Identification of a macrophage-activating factor in granules of the RNK large granular lymphocyte leukemia

Recent work from our laboratory has shown that NK cells rapidly release preformed factor(s) that stimulate monocyte oxidative metabolism and microbicidal activity. We have hypothesized that such factors could also activate macrophage (M phi) tumor lysis and might be stored in the cytoplasmic granules. Granules were isolated from the RNK large granular lymphocyte leukemias by nitrogen cavitation and Percoll fractionation of the cell homogenate. Utilizing CSF-1 differentiated murine bone marrow-derived M phi and P815 tumor target cells, a M phi-activating factor (MAF) was found. The MAF activity was identified in two peaks, the first was coincident with dense granule enzymes and was 60 times more concentrated per mg protein than a second peak in the cytosol fractions. Solubilization in 2 M NaCl was necessary to recover activity from both peaks. Granule NK-MAF required the simultaneous presence of LPS in order to induce tumoricidal activity. Kinetics of NK-MAF activation peaked after 12 h of exposure. The NK-MAF was short lived in the solubilized granules; however, its heat resistance allowed us to prepare enriched and stable preparations. Treatment of NK-MAF with pepsin but not trypsin completely abrogated its activity. The NK-MAF passed through an ultrafiltration membrane with a nominal cut-off of 10 kDa. This work indicates that NK cell granules contain a small heat-stable peptide capable of activating M phi tumoricidal activity.     

Effect of L-arginine on the retention of macrophage tumoricidal activity

It has been reported that the tumoricidal activity of macrophages (M phi) depends on L-arginine and that L-arginine metabolites such as reactive nitrogen intermediates alter M phi physical capacities. The aim of this report is to investigate the dose-related effect of L-arginine on the expression and retention of M phi tumoricidal activity. Cytotoxicity of M phi activated by IFN-gamma plus LPS was detected in the presence of about 0.1 mM or more of L-arginine. This paralleled the NO2- production in the presence, but not in the absence, of L-arginine. On the other hand, activated M phi were destined to die and lost their tumoricidal activity with time in the presence of 0.3 mM or more L-arginine. They retained, however, considerable activity in the absence or presence of 0.15 mM L-arginine. This retention of M phi cytotoxicity was longer when M phi were preactivated by 100 ng/ml than 10 ng/ml of LPS in combination with IFN-gamma. Addition of indomethacin, an inhibitor of prostaglandin production, did not prevent the decay of M phi cytotoxicity but rather facilitated it even in the absence of L-arginine. Regardless of indomethacin, consecutive stimulation with LPS or LPS plus IFN-gamma during culture was effective in maintaining the tumoricidal activity at a high level. In addition, we found that M phi which had lost tumoricidal activity during culture in L-arginine deficient medium could be reactivated by LPS to attack tumor target cells.     

Antigen-specific activation of effector macrophages by IFN-gamma producing (TH1) T cell clones. Failure of IL-4-producing (TH2) T cell clones to activate effector function in macrophages

IFN-gamma-producing (TH1) and IL-4-producing (TH2) clones were assayed for their ability to directly induce cytostatic activity in macrophages generated from splenic myeloid precursors (M phi-c). In the presence, but not in the absence, of antigen, TH1 clones activated the M phi-c to inhibit the growth of P815 tumor cells in vitro. TH2 clones were not able to activate such effector activity in the M phi-c. The M phi-c did effectively present Ag to the TH2 clones as evidenced by the proliferation of TH2 cells cultured with Ag in the presence, but not in the absence, of M phi-c. Therefore, although both TH1 and TH2 were activated by cognate interaction with antigen presenting M phi-c, only TH1:M phi-c interactions displayed reciprocity resulting in activation of the M phi-c. TH1-derived lymphokines or rIFN-gamma, in the presence of LPS, could activate proteose-peptone elicited M phi, resident peritoneal M phi, and M phi-c whereas neither TH2-derived lymphokines nor rIL4 could induce detectable activity in any of the 3 M phi populations. IFN-gamma, in the absence of LPS, could activate the elicited M phi and to a lesser and more variable degree, the resident M phi Only the M phi-c consistently required both IFN-gamma and LPS for induction of cytostatic activity. Since M phi-c consistently required at least two signals for activation, the ability of TH1-derived lymphokines to synergize with TH2 cells in M phi activation was examined. TH2 could activate the Ag-presenting M phi-c in the presence of IFN-gamma. The ability of added IFN-gamma to synergize with TH2 indicates that the cognate interaction between TH2 and antigen presenting M phi-c does result in delivery of at least one of the signal required for M phi activation.     

Down-regulation of macrophage lysozyme by lipopolysaccharide and interferon

Lipopolysaccharide (LPS) treatment of resident mouse peritoneal macrophages (M phi) was found to suppress intracellular as well as secreted lysozyme (LZM). Interferon (IFN) had a similar effect. LZM was identified by the capacity of cell lysates or medium to lyse Micrococcus lysodeikticus, and by the presence of a 14.5 Kd protein band which co-migrated with human LZM in SDS-PAGE and which reacted positively in Western blots with antiserum to human LZM. The size of the 14.5 Kd band decreased sequentially with increasing concentrations of LPS to which the cells were exposed. Although the LPS influence on LZM levels was dose-dependent, the intracellular LZM pool responded more readily than secreted LZM. Maximal intracellular LZM suppression of 80% was obtained with 10 micrograms LPS, whereas secreted LZM was reduced by only 66%. An IFN concentration of 100 U reduced secreted LZM by 24%, whereas 10,000 U of IFN decreased the amount of LZM secreted by 71%. Thioglycolate-elicited M phi had 75% less intracellular LZM than untreated resident M phi. Moreover, thioglycolate-elicited M phi were hyporesponsive to the suppressive effects of LPS added in vitro. Because both LPS and IFN have been shown to stimulate numerous M phi functions, the data are of interest because they support the concept, based on other studies, that agents which are capable of enhancing some M phi activities may concomitantly down-regulate other functions.     

Characterization of lptA and lptB, two essential genes implicated in lipopolysaccharide transport to the outer membrane of Escherichia coli

The outer membrane (OM) of gram-negative bacteria is an asymmetric lipid bilayer that protects the cell from toxic molecules. Lipopolysaccharide (LPS) is an essential component of the OM in most gram-negative bacteria, and its structure and biosynthesis are well known. Nevertheless, the mechanisms of transport and assembly of this molecule in the OM are poorly understood. To date, the only proteins implicated in LPS transport are MsbA, responsible for LPS flipping across the inner membrane, and the Imp/RlpB complex, involved in LPS targeting to the OM. Here, we present evidence that two Escherichia coli essential genes, yhbN and yhbG, now renamed lptA and lptB, respectively, participate in LPS biogenesis. We show that mutants depleted of LptA and/or LptB not only produce an anomalous LPS form, but also are defective in LPS transport to the OM and accumulate de novo-synthesized LPS in a novel membrane fraction of intermediate density between the inner membrane (IM) and the OM. In addition, we show that LptA is located in the periplasm and that expression of the lptA-lptB operon is controlled by the extracytoplasmic sigma factor RpoE. Based on these data, we propose that LptA and LptB are implicated in the transport of LPS from the IM to the OM of E. coli.     

Relationship between membrane potential changes and superoxide-releasing capacity in resident and activated mouse peritoneal macrophages

In an attempt to understand better the molecular basis for the enhanced respiratory burst of activated macrophages (M phi), we investigated the relationship between stimulus-induced changes in membrane potential and release of superoxide anion (O2-) in mouse peritoneal M phi. Resident M phi and M phi elicited by injection of lipopolysaccharide (LPS-M phi) or obtained from animals infected with bacille Calmette-Guérin (BCG-M phi) were used. LPS-M phi and BCG-M phi showed more pronounced changes in membrane potential (depolarization) and greater release of O2- on contact with phorbol myristate acetate (PMA) than did resident macrophages. The lag time between addition of stimulus and onset of release of O2- was reduced in activated compared with resident cells. Membrane potential changes began 60 to 90 sec before release of O2- could be detected in each cell type. The dose-response curves for triggering of membrane potential changes and O2- release by PMA were identical. The magnitude of membrane potential changes and of O2- release in LPS-M phi and BCG-M phi declined progressively during in vitro culture, and values on day 3 approached those in resident macrophages ("deactivation"). Extracellular glucose was required for effective stimulated change in membrane potential and O2- release. These findings indicate that membrane potential changes are closely associated with O2- -releasing capacity in macrophages, and that the systems that mediate membrane potential changes and production of O2- develop or decline concomitantly during activation or deactivation of the cells. Although the plasma membrane was highly depolarized by high extracellular K+ or by the sodium ionophore gramicidin, O2- release was not induced by these maneuvers, indicating that changes in membrane potential by themselves are not sufficient to trigger the respiratory burst in macrophages. Release of O2- was not impaired in buffers in which Na+ was completely replaced with equimolar concentrations of K+ or choline+; thus, induction or maintenance of the respiratory burst in M phi does not require an influx of Na+.     

Macrophage-mediated tumor cell killing: lack of dependence on the cyclooxygenase pathway of prostaglandin synthesis.

M phi obtained directly from disaggregated murine Moloney sarcomas produced PGE2 and a hydroxy fatty acid derivative as the major products of arachidonic acid metabolism. M phi-immunoreactive PGE synthetic rates decreased substantially and cytotoxic activity was lost when freshly explanted tumor M phi were held in culture 24 hr. Such cultured M phi remained in a partially activated "primed" state, however, wherein the addition of minute (ng) amounts of bacterial lipopolysaccharide (LPS) returned cytolytic activity and PGE synthesis to original levels. Indomethacin-induced blockade of the M phi cyclooxygenase pathway inhibited PG synthesis by LPS-stimulated, primed M phi without affecting the return of cytolytic activity. We conclude, therefore, that the production of PG had no direct role in the mediation of tumor cell killing by activated M phi isolated from these neoplasms.     

The effect of dexamethoxin on the integrity of cytoplasmic membrane in gram-positive and gram-negative microorganisms

Decamethoxin is shown to be able to increase membrane permeability of Pseudomonas aeruginosa, Escherichia coli and Micrococcus lysodeikticus, that is confirmed by a loss of compounds with the absorption maximum at 260 nm by cells. Parallel with this the number of viable individuals has fallen and activity of dehydrogenases has been inhibited. The aspartate and alanine aminotransferase activity was not inhibited by decamethoxin and even increased. Decamethoxin lysed the protoplasts of the tested microorganisms. At high decamethoxin concentrations (over 500 micrograms/ml for P. aeruginosa and over 200 mu/ml--for E. coli) the outflow of components from the cells of gram-negative bacteria ceased, that may be associated with the coagulation changes in the cytoplasm. A loss of the low-molecular components by M. lysodeikticus cells and lysis of protoplasts proceeded less intensely than the same processes in the gram-negative microorganisms, that is explained by a less resistance of M. lysodeikticus to decamethoxin and earlier coagulation of the cytoplasm preventing lysis.     

Lipopolysaccharide-mediated macrophage activation: the role of calcium in the generation of tumoricidal activity

As we have reported, calcium ionophore A23187 activates macrophages for tumor cell killing, and the activated macrophages produced a soluble cytotoxic factor (M phi-CF) that is similar, if not identical, to tumor necrosis factor. Based on these observations, we have investigated whether calcium is involved in the activation mediated by another potent macrophage activator, namely lipopolysaccharide (LPS). We first showed that A23187 caused uptake of extracellular calcium-45 by macrophage monolayers, whereas LPS did not. Because in this system rapid changes would not have been detected, several other approaches also have been used. We have examined the effect of depleting extracellular calcium by using medium containing no added calcium, supplemented with 1 mM EGTA. In no case did depletion result in decreased M phi-CF production by LPS-treated macrophages. Measurements using the fluorescent intracellular calcium indicator Quin 2 have also been performed. The calcium ionophore ionomycin caused a rapid change in the intracellular Quin 2 signal. LPS, even at a concentration in vast excess of that required to activate the macrophages, caused no change in the signal during a 2-hr period. If the macrophages were loaded with high doses of Quin 2 or another intracellular chelator, TMB-8, M phi-CF production decreased and cytotoxic activity was impaired. These data indicate that one or more of the processes involved in M phi-CF production does require calcium, but that activation mediated by LPS occurs without the influx of extracellular calcium or redistribution of intracellular calcium.     

Macrophage membrane glycoprotein binding of Griffonia simplicifolia I-B4 induces TNF-alpha production and a tumoricidal response.

Thioglycollate-elicited macrophages (m phi), upon binding the lectin Griffonia simplicifolia IB4 (GSIB4) at the plasma membrane, are induced to secrete several low molecular weight proteins. In this investigation, results from specific ELISA and immunoprecipitation analysis of these molecules confirmed that the cytokine, tumor necrosis factor-alpha (TNF-alpha), belongs to the group of elicited proteins. This specific m phi response is directly influenced by the dose of GSIB4 used and the time in contact with the cells. At 40 micrograms/ml GSIB4, the maximum dose of lectin used, the m phi activity was equal to that achieved when the cells were incubated with an interferon-gamma/lipopolysaccharide (IFN/LPS) stimulus alone. Moreover, the data showed that TNF-mediated tumoricidal activity was significantly influenced by GSIB4 binding to the m phi membrane. When the lectin was incubated alone or in sequence with IFN/LPS, this ligand-receptor binding promoted the lysis of WEHI 164 tumor target cells. However, concurrent incubation of both IFN/LPS and GSIB4 with m phi significantly diminished the tumoricidal response. This suggested that one of the metabolic pathways utilized subsequent to receptor-ligand binding was altered by these interactions. When cyclic AMP (cAMP) and inositol triphosphate (IP3) levels were examined, the results showed that the concentration of cAMP was unchanged despite the fact that IP3 levels were significantly enhanced upon m phi-GSIB4 binding. Collectively, the data show that GSIB4 binding to specific glycoproteins in the m phi membrane induces TNF-alpha production and facilitates TNF-alpha dependent tumoricidal responses. It also appears that the transduction of the signal, in part, at least utilizes the phosphatidyl inositol pathway. Finally, it is noteworthy that m phi activity is influenced by the sequence in which GSIB4 is presented to the m phi relative to the IFN/LPS treatment.     

Activated T lymphocytes and macrophages secrete fibronectin which strongly supports cell adhesion.

Matrix-bound fibronectin (FN) appears to be involved in cell adhesion and motility mediated by integrin receptors. Although lymphoid cells and other cell types are capable of producing and secreting FN, the precise role of this secreted FN-like factor in regulating immune reactions is unclear. In the present study we analyzed the adhesive properties of FN secreted by rat CD4+ T cells and clone cells activated by the T cell mitogen concanavalin A (Con A), antigen, or via the CD2 pathways, or by macrophages (M phi) activated by lipopolysaccharide (LPS). Immobilized culture supernatant (CS) from the activated T cells or M phi supports the adhesion of activated rat or human CD4+ T cell or murine tumor cell. These CS contained FN and were more potent at facilitating cell adhesion then plasma FN. The adhesion activity of CS was attributed to FN because (a) gelatin columns depleted the FN present in the CS and (b) pretreating the cells with peptides of the cell-binding domain of FN abrogated their ability to bind CS. CS-mediated adhesion appears to occur primarily via the recognition of the Arg-Gly-Asp (RGD) by the beta 1-integrin-specific receptors of the adhesive cells. Thus, we postulate that FN secreted by various types of leukocytes is involved in promoting essential cell-matrix interactions, possibly affecting cell-adhesive and migratory processes at inflammatory or extravasation sites.     

Dynamics of cytochemically distinct subpopulations of macrophages in elicited rat peritoneal exudates

Qualitative and quantitative changes in rat peritoneal macrophage (M phi) subpopulations, differing in their ultrastructural peroxidatic staining characteristics were followed over the course of a thioglycollate (TG) broth-induced inflammatory response. In addition, selected functional features of the normal steady-state and 4-day TG-induced populations of M phi were compared. The steady-state population consisted primarily of M phi with peroxidatic staining limited to the nuclear envelope (NE) and rough endoplasmic reticulum (RER); such cells are called resident M phi. Within hours of TG injection, there was an influx of monocyte-derived exudate M phi, the number of which reached a maximum, by 24 hr. During the next 24 hr, the proportion of exudate M phi decreased with a concomitant increase in peroxidatic activity (PA)-negative M phi. These two cell types continued to predominate for the next 48 hr during which there was a gradual increase in resident M phi and so-called "exudate-resident" M phi, the latter of which exhibits both exudate and resident PA patterns. Thus, the 4-day TG-induced population consisted of four cytochemically distinct M phi subpopulations: approximately 50% PA-negative M phi, approximately 25% exudate M phi, approximately 15% resident M phi, and approximately 10% exudate-resident M phi. Differences in Fc receptors and complement receptors 1 and 3 were noted between the two populations in the presence of progenitors that give rise to colonies of M phi in liquid culture in response to murine-derived colony-stimulating factor 1. The implications of these results in regard to the origin(s) of M phi diversity are discussed.     

Nicotine and lipopolysaccharide stimulate the formation of osteoclast-like cells by increasing macrophage colony-stimulating factor and prostaglandin E2 production by osteoblasts

Several studies have indicated that one of the causes of alveolar bone destruction with periodontitis is lipopolysaccharide (LPS) from the cell wall of Gram-negative bacteria in plaque and that tobacco smoking may be an important risk factor for the development and severity of periodontitis. The present study was undertaken to determine the effect of nicotine and LPS on the expression of macrophage colony-stimulating factor (M-CSF), osteoprotegerin (OPG), and prostaglandin E2 (PGE2) in osteoblasts, and the indirect effect of nicotine and LPS on the formation of osteoclast-like cells. Saos-2 cells were cultured with 10(-3) M nicotine, or 1 or 10 microg/ml LPS and 10(-3) M nicotine, for up to 14 days. The gene and protein expression of M-CSF and OPG were determined using real-time PCR and ELISA, respectively. PGE2 expression was determined using ELISA. The formation of osteoclast-like cells was estimated using tartrate-resistant acid phosphatase (TRAP) staining of osteoclast precursors in culture with conditioned medium from nicotine and LPS-treated Saos-2 cells and the soluble receptor activator of NF-kappaB ligand (RANKL). M-CSF and PGE2 expression increased markedly in cells cultured with nicotine and LPS compared with those cultured with nicotine alone. OPG expression increased in the initial stages of culture with nicotine and LPS but decreased in the later stages of culture. The conditioned medium containing M-CSF and PGE2 produced by nicotine and LPS-treated Saos-2 cells with soluble RANKL increased the TRAP staining of osteoclast precursors compared with that produced by nicotine treatment alone. These results suggest that nicotine and LPS stimulate the formation of osteoclast-like cells via an increase in M-CSF and PGE2 production and that the stimulation is greater than with nicotine treatment alone.     

Dendritic cell activating factor produced by mouse macrophage hybridoma

A mouse macrophage (M phi) hybridoma which produces a soluble factor responsible for the cooperation between M phi and spleen dendritic cells (DC) was constructed. The antigen-presenting activity and the stimulator cell activity in the allogeneic or syngeneic mixed leukocyte reaction of DC were significantly augmented when DC were incubated with the culture supernatant of the hybridoma treated with various stimulants including latex beads. The monokine present in the culture supernatant of the hybridoma, called dendritic cell-activating factor (DCAF), augmented the production of lymphocyte-activating factor by DC while Ia expression of DCAF-treated DC was not altered. DCAF had no effect on the antigen-presenting activity of peritoneal resident M phi or B cell blasts while the antigen-presenting activity of spleen M phi was enhanced, but the degree of the enhancement was much less than that of spleen DC. DCAF was found to have the following properties: its pI value is between pH 4 and 5; it is stable at pH 2 to 10; and it loses its activity on incubation at 75 C for 30 min. When the culture supernatant of the hybridoma stimulated with latex beads was subjected to gel filtration, the DCAF activity was detected in the 20 Kd to 25 Kd, 30 Kd to 40 Kd, and 50 Kd to 60 Kd molecular weight regions. The 30 Kd to 40 Kd fraction, which is the major peak fraction, was further purified by ion-exchange chromatography followed by gel-filtration chromatography. When each fraction was subjected to SDS-PAGE, a 30 Kd band corresponding to the DCAF activity was observed and DCAF was purified to about 90% purity.     

Identification and molecular characterization of macrophage-associated antigens of the rat

Rabbit antiserum to rat peritoneal exudate (PE) macrophage (M phi) antigens was prepared and its reactivity with cell surface proteins of M phi, granulocytes, and lymphocytes was studied by one- and two-dimensional polyacrylamide gel electrophoresis (PAGE). A total of 14 membrane antigens were identified of which three were found to be expressed only by M phi and granulocytes. By one-dimensional analysis, a protein with an approximate m.w. of 105,000 was present on splenic and PE M phi and on splenic lymphocytes. Two-dimensional analysis revealed that this band was heterogeneous and contained at least three species, one of which was restricted to expression on M phi and granulocytes. A second protein of 150,000 daltons was resolved into two species by two-dimensional analysis. Both of these species were present on M phi and granulocytes but not on lymphocytes. Both the 105,000- and 150,000-dalton proteins were glycosylated. Because the 105,000- and 150-000-dalton proteins expressed by M phi were also expressed by granulocytes, is is likely that these are differentiation antigens whose expression is a characteristic property of cells within both monocytoid and myeloid lineages. All three 105,000-dalton species and one of the two 150,000-dalton species were detected on mouse M phi, indicating their expression is not unique to the rat.     

Plasma lipopolysaccharide (LPS)-binding protein. A key component in macrophage recognition of gram-negative LPS.

LPS-binding protein (LBP) binds with high affinity (Kd approximately equal to 10(-9) M) to lipid A of LPS isolated from rough (R)- or smooth (S)-form Gram-negative bacteria as well as to lipid A partial structures such as precursor IVA. To define the role of LBP in regulating responses to LPS we have examined TNF release in rabbit peritoneal exudate macrophages (M phi) stimulated with LPS or with complete or partial lipid A preparations in the presence or absence of LBP. In the presence of LBP, M phi showed increased sensitivity to S- and R-form LPS as well as synthetic lipid A. Compared with LPS or lipid A, up to 1000-fold greater concentrations of partial lipid A structures were required to induce TNF production. However, consistent with our previous observations that these structures bind to LBP, TNF production was increased in the presence of LBP. In contrast, LBP did not enhance or inhibit TNF production produced by heat-killed Staphylococcus aureus, peptidoglycan isolated from S. aureus cell walls, or PMA. Potentiated M phi responsiveness to LPS was observed with as little as 1 ng LBP/ml. Heat-denatured LBP (which no longer binds LPS), BPI (an homologous LPS-binding protein isolated from neutrophils), or other serum proteins were without effect. LBP-treated M phi also showed a more rapid induction of cytokine mRNA (TNF and IL-1 beta), higher steady-state mRNA levels and increased TNF mRNA stability. These data provide additional evidence that LBP is part of a highly specific recognition system controlling M phi responses to LPS. The effects of LBP are lipid A dependent and importantly, extend to LPS preparations isolated from bacteria of R- and S-form phenotype.     

Docosahexaenoic acid, a constituent of rodent fetal serum and fish oil diets, inhibits acquisition of macrophage tumoricidal function

Macrophage (M phi) activation is deficient in the fetus and neonate, at times when the serum concentration of docosahexaenoic acid (DHA; 22:6n3) is approximately 10-fold higher than in the adult. We tested the effects of highly purified DHA on M phi activation in vitro. M phi were stimulated with rIFN-gamma plus either of two second or "triggering" signals, LPS or heat-killed Listeria monocytogenes. M phi activation was assayed as the lysis of P815 mastocytoma cells, which are resistant to TNF-alpha. DNA inhibited the activation of peritoneal M phi and the M phi line RAW264.7 in a dose-dependent manner at concentrations between 20 and 160 microM. These concentrations are found in fetal and neonatal rodent sera. Another polyunsaturated fatty acid, arachidonic acid (20:4n6), was much less inhibitory. In contrast to its profound effect on tumoricidal activation, DHA did not inhibit phagocytosis and catabolism of 125I-heat-killed Listeria monocytogenes. Increasing the rIFN-gamma or second signals reduced the inhibition of tumoricidal activation by DHA but not M phi incorporation of 14C-DHA. When the rIFN-gamma and second signals were separated in time, DHA was far more inhibitory if delivered with the triggering signal than if delivered with the rIFN-gamma. However, the incorporation of 14C-DHA was the same under these two conditions. In M phi treated with DHA during LPS stimulation, the inhibition was time-dependent, requiring more than 2 h. Although DHA inhibits cyclooxygenase activity, its inhibition of M phi activation was not reversed with the following cyclooxygenase products: PGE2, a stable TXA2 analog (U-46, 619) or a stable PGI2 analog (Iloprost). Although DHA is metabolized by lipoxygenases, the inhibition was not reversed by the lipoxygenase inhibitors 5, 8, 11, 14-eicosatetraynoic acid and nordihydroguaiaretic acid. Altogether, the data indicate that DHA, at concentrations present in fetal and neonatal sera, inhibits M phi activation and may contribute to the previously observed deficits in M phi function in the fetus and neonate.     

Activation of terminally differentiated human monocytes/macrophages by dengue virus: productive infection,hierarchical production of innate cytokines and chemokines,and the synergistic effect of lipopolysaccharide

Dengue virus (DV) primarily infects blood monocytes (MO) and tissue macrophages (M phi). We have shown in the present study that DV can productively infect primary human MO/M phi regardless of the stage of cell differentiation. After DV infection, the in vitro-differentiated MO/M phi secreted multiple innate cytokines and chemokines, including tumor necrosis factor alpha, alpha interferon (IFN-alpha), interleukin-1 beta (IL-1 beta), IL-8, IL-12, MIP-1 alpha, and RANTES but not IL-6, IL-15, or nitric oxide. Secretion of these mediators was highlighted by distinct magnitude, onset, kinetics, duration, and induction potential. A chemokine-to-cytokine hierarchy was noted in the magnitude and induction potential of secretion, and a chemokine-to-cytokine-to-chemokine/Th1 cytokine cascade could be seen in the production kinetics. Furthermore, we found that terminally differentiated MO/M phi cultured for more than 45 days could support productive DV infection and produce innate cytokines and chemokines, indicating that these mature cells were functionally competent in the context of a viral infection. In addition, DV replication in primary differentiated human MO/M phi was enhanced and prolonged in the presence of lipopolysaccharide (LPS), and LPS-mediated synergistic production of IFN-alpha could be seen in DV-infected MO/M phi. The secretion of innate cytokines and chemokines by differentiated MO/M phi suggests that regional accumulation of these mediators may occur in various tissues to which DV has disseminated and may thus result in local inflammation. The LPS-mediated enhancement of virus replication and synergistic IFN-alpha production suggests that concurrent bacterial infection may modulate cytokine-mediated disease progression during DV infection.     

Enhancement of granulocyte luminescence by murine macrophage secretory products: Suggestive evidence for the release of a new activator

The incubation of macropages (MΦ) in the presence of lipopolysaccharides (LPS) usually results in the release of a variety of immunoregulatory cytokines such as interleukins (IL), tumour necrosis factor (TNF) and colony stimulating factors (CSF). We recently observed that conditioned media (CM) from LPS-treated murine MΦ lines probably contain another protein endowed with granulocyte stimulatory activity. This cytokine, which has an apparent MW of about 55 kDa enhances the PMA-induced luminescence of granulocytes and also stimulates their degranulation as measured by lactoferrin release. In contrast to IL₁ and IL₆ this factor is destroyed by brief treatment at pH 2, but is stable for 60 minutes at 65°C. Unlike CSF, its activity is unchanged by reducing agents such as beta-mercaptoethanol. Furthermore, pretreatment of the MΦ with dexamethasone, in order to reduce the release of IL₁ and TNF, hardly reduces the effect on granulocyte activation. Finally, treatment with a neutralizing polyclonal anti-murine TNF antiserum only partly abolishes its activity. These results show that, in addition to the already well-described cytokines, LPS-treated murine MΦ lines most probably secrete another granulocyte activator.