Biomedical subjects
Publications and source records attributed to J F Petit.
Two 125I-labelled aryl-azide derivatives of MDP have been synthesized. Photoaffinity labelling experiments demonstrated cell surface binding sites for muramylpeptides on activated B-lymphocytes and intracellular muramylpeptides binding protein(s) of 40-45 kD in rat alveolar macrophages.
Murine peritoneal macrophages activated for cytotoxicity by trehalose dimycolate in vivo and lipopolysaccharide in vitro released cytostatic factor(s) against EMT6 target cells, in 8-hr conditioned medium (CM). The cytostatic factor(s) completely blocked DNA synthesis by EMT6 cells within 16 hr. Other cell lines are less sensitive (P815 and R-L929) or resistant (KB and HT29) to the cytostatic effect of CM. The anti-proliferative activity of CM had a MW greater than 10,000 Da, as judged by ultrafiltration. It was destroyed by proteases and strongly inhibited by P815 cell product(s). Conditioned media from nonactivated macrophages were not cytostatic against EMT6 cells. No relationship was found between cytostatic factor(s) in CM and interleukin 1 (IL-1), tumor necrosis factor alpha (TNF-alpha), and interferon-alpha/beta (IFN-alpha/beta): the growth of EMT6 cells was unaffected by Hu.r.IL-is and Hu.r.TNF-alpha and was only slightly inhibited by IFN-alpha/beta. Furthermore, cytostatic CM contained low levels of TNF and IFN activities. Finally, antibodies raised against murine IFN-alpha/beta had no effect on the cytostatic activity of CM.
The axial organ (AO-cells) of the starfish Asterias rubens is a primitive immune organ. The total population was fractionated into two populations: adherent (B-like) and non-adherent (T-like) to nylon wool. Nocardia-delipidated cell mitogen (NDCM), Nocardia water soluble mitogen (NWSM), NWSM-pellet (NWSMP) and purified cell walls (CW) from Nocardia opaca induce the proliferation of B-like cells from the axial organ. The Nocardia immunomodulators studied are inactive on the total and T-like population. The mitogenic activity of B-like cells requires the presence of phagocytic cells.
A novel analog of MDP, the 3'-iodo-4'-azido-L-phenylalanine methyl ester derivative of N-acetyl-L-alanyl-D-isoglutamine, has been prepared. This compound is capable of activating macrophages to the tumoricidal state and increasing the specific immune response of B cells. It thus appears to exhibit similar biological activities to MDP. Moreover, this compound is of potential interest for receptor photolabelling studies.
We have prepared liposomes from mannosylated phosphatidylmyo-inositol, derived from mycobacteria, and cholesterol. The size of the particles so formed could be controlled by membrane filtration. The vesicles encapsulated a significant amount of aqueous phase (about 8 microliter per mg phospholipid). Markers of the liposomal membrane and aqueous phase rapidly associated with mouse peritoneal macrophages and, more slowly, with rat alveolar macrophages. The uptake was saturable at high liposome concentrations, although phagocytosis of latex particles of the same mean diameter was not saturable at these concentrations. An excess of unlabelled liposomes composed of phosphatidylcholine and phosphatidylserine, which were also taken up readily by macrophages, did not inhibit the uptake of mannosylated liposomes. The uptake of fluorescent mannosylated bovine serum albumin was inhibited by these liposomes, suggesting a specific interaction with the macrophage mannose-fucose receptor. We conclude that this type of liposome would be useful for the delivery of immunomodulators to reticuloendothelial cells.
It was possible to define the effects of trehalose dimycolate (TDM), a glycolipid extracted from Mycobacterium tuberculosis, on mouse peritoneal macrophages more precisely using endotoxin-free culture conditions. TDM-elicited macrophages, when assayed in vitro in the absence of endotoxin, were unable to limit tumor growth; however, after a short treatment (4 h) with low doses of lipopolysaccharide (LPS; 1-10 ng/ml), they exhibited a strong cytostatic capacity against P815 mastocytoma cells. Thus, TDM injected in vivo did not activate macrophages fully but it primed them to respond in vitro to low doses of LPS, which provided the final stimulus for activation to antitumor competence. Macrophages elicited by an injection of killed group C Streptococci were also in a primed state; in contrast, thioglycollate-elicited macrophages were in a nonreceptive state. Besides LPS, concanavalin A (5 micrograms/ml), MDP (0.2-1 microgram/ml) and the ionophore A23187 (5 microM) can deliver the activation signal to TDM-primed macrophages. Primed macrophages were found to express several biochemical markers previously described as specific for activated macrophages (low levels of alkaline phosphodiesterase and beta-galactosidase, for example) and, although they were not cytotoxic for tumor cells, they had the capacity to release large amounts of H2O2. However, when pulsed by LPS or MDP, primed macrophages responded by further modifications in their metabolism: the rate of glucose consumption and the labeling of glycoproteins by D-[2-3H]mannose were greatly increased and the secretion of a polypeptide of 22 kDa was enhanced. The activation-associated biochemical markers are thus acquired in two steps. The ability to produce activated oxygen species is expressed earlier than the antitumoral activity.
After biosynthetic radiolabeling, polypeptides secreted by macrophages were analyzed by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate and fluorography. Comparative studies of various macrophage populations (inflammatory macrophages, primed and activated macrophages, resident macrophages) showed that secretory activity is elaborately controlled according to macrophage developmental and functional state. When young macrophages activated to cytotoxicity against tumor cells by an appropriate eliciting agent and culture in the presence of lipopolysaccharide were compared to mature resident macrophages devoid of antitumor activity, the following changes were observed in the protein secretion phenotype: (1) New polypeptides are expressed: the intensity of four bands was increased more than five-fold on one-dimensional gels and 17 new spots were detected on two-dimensional gels. (2) The production of several major polypeptides is repressed: six bands were strongly decreased on one-dimensional gels, and 11 major spots (or groups of spots) were lost on two-dimensional gels. (3) The charge microheterogeneity pattern exhibited by several polypeptides on two-dimensional gels is shifted towards less acidic species.
Intratracheal (i.t.) administration of trehalose dimycolate (TDM) in saline as liposomes induces a transient inflammatory effect. Limited granulomas appeared in some peribronchial areas but most subsided after a few weeks. The alveolar macrophages were activated as judged by their cytostatic activity against the syngeneic P77 fibrohistiocytoma 3 days after administration of 0.2 mg TDM. The NK activity of the lymphocytes of the lung microcirculation did not increase and diminished slightly between 1 and 3 days after TDM administration, thus suggesting that macrophages might be the main effector cells responding to TDM. Repeated i.t. TDM administration protected rats against the development of colonies in the lung after i.v. injection of 5 X 10(5) P77 cells. The survival of the rats was significantly increased. Thus, in this system, a relationship exists between activation of alveolar macrophages and protection against colonies arising from i.v. injected tumor cells.
Macrophage-mediated antitumor activity is believed to be regulated by E-type prostaglandins released by target cells or even by macrophages themselves. In these studies we showed that a subcutaneous injection of polyacrylamide beads (Biogel P100), induced in mice, a population of immature macrophages which became fully cytostatic to syngeneic P815 mastocytoma when pulsed in vitro with lipopolysaccharide (LPS). Blockade of prostaglandin synthesis by indomethacin prevented LPS effect and led to a substantial resumption of target growth. Addition of PGE2 did not reverse the indomethacin effect but inhibited macrophage-mediated cytostatic activity. These findings suggest that acquisition of cytostatic properties by macrophages is, at a certain stage of their maturation, under the control of both PGE2 and another endogenously produced eicosanoid.
Two populations of phagocytic cells from trehalose dimycolate-elicited mouse peritoneal cells are demonstrated by flow cytofluorometry, using two fluorescent probes excited at the same wavelength (488 nm). Liposomes containing diethylenetriaminepentaacetate daunorubicin conjugate (maximum emission wavelength: 590 nm) allow the discrimination of phagocytes and non-phagocytic cells. Among the phagocytes, an activated population is revealed by a cell-associated fluorescence of the oxidation product of dichlorofluorescein diacetate (maximum emission wavelength: 520 nm).
Nocardia-delipidated cell mitogen (NDCM), a particulate fraction prepared from Nocardia opaca, is able to stimulate the proliferation of small resting human B lymphocytes and their differentiation into Ig-secreting cells. This fraction contains two active structures: the cell wall peptidoglycan (PG) and a fraction (Cy I) derived from the cytoplasmic compartment. Treatment of insoluble PG with various bacteriolytic enzymes showed that the minimal structure required for mitogenic activity is more complex than that required for the differentiation of human lymphocytes. The mitogenic activity of cell wall fractions varies in different bacterial species; that prepared from N. opaca is the more potent. Both mitogenic structures of N. opaca induce higher responses in infant and adult PBL as compared to cord lymphocytes. The differentiation of B lymphocytes into Ig-secreting cells induced by PG fractions is T-dependent.
The ability of liposomes containing a new lipophilic muramyl peptide derivative, MDP-L-alanyl-cholesterol (MTP-CHOL), to induce peritoneal macrophage cytostatic activity and alveolar macrophage cytotoxic activity toward tumor cell targets in vitro was determined. MTP-CHOL was shown to be efficiently incorporated and subsequently retained in distearoylphosphatidylcholine/phosphatidylserine liposomes (DSPC/PS; 7:3 molar ratio), whereas hydrosoluble muramyl dipeptide (MDP) was rapidly lost due to leakage. Liposomes containing MTP-CHOL were able to stimulate mouse peritoneal macrophage cytostatic activity under conditions where free MDP was without effect. MTP-CHOL incorporated into liposomes was approximately eightfold more effective than liposomes containing entrapped MDP and 7,400-fold more effective than free MDP in inducing rat alveolar macrophage cytotoxic activity. These results provide evidence that the coupling of MDP to a lipophilic molecule, cholesterol, results in the formation of a viable liposome formulation that is a potent inducer of macrophage-mediated antitumor activity.
We have previously shown that production of plasminogen activator by inflammatory macrophages can be inhibited by immunomodulators of bacterial origin, especially LPS. In these experiments, the contribution of prostaglandins to this control was investigated. We examined the effect of two prostaglandin synthetase blockers, indomethacin and diclofenac. These drugs do not modify per se the production of plasminogen activator but they can partially prevent the inhibitory effect of LPS. Since restoration was not complete when plasminogen activator production was strongly inhibited, it is suggested that the immunomodulator acts through a mechanism involving more than one pathway only one of which would be mediated by prostaglandins.
There is evidence indicating that bioreactive lipid mediators, PAF-acether (platelet-activating factor: 1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine) and arachidonic acid (AA) metabolites, are formed upon deacylation of ether lipids. M phi obtained from mice treated with the sterile irritant thioglycolate exhibited an impaired formation of PAF-acether, leucotrienes (LT) C4 and prostaglandins (PG). In order to assess whether the impaired formation in lipid mediators is a general feature of M phi found at inflammatory sites, we have compared the capacity of resident (R-M phi) and immunostimulant-activated M phi to release, upon a zymosan challenge, PAF-acether and cyclooxygenase and lipoxygenase products. Activated M phi was obtained from mice injected intraperitoneally with non-viable C74 streptococci (St-M phi), bacilli Calmette-Guerin (BCG-M phi) or trehalose dimycolate (TDM-M phi), a defined immunostimulant isolated from Mycobacterium tuberculosis. All populations were capable of releasing PAF-acether. However, the amount of cell-associated PAF-acether was reduced by 75-90% in activated M phi populations as compared to R-M phi. Although the acetyltransferase level was comparable, acetyl-CoA supplementation restored the formation of PAF-acether by activated M phi to control (R-M phi) level. The amount of 14C-AA metabolites released by St-M phi was much lower compared with TDM-M phi or R-M phi, as was the amount of LTC4 detected as SRS contractile activity after HPLC.(ABSTRACT TRUNCATED AT 250 WORDS)
The metabolism of bioreactive lipid mediators was studied in two types of activated macrophages (M phi). We compared the capacity of resident and activated M phi to release, upon a zymosan challenge, cyclooxygenase and lipoxygenase products as well as PAF-acether (platelet-activating factor) and its 2-lyso precursor. Activated M phi were obtained from mice injected intraperitoneally either with nonviable C74 streptococci (St-M phi) or with trehalose dimycolate, a defined immunostimulant isolated from Mycobacterium tuberculosis (TDM-M phi). Both activated populations exhibited common features: conversion of endogenous [14C]arachidonic acid into prostaglandin E2 and thromboxane A2 rather than into prostaglandin I2 and low biosynthesis of PAF-acether, probably due to an impairment of the acetylation step. However, contrary to St-M phi, TDM-M phi did not display a marked overall reduction of arachidonate metabolism. In addition, as compared to resident M phi, TDM-M phi presented a ratio of thromboxane B2/6-ketoprostaglandin F1 alpha 30-fold higher, a better conversion of leukotriene C to leukotriene D and a higher capacity to release the PAF-acether they synthesize. These macrophages thus seem to be valuable tools for studying the formation of mediators and for determining specific markers of an activated state.
In inflammatory macrophages, plasminogen activator exists in two active forms, a soluble form released into the extracellular medium and a cell-associated form. This communication describes some properties of the cellular form of plasminogen activator, in intact macrophages and in cell lysates. Cellular plasminogen activator is a membrane protein, associated with the outer face of the plasma membrane; in intact macrophages, it participates in the activation of exogenous plasminogen and, thus, has to be considered as an ectoenzyme. A plasminogen activator activity can be detected in cell lysates (macrophage monolayers lysed in 0.1% Triton X-100) only when plasmin production is followed by the use of small synthetic substrates because a soluble inhibitor, released during extraction, blocks plasmin fibrinolytic activity. In these lysates, plasminogen activator molecules exist as high molecular weight unstable complexes exhibiting a high affinity for plasminogen.
The antitumor activity of trehalose-6,6'-dimycolate (TDM) dispersed in saline was studied in vitro and in vivo. In vitro, macrophages from TDM-treated mice entirely abolish, even at a 1.25:1 effector/target cell ratio, the (3H)TdR incorporation of P815 mastocytoma cells to the culture in which they were added. In vivo, the number of P815 cells harvested 10 days after their injection into TDM-treated mice was reduced by a factor higher than 50. The slopes of the radiolabel elimination of TDM-treated mice injected with 10(7) 125I-UdR-labelled L1210 leukemic cells were greater than those of untreated controls. The interest of such a compound which seems to be one of the active structures responsible for the antitumor activity of BCG is its efficiency when administered dispersed in saline.
Stimulation by PMA of Streptococci-elicited macrophages induced a transient membrane depolarization preceding the onset of detectable O-2 production. Mice-resident peritoneal macrophages were unresponsive to PMA for both activities. The PMA-triggered membrane depolarization seemed to be independent from O-2 production because inhibition of membrane depolarization by EGTA had no effect on rates of O-2 or H2O2 release and rate of antimycin A insensitive O2 uptake by Streptococci-elicited macrophages. The portion of O2 uptake recovered as O-2 was found to be 1/3. The rate of O-2 release was twice the rate of H2O2 production (1.1 nmol H2O2.min-1 X 10(6) macrophages-1).