosol fractions was independent of charcoal treatment. It was concluded that serum albumin with Zn2+ induces the activation of apoalkaline phosphatase due to Zn2+ binding.     

Identification of the serum factor required for in vitro activation of macrophages. Role of vitamin D3-binding protein (group specific component, Gc) in lysophospholipid activation of mouse peritoneal macrophages

In vitro treatment of mouse peritoneal cells (mixture of adherent and nonadherent cells) with lysophosphatidylcholine (lyso-Pc) in 10% FCS supplemented medium RPMI 1640 results in a greatly enhanced FcR-mediated phagocytic activity of macrophages. This macrophage-activation process requires a serum factor. Fractionation studies with starch block electrophoresis of fetal calf and human sera revealed that alpha 2-globulin fraction contains a serum factor essential for macrophage activation. To identify the serum factor, human serum was precipitated with 50% saturated ammonium sulfate and fractionated on a Sephadex G-100 column. A protein fraction with a lower m.w. than albumin had the capacity to support activation of macrophages. The active serum factor in this protein fraction was analyzed by immunoabsorption by using rabbit antisera against three major proteins of human alpha 2-globulin. This active serum factor was shown to be a vitamin D3-binding protein (group specific component, Gc). By using a monoclonal anti-Gc-absorbed active column fraction of human serum, we observed no enhanced macrophage activation over the results with serum fraction-free cultivation of lyso-Pc-treated peritoneal cells. Cultivation of lyso-Pc-treated peritoneal cells in a medium containing a low concentration of purified human Gc protein (0.1 to 2.6 ng/ml) produced a greatly enhanced phagocytic activity of macrophages. When purified human Gc protein was used in a serum-free medium for stepwise cultivation of lyso-Pc-treated nonadherent cell types, a macrophage-activating factor was efficiently generated. Therefore, it is concluded that the vitamin D3-binding protein is the essential serum factor for the lyso-Pc-primed activation of macrophages.     

A solid-phase assay for beta-1,4-galactosyltransferase activity in human serum using recombinant aequorin

We have developed a sensitive and rapid solid-phase assay for the serum enzyme UDPGal:beta-D-GlcNAc beta-1,4-galactosyltransferase (beta 1,4-GT) (EC 2.4.1.38) that employs the recombinant bioluminescent protein aequorin as the enzyme label for product detection. The substrate for beta 1,4-GT is a neoglycoprotein, bovine serum albumin containing covalently attached GlcNAc residues (GlcNAc-BSA), and it was immobilized by adsorption in microtiter plate wells. Serum samples were added to each well along with saturating levels of UDPGal and Mn2+. Galactosylation of the neoglycoprotein acceptor by the serum beta 1,4-GT produces the N-acetyllactosamine derivative Gal beta 1, 4GlcNAc-BSA. The product formed is quantified by adding the biotinylated plant lectin Ricinus communis agglutinin-I, which binds specifically to N-acetyllactosamine, followed by the addition of streptavidin and the biotinylated aequorin. Aequorin produces a flash of light in response to Ca2+ and is detectable to 10(-19) mol in a luminometer. Using this assay, the beta 1,4-GT activity in human serum and the activity of a semipurified beta 1,4-GT are linear with time and serum concentration over a wide range. The reaction is dependent on UDPGal and Mn2+, is highly reproducible with a low background, and can be performed in a few hours. Assays employing aequorin have a wider range of linearity than those employing horseradish peroxidase as an enzyme label. These results demonstrate that the assay for beta 1,4-GT is useful for determining activity in heterogeneous samples and also demonstrate the utility of the recombinant protein aequorin for solid-phase assay methods.     

Protein turnover in pulmonary macrophages. Utilization of amino acids derived from protein degradation.

Conditions were defined under which rates of protein synthesis and degradation could be estimated in alveolar macrophages isolated from rabbits by pulmonary lavage and incubated in the presence of plasma concentrations of amino acids and 5.6 mM-glucose. Phenylalanine was validated as suitable precursor for use in these studies: it was not metabolized appreciably, except in the pathways of protein synthesis and degradation; it entered the cells rapidly; it maintained a stable intracellular concentration; and it was incorporated into protein at measurable rates. When extracellular phenylalanine was raised to a concentration sufficient to minimize dilution of the specific radioactivity of the precursor for protein synthesis with amino acid derived from protein degradation, the specific radioactivity of phenylalanyl-tRNA was only 60% of that of the extracellular amino acid. This relationship was unchanged in cells where proteolysis increased 2.5-fold after uptake and degradation of exogenous bovine serum albumin. In contrast, albumin prevented the decrease in phenylalanine incorporation observed in macrophages deprived of an exogenous source of amino acids. These observations suggested that macrophages preferentially re-utilized amino acids derived from the degradation of endogenous, but not from exogenous (albumin), protein. However, when the extracellular supply of amino acids was restricted, substrates derived from albumin catabolism could support the protein-synthetic pathway.     

Lectin-like molecules on the murine macrophage cell surface

Lectin-like molecules on the surface of murine peritoneal exudate macrophages induced by thioglycolate or an anti-tumor streptococcal preparation, OK-432, were investigated and isolated. Furthermore, their sugar-binding specificities and their role in macrophage-mediated tumor cytotoxicity were examined. A neoglycoprotein, D-galactose (Gal)-bovine serum albumin, bound to these murine peritoneal macrophages. This binding of Gal-bovine serum albumin was inhibited by D-galactose, and by complex-type oligosaccharides (unit B) and high mannose-type oligosaccharides (unit A) prepared from porcine thyroglobulin. When thioglycolate-elicited macrophages were activated by lipopolysaccharide and/or the culture supernatant of concanavalin A-activated mouse spleen cells, they became tumoricidal and the number of the lectin-like molecules on the macrophage surface was found to increase. Since the binding and cytotoxic activities of these tumoricidal macrophages toward tumor cells were partially inhibited by D-galactose, the D-galactose-binding lectin-like molecules on the surface of tumoricidal macrophages might play an important role in macrophage-mediated cytotoxicity. These lectin-like molecules were then isolated from solubilized murine peritoneal exudate cells labeled with pyridoxal 5'-phosphate and sodium [3H]borohydride by affinity chromatography on columns of asialo unit B oligosaccharide-Sepharose 4B and/or beta-D-galactose-Bio-Gel P-100. The proteins bound to the asialo unit B oligosaccharide-Sepharose 4B column and eluted specifically were found to have approximate molecular weights of 79 000 and 18 000, and the protein bound to and eluted from the beta-D-galactose-Bio-Gel P-100 column had an approximate molecular weight of 77 000. These isolated proteins bound to the surface of glutaraldehyde-fixed tumor cells, and their binding was inhibited by D-galactose and also by D-mannose. Since most of the 77 kDa protein bound to the asialo unit B oligosaccharide-Sepharose 4B, this protein was assumed to be identical with the 79 kDa protein. These results suggest that the lectin-like molecules on murine macrophages have wide specificity and that one lectin-like molecule can bind both D-galactose and D-mannose.     

Induction, in vivo and in vitro, of macrophage membrane interleukin-1 by adjuvant-active synthetic muramyl peptides

Recent evidence has shown that a membrane form of interleukin-1 (IL-1) serves as a necessary signal for antigen presentation, leading to T-cell activation. The synthetic immunostimulant muramyl dipeptide (MDP) is known to induce secretion of IL-1 and its adjuvant effect was found to be mediated through enhancement of T-helper cells. We have investigated the ability of MDP and 19 other adjuvant-active or -inactive MDP analogs and derivatives to induce membrane IL-1 in mouse peritoneal macrophages. Enhancement in vitro of membrane expression and secretion of IL-1 in fresh or aged cultures of macrophages was observed after stimulation with MDP or with adjuvant-active but not with adjuvant-inactive muramyl peptides. Administration in vivo of adjuvant-active doses of MDP or of any of 12 other active analogs induced high levels of macrophage membrane IL-1 detected by the lymphocyte-activating factor assay. This effect was not observed when 7 other adjuvant-inactive derivatives were used. Moreover, under conditions where MDP did not exert an adjuvant effect, this immunomodulator was found to be incapable of inducing the expression of macrophage membrane IL-1. These results demonstrate a very high correlation between the ability to induce membrane IL-1 and the adjuvant activity of muramyl peptides. The correlation was observed irrespective of other biological effects of the synthetic adjuvants such as pyrogenicity and/or anti-infectious activity.     

Synergistic activation by recombinant mouse interferon-gamma and muramyl dipeptide of tumoricidal properties in mouse macrophages

Mouse peritoneal macrophages were activated to become cytotoxic against B16-BL6 melanoma cells by the combination of subthreshold amounts of murine interferon-gamma (IFN-gamma; 0.1 to 10 U/ml) and N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP; 0.001 to 10 micrograms/ml), but not by the combination of pH 2-treated IFN-gamma and MDP, heat-treated IFN-gamma and MDP, or IFN-gamma and the inactive stereoisomer of MDP, N-acetyl-muramyl-D-alanyl-D-isoglutamine (MDP-D). The encapsulation of intact IFN-gamma and MDP within the same liposome preparation was synergistic for macrophage activation. In contrast, the presentation of identical concentrations of IFN-gamma and MDP in separate liposome preparations did not activate macrophages. These data allow us to conclude that the encapsulation of genetically engineered IFN-gamma and synthetically produced MDP within the same liposome is highly efficient in producing synergistic activation of tumoricidal properties in mouse macrophages.     

Activation of delta5-3-ketosteroid isomerase of bovine adrenal microsomes by serum albumins.

The Δ⁵-3-ketosteroid isomerase (EC 5.3.3.1) of bovine adrenal microsomes is activated as much as 10- to 20-fold by micromolar concentrations of bovine serum albumin. Comparable activations are observed with the serum albumins of 10 other mammalian species, but are not seen with ovalbumin or conalbumin. Evidence that the activation is attributable to the serum albumins, rather than to a small, firmly-bound ligand, is based on: (1) Failure to remove the stimulatory activity from the albumin by chloroform extraction, dialysis, or gel filtration; (2) Destruction of the activity by heating or by trypsin digestion; (3) Precipitation of the stimulatory activity of bovine serum albumin by specific antibody. Bovine serum albumin induces small decreases in the Michaelis constant for Δ⁵-androstene-3,17-dione, but most of the activational effect reflects an increase in the maximum velocity. Low concentrations of Triton X-100, which are without effect on the isomerase activity, prevent the activation by bovine serum albumin.     

Effect of Muramyldipeptide,a Synthetic Bacterial Adjuvant,on Enzyme Release from Cultured Mouse Macrophages

Monolayer cultures of macrophages obtained by peritoneal lavage of normal or thioglycollate-stimulated mice spontaneously secreted lysosomal enzymes into the culture medium. When the elicited macrophages were cultured in the presence of muramyldipeptide (MDP), a 20–30% increase in the release of β-glucuro-nidase was consistently observed and the intracellular activity decreased to about 45% of that of control cells after 6–8 days' culture. A stimulatory effect of MDP on lysozyme secretion, though less profound, was also observed. In contrast, release of neither enzyme was stimulated in resident macrophages by the addition of MDP. A neutral α-glucosidase, which has recently been found to localize also in granules of macrophages, remained inside the cells and neither its activity nor its release was affected by the addition of MDP to either type of macrophages. A large amount of lactic dehydrogenase was released only when the resident, not the elicited, macrophages were cultured for 3–4 days and then phagocytosed zymosan.     

Role of macrophage destruction products in the alveolar phagocytosis reaction]

Peritoneal macrophages (PM) of Wistar rats harvested after the intraperitoneal injection of paraffin oil were destroyed by repeated freezing-thawing. When injected intratracheally to control rats or to those after 4 daily exposured to TiO2 dust, these macrophage destruction products (MDP) caused a significant rise of both the alveolar macrophages (AM) and the neutrophilic leukocytes (NL) counts in the pulmonary washing-outs; the mean NL/AM ratio increased several times as compared to rats injected with normal saline intratracheally. Thus, the response to the inert dust particles plus the exogenous MDP became similar to the one observed after the cytotoxic (for instance silica) particles inhalation. Enhancing the NL contribution to the inhaled particles phagocytosis, the MDP led to a significant decrease of the mean "Dust load" of a single AM, although the total number of the engulfed particles increased. The predominant attraction of granulocytes and particularly of the NL as compared to the peritoneal macrophages was also found in the peritoneal exudates of rats injected with the MDP or silica suspension intraperitoneally, while the alveolar phagocytosis was not influenced. In vitro the MDP was shown to stimulate the NL migration and to facilitate the O2 consumption by PM. A possible role of the MDP as a multipotent controlling factor of phagocytosis response is briefly discussed.