Thermal injury-induced enhancement of oxidative metabolism by mononuclear phagocytes.

C57BL/6 female mice were subjected to full-thickness scald thermal injuries covering 25% of their total body surface areas, and thioglycollate-elicited peritoneal macrophages were isolated 4 days later. Macrophages from burned mice displayed a 2-fold increase in phorbol ester-stimulated superoxide production, whereas superoxide production in response to zymosan or immune complexes was unaltered. Maximal levels of superoxide production by thermal injury macrophages were comparable for all stimuli. Reduction of injury size to 12.5% total body surface area abrogated the enhancement of superoxide production. Protein kinase C activity and protein phosphorylation was not altered after thermal injury. These results indicate that the enhancement of macrophage-oxidative metabolism after thermal injury is independent of changes in reduced nicotinamide adenine dinucleotide phosphate oxidase or protein kinase C activity and may in part explain the increased susceptibility to multiple organ failure after this form of trauma.

Thermal injury induces macrophage hyperactivity through pertussis toxin-sensitive and -insensitive pathways.

C57BL/6 mice were subjected to a full thickness scald thermal injury covering 25% of their total body surface area, and thioglycollate elicited peritoneal macrophages (Mphi were isolated 4 days later. Mphi from injured mice produced significantly greater amounts of reactive nitrogen intermediates and tumor necrosis factor-alpha in response to lipopolysaccharide and lipid A. Pertussis toxin (PTX) treatment of Mphi dose-dependently inhibited reactive nitrogen intermediate production in Mphi from sham-treated mice; however, Mphi from injured mice were insensitive to PTX-mediated inhibition. Conversely, tumor necrosis factor-alpha production was enhanced by PTX treatment, with Mphi from injured mice being more sensitive than Mphi from sham-treated mice to this effect of PTX. These results indicate that thermal injury increases Mphi sensitivity to lipopolysaccharide by a mechanism that is both PTX sensitive and PTX insensitive, thereby suggesting a role for G proteins in the modulation of Mphi activity after thermal injury.

Thermal injury-induced immunosuppression in mice: the role of macrophage-derived reactive nitrogen intermediates.

Macrophages (Mphi) have been implicated in the suppression of lymphocyte function following thermal injury. Splenocytes isolated from C57BL/ 6NCR female mice 4-7 days after thermal injury displayed suppressed proliferative responses to Concanavalin A (ConA) and lipopolysaccharide (LPS) and high levels of reactive nitrogen intermediate (RNI) production. Inhibition of nitric oxide synthase activity with NG-monomethyl-L-arginine restored ConA responses but not LPS responses. Surprisingly, ConA-stimulated interferon-gamma (IFN-gamma) production was increased in splenocytes from injured mice. IFN-gamma contributed to the RNI-mediated immunosuppression as antibodies against IFN-gamma reduced RNI production and immunosuppression. ConA-stimulated co-cultures of splenic Mphi from injured mice and normal splenocytes produced high levels of RNI only under conditions of cellular contact and splenic Mphi from injured mice were capable of suppressing normal splenocytes responses in co-culture. These results indicate that Mphi activity and specifically RNI production contribute to the suppression of T lymphocyte function after thermal injury.

Alteration of tumor necrosis factor-alpha production by macrophages from mice fed diets high in eicosapentaenoic and docosahexaenoic fatty acids.

Dietary exposure to n-3 fats found in marine fish oils are known to reduce certain inflammatory conditions. Although depressed prostaglandin E2 (PGE2) production is thought to be a major mechanism of the beneficial effects, the direct effects of n-3 fatty acids on inflammatory macrophage function are not well understood. In this study, production of the inflammatory monokine, tumor necrosis factor-alpha (TNF alpha), by isolated murine macrophages was assessed following a 3-week feeding with diets containing either 10% menhaden fish oil as a source of n-3 fatty acids or, as a control and source of n-6 fatty acids, 10% safflower oil. Cultures of peritoneal macrophages from mice fed diets with n-3 fatty acids had more TNF alpha activity 24 hr after in vitro stimulation with bacterial lipopolysaccharide than did macrophages from mice fed the n-6-containing diet. The onset and maximal synthesis of bioactive TNF alpha and down-regulation of messenger RNA for TNF alpha appeared to be similar for the two diets, suggesting that macrophages from mice fed a diet high in n-6 but not n-3 fatty acids were capable of removing active TNF alpha from culture media. Experiments in which PGE2 was added exogenously indicated that the removal of TNF alpha from culture supernatant by macrophages was induced by lower concentrations of PGE2 than that associated with termination of production, and that n-3 fatty acid diets caused a selective loss in the clearance mechanism. These results demonstrate a specific alteration of PGE2-mediated regulation of macrophage-produced TNF alpha by n-3 fatty acids.

Regulation of murine macrophage function by IL-4: IL-4 and IFN-gamma differentially regulate macrophage tumoricidal activation.

To understand the differential role of interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) in the process of macrophage tumoricidal activation, we investigated the production of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide in activated murine macrophages and the effects of those lymphokines on prostaglandin E2 (PGE2)-mediated down-regulation. IFN-gamma and IL-4 increased lipopolysaccharide (LPS)-induced TNF-alpha production by different mechanisms because IL-4, unlike IFN-gamma, failed to overcome the LPS-hyporesponsiveness in C3H/HeJ mice. Moreover, only IFN-gamma synergized with LPS to induce nitric oxide production and blocked eicosanoid-mediated down-regulation. These differential effects of IFN-gamma and IL-4 on the select efferent cytolytic activities may be the result of an altered or different signal transduction pathway. Because potentiation of protein kinase C (PKC) activity by IFN-gamma has been previously documented, we next studied the role of IFN-gamma and IL-4 in alteration of enzymatic activity of PKC. Two lymphokines caused translocation of PKC from cytosol to membrane with different levels, providing a biochemical basis for explaining how two lymphokines lead to different phenotypic responses. Although treatment of macrophages with IFN-gamma and IL-4 gave rise to a similar enhancing effect on macrophage TNF-alpha production, these two lymphokines appeared to differentially regulate the overall functional state of macrophages for tumour cell killing capability. Additionally, this differential regulation seems to be accomplished in part by different biochemical events.

Effects of in vitro exposure to arachidonic acid on TNF-alpha production by murine peritoneal macrophages.

Modifying the fatty acid composition of macrophages through diet can significantly alter some of their functions, such as tumoricidal capacity and tumor necrosis factor alpha (TNF-alpha) production. The mechanism of that modification, however, is unknown. In this report, we provide evidence that fatty acids added to macrophages in culture can significantly alter macrophage TNF-alpha production. For example when inflammatory macrophages were incubated with various doses of arachidonic acid [20:4(n-6)] during activation with lipopolysaccharide (LPS), we observed a dose-dependent decrease in the level of bioactive TNF-alpha with complete inhibition at 2-5 microM. This inhibition was specific for 20:4(n-6) because in vitro treatment with other fatty acids, such as eicosapentaenoic [20:5(n-3)] or docosahexaenoic [22:6(n-3)] acids, had differential effects. The inhibitory action of 20:4(n-6) did not involve toxicity because cell viability was not affected and in vitro interferon-gamma and lipopolysaccharide (LPS) activation of macrophages for killing of P815 tumor targets was not altered. Inhibition by 20:4(n-6) occurred posttranscriptionally, and could be reversed when macrophages were treated with indomethacin during activation. Arachidonic acid treatment also significantly increased the production of immunoreactive prostaglandin E2 (PGE2) by LPS-treated and untreated macrophages. These results suggest that in vitro treatment of macrophages with 20:4(n-6) may inhibit TNF-alpha production through an alteration in the levels of PGE2 at a posttranscriptional level. These results provide evidence that some dietary fats may affect macrophage activity through modification of eicosanoid synthesis.

Down-regulation of chemotaxis of polymorphonuclear leukocytes following thermal injury involves two distinct mechanisms.

Thermal injury induces a depression of major effector functions of polymorphonuclear leukocytes (PMNL) that contributes to the increased susceptibility to bacterial infection associated with severe injury. In a study on chemotactic alterations in PMNL induced by thermal injury in a well-characterized guinea pig model, a concomitant reduction in the chemotactic response of PMNL to zymosan-activated serum (ZAS) and FMLP was seen early after thermal injury in temporal association with the previously reported bactericidal defect and depression of superoxide anion production. Unlike the bactericidal defect, the chemotactic alterations were not directly linked to the marked elevation of intracellular cAMP in PMNL associated with thermal injury. Two mechanisms (adaptation and desensitization) were shown to be involved in the reduction of chemotactic responses of PMNL to FMLP and ZAS, respectively. Adaptation appears to be a protective response of PMNL to thermal injury unassociated with receptor down-regulation.

Circulating factors contribute to elevation of intracellular cyclic-3',5'-adenosine monophosphate and depression of superoxide anion production in polymorphonuclear leukocytes following thermal injury.

We have previously demonstrated that bactericidal activity and superoxide anion (O2-) production are depressed concomitantly in polymorphonuclear leukocytes (PMNs) following thermal injury in a guinea pig model, and the bactericidal defect is related to elevation of intracellular cyclic-3',5'-adenosine monophosphate (cAMP). The purpose of the present investigation was to determine the relationship between elevation of intracellular cAMP and depression of O2- production in PMNs following thermal injury and determine the involvement of circulating factors in the development of these alterations. The kinetics of O2- production and dose responses to formylmethionyl-leucyl-phenylalanine (fMLP) and phorbol myristate acetate (PMA) were depressed in peripheral PMNs following thermal injury in this experimental model. Sera obtained during the period of PMN dysfunction induced depression of O2- production in response to fMLP and elevation of intracellular cAMP in normal PMNs. Pretreatment of normal PMNs with nonsteroidal anti-inflammatory drugs (NSAID; indomethacin or piroxicam) inhibited the elevation of intracellular cAMP mediated by sera from the injured animals but had no effect on the depression of O2- production observed under similar conditions. Treatment of PMNs from injured animals with NSAID under conditions known to reduce the cAMP content of the cells and correct the bactericidal defect did not normalize O2- production. Studies utilizing sera from two thermally injured patients confirmed findings in the guinea pig model of serum-mediated elevation of intracellular cAMP and depression of O2- production in normal PMNs and effects observed with NSAID. These results suggest that circulating factors contribute to the elevation of intracellular cAMP and depression of O2- production in PMNs following thermal injury. Whereas the increase in intracellular cAMP may be involved in the depression of O2- production, our results suggest that there is not a direct link between these alterations.

Effect of dietary fish oil on development and selected functions of murine inflammatory macrophages.

Inflammatory macrophages from mice fed diets containing menhaden fish oil (MFO) have a reduced capacity for cytotoxicity of mastocytoma cells upon activation with interferon-gamma (IFN gamma) and lipopolysaccharide due to an altered responsiveness to IFN gamma. In an effort to elucidate further how dietary MFO effects macrophage function, we have studied the maturation of inflammatory macrophages from mice fed MFO compared with mice fed safflower oil (SFO) using several processes that serve as markers of the activational state. No significant differences in the recruitment or percentage of peritoneal exudate cells as macrophages after thioglycollate injection and no differences in spreading, binding, or phagocytosis of sheep erythrocytes or phagocytosis of yeast by inflammatory macrophages were observed when the dietary groups were compared. However, MFO macrophages had an altered capacity for peroxide release when stimulated with unopsonized zymosan (10-200 micrograms/ml). Furthermore, to elucidate how MFO feeding could alter IFN gamma-induced responses of inflammatory macrophages, we assessed phorbol-12-myristate-13-acetate-induced hydrogen peroxide production and expression of class II MHC determinants (Ia). There were no differences between macrophages from mice fed the two diets with respect to the production of peroxide when they were preincubated with 0.1-10 U/ml of IFN gamma. However, MFO macrophages had greater peroxide production after enhancement with 100 U/ml of IFN gamma. With respect to Ia induction, the percentage of macrophages responding to IFN gamma was not altered by diet, and there were no differences in expression of Ia induced by 24 hr exposure to IFN gamma. Thus the differential effect of MFO compared with SFO is probably mediated not by an alteration in the maturation of inflammatory macrophages but rather through the alteration of IFN gamma-induced functions such as peroxide production.

Alteration of in vitro murine peritoneal macrophage function by dietary enrichment with eicosapentaenoic and docosahexaenoic acids in menhaden fish oil.

The effects of diets containing menhaden fish oil (MFO), compared with those of diets containing safflower oil (SAF) or an essential fatty acid deficient hydrogenated coconut oil (HCO), on in vitro activation of tumoricidal capacity by murine macrophages were assessed. Mice fed the experimental diets for 4 weeks were injected intraperitoneally with sterile thioglycollate broth 3 days before use. There was no difference between any of the groups with respect to total peritoneal exudate cells or the percentage of macrophages, although the fatty acid profile of purified adherent macrophages closely paralleled that of the diets. Macrophages from mice fed MFO killed fewer P815 mastocytoma cells upon activation with recombinant interferon gamma (IFN gamma) and lipopolysaccharide. Macrophages from all diets were equally competent for tumoricidal capacity when activated pharmacologically with calcium ionophore, phorbol 12-myristate 13-acetate, and lipopolysaccharide (LPS), suggesting that MFO diet macrophages were hyporesponsive to IFN gamma. Priming with higher concentrations of IFN gamma restored the partial defect in activation of MFO macrophages. When activated for 24 hr with high levels of LPS, macrophages from mice fed SAF displayed little cytolytic capacity; addition of indomethacin. (1 microM) resulted in enhanced levels of P815 kill. In contrast, MFO and HCO diet macrophages were highly cytolytic with similar LPS treatment with or without indomethacin. Macrophages from mice fed SAF produced threefold more prostaglandin E in response to LPS than did MFO and HCO diet macrophages. These results suggest that dietary manipulation of fatty acids can alter activation of tumoricidal capacity of macrophages, possibly both dependent and independent of changes in eicosanoid synthesis.

Regulation of murine macrophage function by IL-4. I. Activation of macrophages by a T-T cell hybridoma is due to IL-4.

A T-cell hybridoma produced by fusion of concanavalin A-stimulated murine splenocytes produced a factor (MAFH) capable of activating tumoricidal capacity by responsive murine peritoneal macrophages. Macrophages treated with the MAFH required an additional trigger signal of bacterial lipopolysaccharide (LPS) for maximal activity. In contrast to interferon-gamma (IFN gamma), which induced tumoricidal activity against all tumor cells tested, MAFH only induced macrophage-mediated kill of the BI6P51 and 168 lines, and not of the P815 or B16BL6 lines. An identical pattern of tumoricidal activity was obtained by treating macrophages with recombinant interleukin-4 (IL-4). The active moiety of MAFH appeared to be IL-4 as (i) monoclonal antibody against IL-4 blocked MAFH, but not IFN gamma, activity, and (ii) the T-cell hybridoma contained large amounts of mRNA for IL-4 and no detectable mRNA for IFN gamma (as determined by Northern dot analysis). The pattern of tumoricidal activity observed may be due to an IL-4 mediated enhancement of tumor necrosis factor production by LPS-triggered macrophages.

Dietary manipulation of macrophage phospholipid classes: selective increase of dihomogammalinolenic acid.

Because alterations in the dietary content of fatty acids are an important method for modulating macrophage eicosanoid production, we have quantitated the levels of n-6 and n-3 polyunsaturated fatty acids in peritoneal macrophage individual phospholipids from mice fed diets (3 wk) with either safflower oil (SAF), predominantly containing 18:2n-6, borage (BOR) containing 18:2n-6 and 18:3n-6, fish (MFO) containing 20:5n-3 and 22:6n-3, and borage/fish mixture (MIX) containing 18:2n-6, 18:3n-6, 20:5n-3 and 22:6n-3. Dietary n-3 fatty acids were readily incorporated into macrophage phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylinositol (PI). The increase in n-3 fatty acid levels was accompanied by a decrease in the absolute levels of 18:2n-6, 20:4n-6 and 22:4n-6 in PC, PE and PS. Interestingly, PI 20:4n-6 levels were not significantly lowered (P greater than 0.05) in MIX and MFO macrophages relative to SAF and BOR. These data demonstrate the unique ability of this phospholipid to selectively maintain its 20:4n-6 levels. In BOR and MIX animals, 20:3n-6 levels were significantly increased (P less than 0.05) in all phospholipids relative to SAF and MFO. The combination of borage and fish oils (MIX diet) produced the highest 20:3n-6/20:4n-6 ratio in all phospholipids. These data show that the macrophage eicosanoid precursor levels of 20:3n-6, 20:4n-6 and n-3 acids can be selectively manipulated through the use of specific dietary regimens. This is noteworthy because an increase in phospholipid levels of 20:3n-6 and 20:5n-3, while concomitantly reducing 20:4n-6, may have therapeutic potential in treating inflammatory disorders.

Ability of 15-hydroxyeicosatrienoic acid (15-OH-20:3) to modulate macrophage arachidonic acid metabolism.

Mouse peritoneal macrophages metabolize dihomogammalinolenic acid (20:3n-6) primarily to 15-hydroxy-8,11,13-eicosatrienoic acid (15-OH-20:3). Since the biological properties of this novel trienoic eicosanoid remain poorly defined, the effects of increasing concentrations of 15-OH-20:3 and its arachidonic acid (20:4n-6) derived analogue. 15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE), on mouse macrophage 20:4n-6 metabolism were investigated. Resident peritoneal macrophages were prelabeled with [3H]-20:4n-6 and subsequently stimulated with zymosan in the presence of either 15-OH-20:3 or 15-HETE (1-30 microM). After 1 hr, the radiolabeled soluble metabolites were analyzed by reverse phase high performance liquid chromatography. 15-OH-20:3 inhibited zymosan-induced leukotriene C4 (IC50 = 2.4 microM) and 5-HETE (IC50 = 3.1 microM) synthesis. In contrast to the inhibition of macrophage 5-lipoxygenase, 15-OH-20:3 enhanced 12-HETE synthesis (5-30 microM) and had no measurable effect on cyclooxygenase metabolism (1-10 microM) i.e., 6-keto-prostaglandin F1 alpha and prostaglandin E2 synthesis. Addition of exogenous 15-HETE produced similar effects. These results suggest that the manipulation of macrophage 15-OH-20:3n-6 levels may provide a measure of cellular control over 20:4n-6 metabolism, specifically, leukotriene production.

Fatty acid composition of macrophage phospholipids in mice fed fish or borage oil.

The polyunsaturated fatty acid (PUFA) composition of murine peritoneal macrophage phospholipids was dramatically altered in vivo following the four-wk feeding of specific dietary oils. Fish oil (containing 20:5n-3 and 22:6n-3) feeding significantly increased macrophage 20:5n-3, 22:5n-3, and 22:6n-3 (P less than 0.05), while borage oil (containing 18:2n-6 and 18:3n-6) increased (P less than 0.05) the macrophage 20:3n-6/20:4n-6 ratio, relative to safflower oil (containing 18:2n-6) and hydrogenated coconut oil (containing 12:0)-fed animals. The macrophage phospholipid PUFA profiles were compared with those of the liver, lung and spleen. The significance of the PUFA alterations is discussed.

Utilization of dihomo-gamma-linolenic acid (8,11,14-eicosatrienoic acid) by murine peritoneal macrophages.

This study examined the metabolism of dihomo-gamma-linolenic acid (20:3(n-6] in casein-elicited murine peritoneal macrophages. Cells were incubated with [14C]20:3(n-6) in the presence of 1% fetal bovine serum (FBS) or 0.025% bovine serum albumin (BSA), and the distribution and identity of membrane-bound and soluble products were determined. Approx. 70-80% of the [14C]20:3(n-6) was recovered in membrane phospholipids. The distribution of radiolabel in individual cellular phospholipids revealed a time-dependent (6 vs. 16 h) increase in the percentage of radiolabel esterified to phosphatidylethanolamine (PE). Analysis of cellular total lipids following transmethylation indicated that approx. 4, 2 and 9% of the incorporated 20:3(n-6), respectively, had been desaturated and elongated into 20:4(n-6), 22:4(n-6) and 22:3(n-6). When cells prelabeled for 16 h were incubated in the presence of the divalent cation ionophore, A23187, or zymosan for 30-60 min, two radiolabeled metabolites were isolated in the incubation supernatant. These metabolites were identified as 12-hydroxy-8,10,14- and 15-hydroxy-8,11,13-eicosatrienoic acids, as determined by reverse-phase and normal-phase high-performance liquid chromatography. The generation of monohydroxy fatty acids was notably absent in prelabeled quiescent cells and A23187-stimulated cells incubated with BW755C, a dual cyclooxygenase and lipoxygenase inhibitor. We conclude that casein-elicited murine peritoneal macrophages can extensively metabolize 20:3(n-6) through delta 5-desaturase, elongase and oxygenation reactions.

Inability of murine peritoneal macrophages to convert linoleic acid into arachidonic acid. Evidence of chain elongation.

Various murine macrophage populations synthesize and secrete large amounts of arachidonic acid (20:4n-6) derived eicosanoids (cyclo-oxygenase and lipoxygenase products). These metabolites are known to possess a wide variety of functions with regard to the initiation and regulation of inflammation and tumorigenesis. Because the dietary intake of 20:4n-6 is usually low, tissues are largely dependent upon dietary linoleic acid (18:2n-6) as an initial unsaturated precursor for the biosynthesis of 20:4n-6. The purpose of these experiments was to determine whether resident or responsive murine macrophages possess desaturase and elongase activities capable of in vitro conversion of 18:2n-6 into 20:4n-6. Peritoneal exudate macrophages were purified by adherence and incubated in serum-free medium containing fatty acid-free BSA with [1-14C] 18:2n-6. Approximately 90 to 98% of the [14C]18:2n-6 at 4 and 16 h was recovered in phosphatidylcholine and phosphatidylethanolamine. The metabolism of [14C]18:2n-6 was determined after transesterification and separation of the 14C-fatty acid methyl esters by argentation TLC, reverse phase HPLC, and electron impact gas chromatography/mass spectrometry. Resident and responsive macrophages lacked the capacity to transform [14C]18:2n-6 into 20:4n-6. In addition, prelabeled macrophages incubated with soluble, calcium ionophore A23187 or phorbol myristate, or particulate, zymosan, membrane perturbing agents also lacked delta 6 desaturase activity. All macrophages tested were capable of elongating [14C]18:2n-6 into [14C]20:2n-6. These observations suggest that 20:4n-6, present in macrophage phospholipids, is biosynthesized elsewhere and transported to the macrophage for esterification into the phospholipids. In addition, these findings demonstrate that elongase activity is present in both the resident and responsive peritoneal macrophage.

Maleylated bovine serum albumin triggers cytolytic function in selected populations of primed murine macrophages.

The complex algal polysaccharide fucoidan has been reported as serving as a second signal for activation of macrophages primed in vivo by BCG. To assess the potential utility of this observation in analyzing biochemical mechanisms involved in macrophage activation, we examined the triggering effects of maleylated bovine serum albumin (maleylated-BSA), a defined molecule that clears via similar mechanisms. Cytolysis of P815 mastocytoma targets was triggered by maleylated-BSA, in a dose-dependent manner, in murine peritoneal macrophages primed in vivo by BCG. Unlike bacterial LPS, which triggered cytolysis when used to pretreat the macrophages, maleylated-BSA was only effective if present throughout the period of macrophage-target cytolytic interaction. Maleylated-BSA alone did not lyse the P815 targets and did not affect the binding of such targets by macrophages. Maleylated-BSA was equally effective in triggering cytolysis in BCG-primed macrophages from C3H/HeJ or C3H/HeN mice. Macrophages primed in vitro with IFN-gamma, however, could not be triggered by maleylated-BSA, even though these macrophages bound maleylated-BSA comparably to the BCG-primed macrophages. When responsive macrophages were fully activated in vitro by IFN-gamma and LPS and then allowed to decay to the primed state, maleylated-BSA was then as effective as LPS in triggering cytolysis. Taken together, the results indicate that maleylated-BSA can trigger cytolysis in certain populations of primed macrophages but not in others.

Effects of bacterial lipopolysaccharide on the hydrolysis of phosphatidylinositol-4,5-bisphosphate in murine peritoneal macrophages.

LPS and lipid A initiated enhanced hydrolysis of PIP2 in macrophages. When murine peritoneal macrophages were labeled with [2-3H]myoinositol and stimulated with either LPS or lipid A, a rapid (within 10 sec) rise in Ins(1,4,5)P3 was observed. The breakdown pattern of Ins(1,4,5)P3 was complex; this included breakdown of Ins(1,4,5)P3 and formation of Ins(1,3,4,5)P4 (approximately 10 to 30 sec), and ultimately formation of Ins(1,3,4)P3 (approximately 60 sec). Within 10 sec after treatment, LPS caused an average increase of about fourfold to fivefold in Ins(1,4,5)P3, which declined over 5 min. When the total isomers of InsP3 were measured, levels rose about twofold in response to LPS or to lipid A and remained elevated for as long as 5 min. Lipid A, in the concentration range of 0.1 to 10 micrograms/ml, induced elevated intracellular levels of Ca2+ as quantified by fluorescence with Quin 2 or with Fura 2. When single, adherent Fura 2-loaded macrophages were treated with lipid A, basal levels of calcium rose over 10 sec from approximately 55 nM to almost 600 nM. LPS, paradoxically, did not cause such substantial increases in intracellular calcium (i.e., increases of approximately 26 nM) when judged by Fura 2 fluorescence. LPS treatment led to enhanced phosphorylation of a characteristic set of proteins, similar to those induced by stimulating protein kinase C (PKC) with phorbol myristate acetate as previously reported. The enhanced phosphorylation of pp28, pp33, and pp67 in macrophages was evident by 15 min and optimal by 30 min. Taken together, these observations indicate that LPS and lipid A cause increased breakdown of phosphatidylinositol 4,5-bisphosphate, which led to enhanced intracellular levels of calcium and also to enhanced protein phosphorylation, presumably mediated by PKC. The data thus suggest that one major intracellular signal transduction mechanism, initiated by LPS and lipid A in macrophages, is the rapid breakdown of PIP2.

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.

Maleyl-BSA and fucoidan induce expression of a set of early proteins in murine mononuclear phagocytes.

We examined the effect of maleyl-BSA on specific protein expression in murine peritoneal macrophages by radiolabeling treated macrophages with [35S]methionine followed by SDS-polyacrylamide gel electrophoresis. Such treatment induces the expression of a set of at least seven proteins (38, 42, 57, 65, 75, 80, and 85 kD). A similar set of proteins is also induced by treatment of macrophages with the algal polysaccharide fucoidan. The proteins resemble those induced in response to treatment of this same cell population with bacterial lipopolysaccharide (LPS), as judged by co-migration in both one- and two-dimensional electrophoresis. Two proteins induced by either LPS or maleyl-BSA (e.g., p57 and p85) show similar primary structure, as assessed by partial proteolytic peptide mapping confirming their identity. The induction of these proteins by maleyl-BSA is a transient phenomenon, being expressed as early as 1 hr after treatment and declining after 8 hr even in the continuous presence of the stimulus. The dose of maleyl-BSA required to induce the response varies to some extent with the protein in question, but agrees with the Kd for ligand-receptor binding. Chloroquine, which blocks the degradation of ligand, does not inhibit the induction of early protein synthesis. Whereas the induction of these proteins is blocked by inhibition of RNA synthesis with actinomycin D, the reversible inhibition of protein synthesis with cycloheximide during the induction phase does not prevent their expression. LPS, maleyl-BSA, and fucoidan previously have been shown to stimulate protease secretion and tumoricidal function in appropriately primed macrophages. The present findings now demonstrate that all three agents can also mediate the expression of early genes which may participate in the acquisition of functional competence.