gamma-Interferon enhances the secretion of arachidonic acid metabolites from murine peritoneal macrophages stimulated with phorbol diesters

Macrophage activation in vivo has been associated with qualitative and quantitative alterations in the release and metabolism of arachidonic acid. In the present study, we examined the effect of in vitro macrophage activation with recombinant gamma-interferon (IFN-gamma) on arachidonic acid secretion induced by exposure to a variety of stimulating agents. Secretion stimulated by challenge with unopsonized zymosan, insoluble immune complexes, the calcium ionophore A23187, or combinations thereof was unaltered in IFN-gamma-treated macrophages. However, when phorbol diesters active as tumor promoters were employed as challenge agents, arachidonate secretion was enhanced as much as 10-fold over that seen in nonactivated controls. The enhanced secretory response to PMA was detectable as early as 1 hr after exposure to IFN-gamma, reached a maximum within 3 to 6 hr, and subsequently declined to control levels even in the continued presence of the agent. Treatment with IFN-gamma did not alter the pattern of individual metabolites produced by macrophages challenged with either zymosan or PMA. Finally, the sensitivity to phorbol diesters was also increased by treatment with IFN-gamma (ED50 reduced from 35 ng/ml to 4 ng/ml). Thus, IFN-gamma could prime macrophages for a substantially amplified response to phorbol esters. Because the cellular mediator of PMA action has been identified as a Ca++, phospholipid-dependent protein kinase, a role for this enzyme in macrophage functional development is indicated.     

Divalent cation requirements for mounting a respiratory burst in response to phorbol diesters by macrophages from SENCAR and C57BL/6 mice

Oxygen radicals are thought to play an important role in the promotion phase of carcinogenesis and the action of phorbol esters. Inflammatory cells are an abundant source of reactive oxygen intermediates (ROI) in the body and release large quantities of ROI when exposed to phorbol esters. Both protein kinase C (PKC), the receptor for phorbol esters, and the NADPH oxidase which generates ROI are Ca2+- and Mg2+-dependent. We investigated the requirements for Ca2+ and Mg2+ of macrophages from strains of mice sensitive and resistant to the promotion of tumors by phorbol esters. Macrophages from SENCAR mice, which are sensitive to phorbol ester promotion, required much lower levels of Ca2+ or Mg2+ to mount a full respiratory burst, as measured by the release of H2O2 in response to phorbol ester stimulation, than macrophages from C57BL/6 mice, which are resistant to promotion by phorbol esters. Conversely, when the particulate stimulus zymosan was used, there was little difference between macrophages from the two strains in requirements for Ca2+ and Mg2+ to release H2O2. Lowering the concentration of either cation in the absence of the other was more inhibitory than in the presence of the other cation. The studies demonstrate that differences in sensitivity to divalent cations by macrophages from these two strains is selective for phorbol ester stimulation and that lower requirements for Ca2+ and Mg2+ for ROI release correlates with sensitivity to the promotion of tumors by phorbol esters.     

Regulation by CRAMP of the responses of murine peritoneal macrophages to extracellular ATP

Peritoneal macrophages were isolated from wild type (WT) mice and from mice invalidated for the P2X₇ receptor (KO) which had been pretreated with thioglycolate. In cells from WT mice, 1 mM ATP increased the intracellular concentration of calcium ([Ca²⁺]_i), the uptake of ethidium bromide, the production of reactive oxygen species (ROS), the secretion of IL-1β, the release of oleic acid and of lactate dehydrogenase; it decreased the intracellular concentration of potassium ([K⁺]_i). In KO mice, ATP transiently increased the [Ca²⁺]_i confirming that the P2X₇ receptor is a major receptor of peritoneal macrophages. WKYMVm, an agonist of receptors for formylated peptides (FPR) also increased the [Ca²⁺]_i in murine macrophages. The slight increase of the [Ca²⁺]_i was strongly potentiated by ivermectin confirming the expression of functional P2X₄ receptors by murine peritoneal macrophages. CRAMP, the unique antimicrobial peptide derived from cathelin in mouse inhibited all the responses coupled to P2X₇ receptors in macrophages from WT mice. Agonists for FPR had no effect on the increase of the [Ca²⁺]_i in response to ATP. CRAMP had no effect on the increase of the [Ca²⁺]_i evoked by a combination of ATP and ivermectin in macrophages from P2X₇-KO mice.In summary CRAMP inhibits the responses secondary to the activation of the murine P2X₇ receptors expressed by peritoneal macrophages. This inhibition is not mediated by FPR receptors and is specific since CRAMP has no effect on the response coupled to P2X₄ receptors. It can thus be concluded that the interaction between P2X₇ receptors and cathelin-derived antimicrobial peptides is species-specific, in some cases (man) positive in others (mouse) negative.     

Regulation of I-A expression by murine peritoneal macrophages: differences linked to the Bcg gene

We previously reported that Mycobacterium bovis (strain BCG) induces continuous I-A expression when injected into BCG-resistant strains of mice. We have extended this observation by showing that Corynebacterium parvum also induces continuous I-A expression by macrophages from BCG-resistant but not BCG-susceptible mice. We have linked continuous expression to BCG resistance by using C.D2Ityr mice, which are congenic with BCG-susceptible BALB/c mice except for genes on a portion of chromosome 1, which contains the gene(s) for BCG resistance. Macrophages from C.D2Ityr mice continuously expressed I-A, whereas macrophages from BALB/c mice transiently expressed I-A. Continuous expression by macrophages from both Bcgr and Bcgs mice could be induced in vitro with rIFN-gamma. However, the continuous expression of I-A by macrophages from Bcgs mice required the continued presence of IFN-gamma, whereas that by Bcgr macrophages did not. The continuous expression of I-A by macrophages from Bcgs mice was also inhibited by hydrocortisone, cyclohexamide, tunicamycin, and monensin, whereas I-A expression by Bcgr macrophages was not affected. The continuous expression of I-A by macrophages from Bcgr mice did not require its continued synthesis. The significance of these findings to the induction of immunity and to antimicrobial resistance are discussed.     

Biochemical and functional responses stimulated by platelet-activating factor in murine peritoneal macrophages

Platelet-activating factor (PAF) is a potent stimulant of leukocytes, including macrophages. To analyze the mechanisms of its effects upon macrophages, we determined whether macrophages bear specific surface receptors for PAF. By competitive radioactive binding assays, we determined two classes of specific receptors to be present on purified membranes derived from murine peritoneal macrophages (one having a Kd of approximately 1 X 10(-10) M and one a Kd of approximately 2 X 10(-9) M). When the macrophages were incubated with PAF, rapid formation of several inositol phosphates including inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate were observed. PAF also elevated intracellular levels of calcium to 290 +/- 27% of basal levels which were 82.7 +/- 12 nM. Increases in calcium were observed first in submembranous areas of the macrophages. PAF also led to increases of 1,2-diacylglycerol of approximately 200 pmol/10(7) cells. A characteristic pattern of enhanced protein phosphorylation, similar to that initiated by both phorbol 12,13-myristate and lipopolysaccharide, was observed and involved enhanced phosphorylation of proteins of 28, 33, 67, and 103 kD. The half-maximal dose of PAF for initiating all the above effects was approximately 5 X 10(-9) M. PAF also initiated significant chemotaxis of the cells; the half-maximal dose for this effect was approximately 1 X 10(-11) M. Taken together, these observations suggest that murine mononuclear phagocytes bear specific membrane receptors for PAF and that addition of PAF leads to generation of break-down products of polyphosphoinositides, subsequent changes in intracellular calcium and protein phosphorylation, and chemotaxis.     

Oxidative and non-oxidative activation of murine peritoneal macrophages by histone H1

The present study aimed at assessing the role of histone H1 in activating macrophages. Histone H1, injected intraperitoneally at a dose of 1 mg/kg body weight as multiple regimens weekly, significantly increased the number of peritoneal macrophages post 21 days of injection. The oxidative and non-oxidative activation of peritoneal macrophages by histone H1 was assessed. For the assessment of oxidative activation the levels of superoxide radical and nitric oxide radical were assessed. The oxidative activation was evident from release of significantly high levels of superoxide and nitric oxide radicals liberated by macrophages of animals treated with histone H1 (P < 0.001) than in untreated animals. In addition, the higher activities of superoxide dismutase indicated protective effect of histone H1, to keep away the macrophages from noxious effects of superoxide. The catalase activity was decreased significantly in macrophages of histone H1 treated animals. The levels of reduced glutathione were significantly (P < 0.001) lowered in treated animals, whereas the levels of lipid peroxides generated were non-significant. The non-oxidative activation was assessed from the activities of lysosomal enzymes released and also from cytolysis of NO-insensitive L929 cells. The activities of lysosomal enzymes-acid phosphatase and beta-glucuronidase released were significantly high in treated animals than in untreated animals (P < 0.001). Histone H1 stimulated the cytolysis of macrophages in L929 cells than in untreated animals. These results suggest that histone H1 stimulates macrophages by oxidative and non-oxidative mechanisms, which favor its future therapeutic prospects.     

Phagocytosis and stimulation of the respiratory burst by phorbol diester initiate S-thiolation of specific proteins in macrophages.

Addition of chemical oxidants to cells in culture has been shown to induce binding of low-molecular-weight thiols to reactive sulfhydryls on proteins in a process termed S-thiolation. We found that stimulation of the respiratory burst in mouse macrophages, with release of O2-, resulted in S-thiolation of several proteins, most prominently three with molecular weights of 74, 33, and 22 kDa. One protein (28 kDa) was S-thiolated without addition of an exogenous stimulus. Exposure of cells to concentrations of hydrogen peroxide like those released in the respiratory burst induced S-thiolation of these same proteins. S-thiolation and release of O2- began at approximately the same time. Stimulation of LPS-elicited macrophages induced prominent S-thiolation of three different proteins (38, 30, and 21 kDa). Under the conditions of these experiments, there was no detectable increase in glutathione disulfide and a negligible decrease in glutathione, which suggests that S-thiolation can occur without significant perturbation of the glutathione peroxidase/reductase cycle. S-thiolation of proteins could help protect the macrophage against the autoxidative damage associated with the respiratory burst. Modification of specific proteins by S-thiolation might serve to modulate cellular metabolic events.     

Expression of the transferrin receptor in murine peritoneal macrophages is modulated in the different stages of activation

A specific high affinity binding site for the serum glycoprotein transferrin was identified on murine peritoneal macrophages. The binding reached equilibrium within 60 min and was reversible, saturable, and specific for transferrin. Although the presence of this receptor was detected on the cell surface by studies carried out using intact cells, the majority (70 to 90%) of the binding activity was detectable only in detergent extracts of such cells. This suggests that a substantial portion of the binding activity is localized within the macrophage. The association constant (Ka) for binding to intact cells (6 to 9 X 10(8) M-1) was comparable to values reported for transferrin receptors described on other cell types. The expression of transferrin binding activity was examined in macrophages exhibiting qualitatively and quantitatively different degrees of functional activation. Resident peritoneal macrophages, exudate macrophages primed by elicitation with pyran copolymer, and activated macrophages induced by chronic infection of mice with bacillus Calmette-Guerin (BCG) or elicitation with heat killed Propionibacterium acnes (P. acnes) had low numbers of binding sites (1000 to 5000 total sites/cell). Macrophages elicited by sterile inflammatory agents (thioglycollate broth, fetal bovine serum, or casein) all exhibited a greater number of transferrin receptors (15,000 to 20,000 total sites/cell). This modulation did not appear to result from differential shifts between surface and internal loci. Our results suggest that the expression of the transferrin receptor may be a useful marker of the responsive stage of macrophage functional activation and the membrane changes that accompany activation.     

Phenotypic and functional analysis of murine resident and induced peritoneal macrophages

Primary macrophages from the peritoneal cavities of mice are commonly used ex vivo to produce inflammatory cytokines and test anti-inflammatory agents. Although approximately 1 million peritoneal macrophages can be obtained from an untreated mouse, more than twice that number can be collected 48 to 72 h after intraperitoneal injection of sterile inducing agents such as Brewer thioglycollate broth, casein, and proteose peptone. However, whether 'induced' macrophages are functionally equivalent to 'resident' peritoneal macrophages has been unclear. Flow cytometric analysis revealed significant phenotypic differences between these 2 macrophage types. Resident and induced peritoneal macrophages also demonstrated markedly different capacities to produce the inflammatory cytokines interleukins 6 and 1beta in response to lipopolysaccharide stimulation in vitro. Increased understanding of the differences between resident and induced peritoneal macrophages likely will help investigators decide which macrophage type is appropriate for their in vitro assay needs.     

Cadmium-induced depression of the respiratory burst in mouse pulmonary alveolar macrophages, peritoneal macrophages and polymorphonuclear neutrophils.

No profound alteration in the resting O₂ consumption of mouse pulmonary alveolar macrophages, polymorphonuclear neutrophils or peritoneal macrophages incubated in media containing either cadmium chloride or cadmium acetate was observed. However, when heat-killed $\text{P. aeruginosa}$, opsonized in autologous serum, were added to the cell suspension a significant depression in the respiratory burst accompanying the phagocytic event was manifested. The suppression of the respiratory burst appeared to be related to the concentration of cadmium. The possible alteration in the relationship between macrophage microtubule assembly and endocytosis is discussed.     

Attachment of Blastomyces dermatitidis conidia to murine bronchoalveolar macrophages: Characterization of binding the elicitation of respiratory burst

We studied the mechanisms of adherence of Blastomyces dermatitidis conidia to murine bronchoalveolar macrophages and the ability of the conidia to elicit an increase in macrophage O _inf2 ^sup- production, using an avirulent fungal strain. The number of cell associated conidia was counted by visual inspection of 2 hour macrophage monolayers incubated with conidia and O _inf2 ^sup- was measured by reduction of ferricytochrome c. Adherence of conidia to bronchoalveolar macrophages was time dependent and reached a plateau after 30 min (36±5%, 51±22%, and 36±17% macrophages with adherent conidia after 15, 30, and 60 min, respectively). Both Ca⁺² and Mg⁺² were required. The carbohydrates mannose, mannan, fucose, alpha-methylmannoside, beta-glucan, galactose, N-acetylglucosamine and chitotriose (100–1000 g/ml) did not inhibit adherence of conidia to macrophages. Trypsin treatment of macrophages or conidia did not affect binding. Conidia did not stimulate bronchoalveolar macrophage production of O _inf2 ^sup- above baseline concentrations (2.0±0.9 vs 0.8±0.5 nmol O _inf2 ^sup- , p>0.05). We conclude that murine bronchoalveolar macrophage-B. dermatitidis conidia interactions occur primarily by a non-lectin-like attachment and do not result in the production of macrophage derived O _inf2 ^sup- .     

Role of the maleyl-albumin receptor in activation of murine peritoneal macrophages in vitro

It has been previously demonstrated that maley-lated-BSA (maleyl-albumin) induces functional activation in murine peritoneal macrophages. Furthermore, maleyl-albumin has been shown to interact with two distinct sites on human monocytes; one site is the scavenger receptor, a 260-kDa oligomeric protein which recognizes modified forms of low density lipoprotein (LDL), and the second is a lower affinity site which has yet to be structurally characterized. In the present study, we wished to quantitatively assess the number and character of maleyl-albumin-binding sites on murine peritoneal macrophages and to determine which site or sites are involved in signaling the macrophage to undergo extensive functional development. Binding studies. demonstrate at least two distinct receptors for maleyl-albumin on murine peritoneal macrophages. Scatchard analyses of the binding isotherms reveal two sites characterized by dissociation constants (Kd) of 17.6 nM and 4.9 microM and maximal binding of 1.2 x 10(5) and 1 x 10(6) sites/cell, respectively. The contribution of the scavenger receptor, determined by binding analyses of malondialdehyde-LDL, is described by two sites with Kd of 39.4 pM and 9.6 nM, and maximal binding of 2.7 x 10(3) and 1.9 x 10(4) sites/cell, respectively. Maleyl-albumin blocks binding of malondialdehyde-LDL, whereas modified LDL fails to inhibit binding of maleyl-albumin. Maleyl-albumin, at concentrations producing lower affinity binding, stimulates tumor cytolysis, expression of mRNA encoding TNF, and suppression of INF-gamma-induced expression of Ia Ag. Malondialdehyde-LDL fails to elicit these responses. We conclude that macrophage activation produced by maleyl-albumin is mediated by interaction with the low affinity, high capacity binding site for maleyl-albumin rather than the scavenger receptor.     

Cyclosporin A inhibits phorbol ester-induced activation of superoxide production in resident mouse peritoneal macrophages.

Peritoneal resident macrophages from mice are sensitive to inhibition by cyclosporin A (CsA) of phorbol 12-myristate 13-acetate (PMA)-stimulated oxidative burst. Inhibition was assessed in terms of superoxide anion (O2.-) and H2O2 production. Key findings were as follows. (a) CsA inhibited in a dose-dependent manner the production of O2.- when cells were stimulated with PMA. CsA did not alter the respiratory burst induced by other stimuli (zymosan, concanavalin A and fMet-Leu-Phe). It was verified that CsA itself had no scavenger effect. (b) A concomitant decrease in H2O2 liberation following CsA exposure was found. This inhibition was observed both in the initial rate of synthesis and in the accumulation after 15 min of incubation. (c) NADPH oxidase activity in the crude supernatant was unaffected by the previous incubation of macrophages with CsA. CsA does not inhibit glucose transport measured as 14CO2 production. (d) The production of O2.- was strongly dependent on the glucose concentration. Sodium oleate also stimulated O2.- production in resident macrophages. These data might be correlated with the inhibitory effect of CsA upon other functions of macrophages.     

Stimulation of dopamine synthesis and activation of tyrosine hydroxylase by phorbol diesters in rat striatum

In rat striatal synaptosomes, 4 beta-phorbol 12-myristate 13-acetate (PMA) and 4 beta-phorbol 12,13-dibutyrate (PDBu), two activators of Ca2+-phospholipid-dependent protein kinase (protein kinase C) increased dopamine (DA) synthesis measured by following the release of 14CO2 from L-[1-14C] tyrosine. Maximal stimulation (21-28% increase of basal rate) was produced by 0.5 microM PMA and 1 microM PDBu. 4 beta-Phorbol and 4 beta-phorbol 13-acetate, which are not activators of protein kinase C, were ineffective at 1 microM. PMA did not change the release of 14CO2 from L-[1-14C]DOPA. Addition of 1 mM EGTA to a Ca2+-free incubation medium failed to affect PMA stimulation. KC1 (60 mM) enhanced DA synthesis by 25%. Exposure of synaptosomes to either PMA or PDBu prior to KC1 addition resulted in a more than additive increase (80-100%) of DA synthesis. A similar synergistic effect was observed when the phorbol diesters were combined with either veratridine or d-amphetamine but not with forskolin and dibutyryl cyclic AMP. Pretreatment of striatal synaptosomes with phorbol diesters produced an activation on of tyrosine hydroxylase (TH) associated with a 60% increase of the Vmax and a decrease of the Km for the pterine cofactor 6-methyl-5,6,7,8-tetrahydropterin. These results indicate that protein kinase C participates in the regulation of striatal TH in situ and that its activation may act synergistically with DA releasing agents in stimulating DA synthesis.     

Tumor-promoting phorbol esters inhibit the binding of colony-stimulating factor (CSF-1) to murine peritoneal exudate macrophages

L-cell colony-stimulating factor (CSF-1) is a sialoglycoprotein of molecular weight 70,000 daltons that specifically stimulates macrophage colony formation by single committed cells from normal mouse bone marrow and by various classes of more differentiated tissue-derived mononuclear phagocyte colony-forming cells (Stanley et al., 1978). CSF-1 interacts with target cells by direct and specific binding to membrane receptors (CSF-1 receptors) that are present only on cells of the mononuclear phagocyte series and their precursors. We studied the effect of tumor-promoting phorbol esters on the binding of 125I-labeled CSF-1 (125I-CSF-1) to murine peritoneal exudate macrophages (PEM). Biologically active TPA (12-O-tetradecanoyl phorbol-13-acetate) inhibits the binding of 125I-CSF-1 to its receptor on PEM. This inhibition exhibits temperature, time, and concentration dependence. At 37 degrees C, maximum inhibition occurred at about 10(-7) M; inhibition was 50% at 5 X 10(-9) M. At 0 degrees C, the inhibitory activity of TPA is diminished. The action of TPA on PEM is transient. Treated cells recover their 125I-CSF-1-binding activity whether TPA is later removed or not. The process of recovering CSF-1-binding activity is completely blocked by the addition of cycloheximide. When several phorbol derivatives were tested for their inhibitory activities, only biologically active phorbol esters were found to possess such activities. Furthermore, the inhibitory activities of various phorbol esters are proportional to their tumor-promoting activities. Inhibition appears to be due to a reduction in the total number of available CSF-1 receptors rather than a decrease in receptor affinity.     

Regulation of oxygen radical release from murine peritoneal macrophages by pharmacologic doses of PGE2

The ability of pharmacologic doses of PGE2 to alter the release of superoxide (O2-) and hydrogen peroxide (H2O2) from elicited peritoneal macrophages (M theta) was studied. Twice-daily administration of 200 or 100 micrograms of PGE2 to mice during accumulation of peritoneal M theta resulted in a significant reduction in M theta recovery and in the triggered release of H2O2, but not O2-. Cultivation of elicited M theta from normal mice with concentrations of PGE2 in excess of 10(-7) M for 24-48 h resulted in a significant reduction in the triggered release of H2O2, but not O2-. Cultivation for shorter periods of time or with lower concentrations of PGE2 failed to alter H2O2 release. This effect of PGE2 was reproduced by the phosphodiesterase inhibitor theophylline. The ability of PGE2 to inhibit H2O2 release in the presence of normal production of O2- was not prevented by the addition of superoxide dismutase. Cultivation of peritoneal M theta with 10(-5) M PGE2 for 48 h failed to increase intracellular catalase, although increased H2O2 scavenger activity was demonstrated. The inhibition of extracellular release of H2O2, but not O2-, by pharmacologic doses of PGE2 may be one mechanism for the anti-inflammatory action of this compound.     

Expression of a 120,000 dalton protein during tumoricidal activation in murine peritoneal macrophages

Modulation of protein expression during interferon-gamma (IFN-gamma)-lipopolysaccharide (LPS)-mediated macrophage tumoricidal activation has been examined by metabolic radiolabeling of various murine peritoneal macrophage populations with [35S]methionine followed by SDS-PAGE analysis. Although both IFN-gamma and LPS are capable of stimulating the expression of several proteins when used independently, combined treatment induced the enhanced or de novo expression of a 120,000 dalton polypeptide. The expression of this protein was synergistically regulated by both IFN-gamma and LPS in a manner strongly reminiscent of the functional synergism that these two agents exhibit with respect to induction of tumoricidal activity. p120 expression could be seen first at approximately 3 hr after the addition of both agents, reached optimal expression by 6 hr, and maintained elevated synthesis for up to 24 hr. This time course corresponds closely to that seen for the acquisition of tumoricidal competence. Macrophages elicited in the primed state of activity in vivo with methyl vinyl ether co-polymer II (MVE-II) did not express p120, but could be induced to do so when treated with low doses of LPS. Under similar conditions, MVE-II-elicited cells also acquire tumoricidal activity. Macrophages obtained from mice chronically infected with bacillus Calmette-Guerin constitutively expressed both p120 and cytolytic activity. If such macrophages were cultured for 24 hr, the expression of both events decayed and was lost, but could be restored by treatment with low doses of LPS. Thus the data support a strong correlation between the expression by macrophages of a novel 120,000 dalton protein and the expression of tumor cytotoxicity.     

Induction of metalloelastase mRNA in murine peritoneal macrophages by diethylmaleate

Macrophage-specific metalloelastase (MME) hydrolyzes elastin and other matrix proteins and plays an important physiological role in tissue remodeling and pathological tissue destruction. We have examined the effects of diethylmaleate (DEM), an electrophilic agent that reacts with sulfhydryls, on the expression of MME mRNA in mouse peritoneal macrophages. Quantification of MME mRNA by Northern blot analysis revealed that basal mRNA levels were quite low in freshly isolated cells, although mRNA levels increased markedly and reached a steady level within 12 h when cells were cultured in a serum-supplemented RPMI 1640 medium. When macrophages were challenged with DEM at 0.05-1.0 mM for 8 h the expression of the MME gene was enhanced further. In the presence of 0.1 mM DEM, the level of the MME mRNA increased 2-fold compared to the control levels after 6-9 h and decreased to control levels in 24 h. Other electrophilic agents, catechol and 1-chloro-2,4-dinitrobenzene, also enhanced MME gene expression. However, oxidative stress agents such as hydrogen peroxide, menadione, paraquat (an O-2 generator), sodium arsenite and cadmium chloride had no effect on MME gene expression. These results indicate that the electrophilic agents selectively enhance the expression of MME mRNA during primary culture of the macrophages.     

Effects of bacterial lipopolysaccharide on protein synthesis in murine peritoneal macrophages: relationship to activation for macrophage tumoricidal function

Early biochemical events in the response of murine peritoneal macrophages to bacterial lipopolysaccharide (LPS) have been examined (i.e., 0-4 hr after initiation of treatment). At concentrations of 10 ng/ml or less, LPS stimulated the new or enhanced synthesis of a series of at least six polypeptides of 85, 80, 75, 65, 57, and 38 kD. This effect was dependent upon the lipid A moiety of LPS as lipid A itself could induce the changes and the effect of LPS could be blocked by inclusion of polymixin B sulfate in the culture medium. The effect was specific for LPS in that other endotoxin-free agents known to alter macrophage physiology could not produce the same changes. The time course of LPS stimulation of macrophage protein synthesis was remarkable in that the synthesis of all six proteins was transient even in the continued presence of LPS, being first detected approximately 1 hr after exposure and no longer apparent by 8-10 hr after treatment was initiated. Furthermore, both pulse-chase and cumulative radiolabeling studies indicated that at least two of the proteins (85 and 38 kD) were short-lived and did not accumulate in LPS-treated cells, suggesting the possibility that they participate in a regulatory rather than a functional role. Macrophage tumoricidal activation involves cooperation in response to two independent signals; interferon gamma and LPS. Pretreatment of macrophages with interferon gamma increased the sensitivity of macrophages to LPS-stimulated protein synthesis by one to two orders of magnitude documenting such cooperativity in molecular terms. The LPS-induced stimulation of specific protein synthesis could be reproduced by treatment of macrophages with heat killed Listeria monocytogenes, a gram-positive, endotoxin-negative bacterial stain which has been shown to substitute effectively for LPS in macrophage tumoricidal activation. Furthermore, reversible inhibition (i.e., treatment with cycloheximide) of protein synthesis during LPS treatment abrogated the acquisition of tumoricidal function. These results identify an early biochemical response to LPS which may be a necessary component of the intracellular transduction of signals which regulate macrophage functional development.