The exposure of murine macrophages to alpha 2-macroglobulin 'fast' forms results in the rapid secretion of eicosanoids.

The exposure of [3H]arachidonate-radiolabelled murine peritoneal macrophages to alpha 2-macroglobulin-methylamine or alpha 2-macroglobulin-trypsin but not native alpha 2-macroglobulin (alpha 2M) results in the rapid secretion of [3H]eicosanoids. Resident peritoneal macrophages stimulated with 0.1 microM alpha 2M-methylamine exhibited an enhanced secretion within 10 min. The ability of alpha 2M 'fast' forms to stimulate secretion of [3H]eicosanoids was similar to that observed in the presence of the murine macrophage chemoattractant platelet-activating factor. As observed for total [3H]eicosanoid secretion, alpha 2M 'fast' forms also rapidly enhanced the secretion of the cAMP-elevating prostanoid, prostaglandin E2, from resident peritoneal macrophages. Stimulated secretion of prostaglandin E2 in response to 0.1 microM alpha 2M-methylamine was less rapid than that observed using 0.1 microM platelet-activating factor. Similar amounts of secreted prostaglandin E2 were present in media of macrophage cultures after 1 h exposure to the two stimuli. In the presence of 0.1 microM alpha 2M-methylamine, secreted prostaglandin E2 remained elevated, compared to the appropriate buffer control, for at least 24 h. The present results indicate that receptor recognition of alpha 2M 'fast' forms by macrophages results in the rapid stimulation of eicosanoid secretion and suggest that secretion of prostaglandin E2 and other eicosanoids may be involved in the ability of alpha 2 M 'fast' forms to regulate various macrophage functional responses.     

α2-macroglobulin ‘fast’ forms inhibit superoxide production by activated macrophages

Mouse peritoneal macrophages activated by bacillus Calmette-Guerin (BCG) were incubated with human α₂-macroglobulin converted to its ‘fast’ form with either trypsin or methylamine before being stimulated with phorbol myrystate acetate. Both α₂-macroglobulin-trypsin and α₂-macroglobulin-methylamine inhibited macrophage production of superoxide anion (O₂⁻) while native α₂-macroglobulin had little effect except at high concentration. The α₂-macroglobulin ‘fast’ forms, which bind with a K_d of about 8 nM, inhibited 50% generation of O₂⁻(ID₅₀) at a concentration of 7 nM while α₂-macroglobulin inhibited O₂⁻ production with an ID₅₀ of 141 nM. The ‘fast’ forms of α₂-macroglobulin may play a role in the feedback regulation of inflammatory reactions.     

Chitinous materials inhibit nitric oxide production by activated RAW 264.7 macrophages

Chitinous materials have been studied in wound healing and artificial skin substitutes for many years. Nitric oxide (NO) has been shown to contribute to cytotoxicity in cell proliferation during inflammation of wound healing. In this study, we examined the effect of chitin and its derivatives on NO production by activated RAW 264.7 macrophages. Chitin and chitosan showed a significantly inhibitory effect on NO production by the activated macrophages. Hexa-N-acetylchitohexaose and penta-N-acetylchitopentaose also inhibited NO production but with less potency. However, N-acetylchitotetraose, -triose, -biose, and monomer of chitin, N-acetylglucosamine and glucosamine had little effect on NO production by the activated cells. These results suggest that the promotive effect of chitinous material on wound healing be related, at least partly, to inhibit NO production by the activated macrophages.     

Kinetic analysis of superoxide anion production by activated and resident murine peritoneal macrophages

Using a continuous spectrophotometric assay, we have monitored the formation of superoxide anion (O₂^?) by activated and resident murine peritoneal macrophages. Macrophages elicited by injection with Corynebacterium parvum, as well as resident macrophages from untreated mice, were kept in suspension culture overnight to eliminate short-lived, contaminating neutrophils. Cytochemical analysis of the cultured macrophages disclosed that essentially all of the activated macrophages reduced nitroblue tetrazolium (NBT) dye vigorously. In contrast, only 18% of the resident macrophages demonstrated vigorous NBT reduction; the remainder of the resident macrophages reduced NBT very weakly. Kinetic analysis of macrophage O₂^? formation revealed that activated macrophages exposed to phorbol myristate acetate (PMA) produced O₂^? at a 13-fold greater maximum rate than resident macrophages. The decline in the rate of O₂^? production with time by activated macrophages was also greater than that of resident macrophages. The data indicate that the greater O₂^? production by activated macrophage populations is due to (i) the presence of an increased percentage of macrophages that respond to PMA with vigorous O₂^? production, and (ii) an increased maximum rate of O₂^? formation by these macrophages.     

Probing the stability of native and activated forms of alpha2-macroglobulin

alpha2-Macroglobulin (alpha2M) is a 718 kDa homotetrameric proteinase inhibitor which undergoes a large conformational change upon activation. This conformational change can occur either by proteolytic attack on an approximately 40 amino acid stretch, the bait region, which results in the rupture of the four thioester bonds in alpha2M, or by direct nucleophilic attack on these thioesters by primary amines. Amine activation circumvents both bait region cleavage and protein incorporation, which occurs by proteolytic activation. These different activation methods allow for examination of the roles bait region cleavage and thioester rupture play in alpha2M stability. Differential scanning calorimetry and urea gel electrophoresis demonstrate that both bait region cleavage and covalent incorporation of protein ligands in the thioester pocket play critical roles in the stability of alpha2M complexes.     

Nitroarachidonic acid prevents NADPH oxidase assembly and superoxide radical production in activated macrophages

Nitration of arachidonic acid (AA) to nitroarachidonic acid (AANO₂) leads to anti-inflammatory intracellular activities during macrophage activation. However, less is known about the capacity of AANO₂ to regulate the production of reactive oxygen species under proinflammatory conditions. One of the immediate responses upon macrophage activation involves the production of superoxide radical (O₂^•−) due to the NADPH-dependent univalent reduction of oxygen to O₂^•− by the phagocytic NADPH oxidase isoform (NOX2), the activity of NOX2 being the main source of O₂^•− in monocytes/macrophages. Because the NOX2 and AA pathways are connected, we propose that AANO₂ can modulate macrophage activation by inhibiting O₂^•− formation by NOX2. When macrophages were activated in the presence of AANO₂, a significant inhibition of NOX2 activity was observed as evaluated by cytochrome c reduction, luminol chemiluminescence, Amplex red fluorescence, and flow cytometry; this process also occurs under physiological mimic conditions within the phagosomes. AANO₂ decreased O₂^•− production in a dose- (IC₅₀=4.1±1.8 μM AANO₂) and time-dependent manner. The observed inhibition was not due to a decreased phosphorylation of the cytosolic subunits (e.g., p40^phox and p47^phox), as analyzed by immunoprecipitation and Western blot. However, a reduction in the migration to the membrane of p47^phox was obtained, suggesting that the protective actions involve the prevention of the correct assembly of the active enzyme in the membrane. Finally, the observed in vitro effects were confirmed in an in vivo inflammatory model, in which subcutaneous injection of AANO₂ was able to decrease NOX2 activity in macrophages from thioglycolate-treated mice.     

IL-10 inhibits cytokine production by activated macrophages

IL-10 inhibits the ability of macrophage but not B cell APC to stimulate cytokine synthesis by Th1 T cell clones. In this study we have examined the direct effects of IL-10 on both macrophage cell lines and normal peritoneal macrophages. LPS (or LPS and IFN-gamma)-induced production of IL-1, IL-6, and TNF-alpha proteins was significantly inhibited by IL-10 in two macrophage cell lines. Furthermore, IL-10 appears to be a more potent inhibitor of monokine synthesis than IL-4 when added at similar concentrations. LPS or LPS- and IFN-gamma-induced expression of IL-1 alpha, IL-6, or TNF-alpha mRNA was also inhibited by IL-10 as shown by semiquantitative polymerase chain reaction or Northern blot analysis. Inhibition of LPS-induced IL-6 secretion by IL-10 was less marked in FACS-purified peritoneal macrophages than in the macrophage cell lines. However, IL-6 production by peritoneal macrophages was enhanced by addition of anti-IL-10 antibodies, implying the presence in these cultures of endogenous IL-10, which results in an intrinsic reduction of monokine synthesis after LPS activation. Consistent with this proposal, LPS-stimulated peritoneal macrophages were shown to directly produce IL-10 detectable by ELISA. Furthermore, IFN-gamma was found to enhance IL-6 production by LPS-stimulated peritoneal macrophages, and this could be explained by its suppression of IL-10 production by this same population of cells. In addition to its effects on monokine synthesis, IL-10 also induces a significant change in morphology in IFN-gamma-stimulated peritoneal macrophages. The potent action of IL-10 on the macrophage, particularly at the level of monokine production, supports an important role for this cytokine not only in the regulation of T cell responses but also in acute inflammatory responses.     

Serine protease inhibitors inhibit superoxide production by human basophils stimulated by anti-IgE

Anti-IgE-induced O₂^? production by human basophils was inhibited by potent inactivators of serine proteases. The inhibitory effect of the inhibitor and substrate for chymotrypsin-type protease was much greater than that of those substances for trypsin-type protease. These findings suggest that chymotrypsin-like serine proteases are involved in basophil O₂^? production.     

Zinc hydroxide stimulates superoxide production by rat alveolar macrophages.

The effect of zinc hydroxide on superoxide (O2-) production by rat alveolar macrophages was determined by chemiluminescence and by cytochrome c reduction. Zinc ions had no effect on the chemiluminescence of unstimulated alveolar macrophages. By contrast, zinc hydroxide (ZnOH2), a neutralized form of zinc ions, increased the chemiluminescence level and O2- release. Increased O2- release was inhibited by pertussis toxin, isoquinoline sulfonamide and pretreatment with EGTA. These findings indicate that zinc hydroxide formation from zinc compounds can stimulate the O2- production by alveolar macrophages by receptor-mediated and Ca(2+)-dependent process.     

Phospholipase A2-mediated superoxide production of murine peritoneal macrophages induced by chrysotile stimulation

In the present study, we investigated how chrysotile-stimulated macrophages generate superoxide using murine peritoneal macrophages, with special attention to the modulatory role of phospholipase A(2) (PLA(2)). We examined the effects of the following inhibitors and antagonists for signaling molecules on the superoxide anion (O(-)(2)) production of chrysotile-stimulated macrophages: p-bromophenacyl bromide (pBPB) and mepacrine for PLA(2); islet-activating protein (IAP) for G-protein; H-7 for protein kinase C (PKC); AA861 for 5-lipoxygenase (5-LO); indomethacin for cyclo-oxygenase (COX); ETYA for both 5-LO and COX; hexanolamine PAF for platelet-activating factor (PAF). The PLA(2) and PKC inhibitors effectively inhibited the chrysotile-induced superoxide anion production of macrophages, but not the G-protein inhibitor, the 5-LO and COX inhibitors, and the PAF antagonist. We also examined the effects of the PLA(2) inhibitors on macrophages stimulated by phorbol 12-myristate 13-acetate (PMA) which directly activates PKC. The two structurally different PLA(2) inhibitors showed differential effects on the PMA-induced superoxide generation: pBPB inhibited it but mepacrine did not. These results suggested that (1) PLA(2) and PKC modulate the chrysotile-induced O(2) production, and (2) two different kinds of PLA(2) work upstream and downstream of PKC, but (3) G-protein, 5-LO and COX metabolites, and PAF have no modulatory role in the reaction.     

Neurotensin enhances IL-1 production by activated alveolar macrophages

Peptides may play a physiologic role in regulating immune responses and in triggering a variety of cellular events that modify the sensitivity of cells in the periphery. Neurotensin (NT) is present in the lung and it has been shown to bind to mouse peritoneal macrophages and influence their phagocytic ability. In this study, the effect of NT on the production of IL-1 by rat alveolar macrophages (AM) has been investigated. Although NT did not stimulate the release of IL-1 or increase the apparent intracellular pool of IL-1 when incubated with AM, there was significant cell changes, such as increased adherence, spreading, and altered shape. Furthermore, when AM were stimulated with LPS, both the intracellular and extracellular pools of IL-1 were significantly increased by NT. This effect was dose dependent and was observed at concentrations ranging from 10(-11) to 10(-6) M. NT did not modify the kinetics of LPS-induced IL-1 release nor the effects of a given suboptimal concentration of LPS. The release of IL-1 by various inducers, including muramyl dipeptide (MDP) and zymosan was also enhanced by NT, suggesting a general modulator role for this neuropeptide. When NT was added concomitantly with other potentiators of IL-1 production, such as IFN-gamma and leukotriene B4, no synergistic effect on IL-1 release was seen. Kinetics experiments showed that optimal enhancement of IL-1 production occurred when AM cultures were preincubated with NT before addition of MDP or when NT and MDP were present together at the initiation of the 24-h AM cultures. Taken together, our data suggest that NT acts early in the induction process of IL-1. Because IL-1 plays an important role both in the initiation of the immune response and in the local manifestations of inflammation, NT released in the vicinity of pulmonary blood vessels and the respiratory epithelium may modulate immunologically relevant responses in the lung microenvironment.     

NPGB-induced inhibition of superoxide anion production by normal Lewis rat macrophages

The treatment of Lewis rat peritoneal macrophages with p¹-nitrophenyl p-guanidinobenzoate (NPGB) inhibited the superoxide anion production stimulated with phorbol myristate acetate (PMA). The addition of NPGB at the time of maximum superoxide generation was still able to block the superoxide release. It appears from these findings that NPGB may block either the activation process of the membrane bound NAD(P)H oxidase or directly on the active enzyme. Other protease inhibitors such as, epsilon-amino caproic acid (EACA), pepstatin, trans aminomethyl cyclohexane carboxylic acid (AMCA), aprotinin, and leupeptin did not inhibit the superoxide release. The superoxide anion release by the xanthine-xanthine oxidase system was not inhibited by NPGB. This finding indicates that NPGB does not itself react with superoxide. It has been also demonstrated that NPGB is a good reactant toward sulfhydryl group. The relevance of these finding to experimental allergic encephalomyelitis (EAE) is discussed.     

Alpha 2-macroglobulin: structure, properties and physiological role

The paper is concerned with the results of recent researches devoted to studies of the structure, properties and physiological role of alpha 2-macroglobulin, one of main inhibitors of blood proteolytic enzymes. Data are presented on its primary and quaternary structure, mechanisms of interaction with proteinases. The role of alpha 2-macroglobulin in regulation of the activity of proteinases participating in blood coagulation, fibrinolysis, kininogenesis, immune reactions is shown. Possibilities of its application in medicine are discussed.     

Alpha 2-macroglobulin and proliferative retinopathy in type 1 diabetes

Serum alpha 2-macroglobulin (alpha 2m) and total glycosylated haemoglobin (HbA1) concentrations were measured in 110 insulin dependent Type 1 diabetics with minimal or no fundoscopic retinopathy, referred to as non-retinopaths, and in 52 proliferative retinopaths. Proteinuria was recorded in 8 (7%) non-retinopaths and 29 (56%) retinopaths and was accompanied by elevated alpha 2m concentrations in both groups of diabetics but only significantly so in the non-retinopaths. Diabetics without proteinuria showed a significant correlation between alpha 2m concentration and duration of diabetes, HbA1 and age (being higher at extremes of age). Alpha 2m concentrations were significantly higher in retinopaths than in non-retinopaths without proteinuria when allowance was made for the influence of age and duration of diabetes on alpha 2m. This difference may be attributed to the higher HbA, levels found in retinopaths than in non-retinopaths and was no longer evident when account was taken of the prevailing HbA1 concentration in individual patients.     

Involvement of alpha-2-macroglobulin receptor in clearance of interleukin 8-alpha-2-macroglobulin complexes by human alveolar macrophages

The purpose of this study was to determine if interleukin 8 (IL-8) in complex with alpha2-macroglobulin (alpha-2-M) can be taken up by human alveolar macrophages. First, we demonstrated that human alveolar macrophages have receptors for alpha-2-M but not IL-8. The binding of(125)I-labeled alpha-2-M to the cells was specific and saturable, whereas(125)I-labeled recombinant human IL-8 (rhIL-8) did not bind to macrophages. However,(125)I-rhIL-8-alpha-2-M complexes bound to macrophages, and unlabeled alpha-2-M competed for the binding. We then cultured the cells in the presence of(125)I-rhIL-8-alpha-2-M complexes,(125)I-rhIL-8 alone or buffer for 24 h. Macrophages were lysed, and the released radioactivity measured. IL-8 concentrations in supernatants and cells were also measured using an IL-8 ELISA. When the macrophages were incubated with(125)I-rhIL-8-alpha-2-M complexes there was a significant amount of IL-8 associated with the cells. However, this was not the case when the cells were incubated with(125)I- rhIL-8 alone suggesting that only these complexes were taken-up by human alveolar macrophages. Furthermore, the clearance of complexes was specifically inhibited by a monoclonal antibody against the 515-kDa subunit of the alpha-2-M receptor (alpha-2-MR) but not by an isotopic mouse IgG1. The study shows an important clearance mechanism for IL-8 in the lung.     

Leukotrienes do not regulate interleukin 1 production by activated macrophages

The objective of this work was to investigate the role of leukotrienes in the production of IL-1 by activated human peripheral blood monocytes and mouse peritoneal macrophages. Using overnight adherent macrophages, stimulation with lipopolysaccharide or zymosan caused a time-dependent increase in IL-1 production. LTC4 was detected and preceded IL-1 production only in zymosan-treated macrophages. Lipopolysaccharide did not stimulate macrophages to produce LTC4. Zymosan-stimulated LTC4 production was inhibited by the lipoxygenase inhibitors, ICI207968 (3.20 microM), nordihydroguaiaretic acid (0.22 microM), phenidone (4.60 microM), REV5901 (0.20 microM), and the Merck 5-lipoxygenase "translocation inhibitor" MK886 (0.02 microM) with IC50 values as shown in parenthesis. However, none of these inhibitors reduced IL-1 production at concentrations which completely inhibited leukotriene synthesis. Taken together, these results do not support a role for leukotrienes in the production of IL-1 by zymosan-activated macrophages.     

Alpha 2-macroglobulin gene expression during rat development studied by in situ hybridization.

The sites of alpha 2-macroglobulin mRNA synthesis during rat development have been localized by in situ hybridization using a rat alpha 2-macroglobulin cDNA probe. Fetal liver was found to be the major site of alpha 2-macroglobulin mRNA synthesis. In addition, alpha 2-macroglobulin mRNA was detected in brain, spinal cord and eye. Alpha 2-Macroglobulin mRNA was quantitated by use of a sensitive RNAse protection assay. Maximal levels of alpha 2-macroglobulin mRNA were found in fetal livers shortly before birth. A rapid decline of alpha 2-macroglobulin mRNA occurred within 1 day after parturition. A similar time course, although at an approximately 20-fold lower level, was observed for alpha 2-macroglobulin mRNA in livers of pregnant rats. Alpha 2-Macroglobulin mRNA could also be detected in placenta. The levels were comparable to those found in maternal livers.     

Unsaturated phosphatidylcholines inhibit superoxide production in human neutrophils.

Unsaturated long chain phosphatidylcholines such as phosphatidylcholine dioleoyl and phosphatidylcholine dilinoleoyl in micromolar concentrations inhibited the superoxide production in neutrophils stimulated by the activators of protein kinase C, phorbol 12-myristate 13-acetate and 1,2-dioctanoyl-sn- glycerol. In contrast, the superoxide production induced by surface receptor agonist, formyl-methionyl-leucyl-phenylalanine, was unaffected by the phospholipids. These data suggest that surfactant phosphatidylcholines may have a modulatory role on neutrophil oxidative burst in the lung during inflammation where there is a preponderance of unsaturated phosphatidylcholines.     

Interferons inhibit LTC4 production in murine macrophages

Mouse resident peritoneal macrophages (M phi) produce the highly bioactive eicosanoid LTC4 when stimulated in vitro with zymosan or with the calcium ionophore A23187. This production was dramatically inhibited in M phi pre-exposed to IFN-alpha, IFN-beta, or IFN-gamma. Although all IFN were able to decrease the availability in M phi of the LTC4 precursor AA, this decrease was not the only cause of the IFN-induced inhibition of LTC4. In fact, further analysis of the different steps of the LTC4 biosynthetic pathway revealed that IFN-gamma could inhibit the formation of LTA4, thus of its derivatives LTC4 and LTB4, possibly acting at the level of the enzyme LTA4-synthetase. In contrast, IFN-alpha and IFN-beta only depressed the ability of M phi to metabolize AA into LTC4, leaving unaltered the synthesis of LTB4. However, IFN-alpha and IFN-beta did not influence directly the activity of any of the enzymes involved in LTC4 biosynthesis, indicating that they may act through some indirect, as yet unidentified regulatory mechanism. These data suggest that IFN-alpha and IFN-beta and, in different situations, IFN-gamma can be potentially useful in vivo in antagonizing localized anaphylactic or inflammatory reactions.