IgE-dependent cytokine production by human peripheral blood mononuclear phagocytes.

These studies demonstrate the IgE-dependent production of IL-1 beta and TNF-alpha by circulating blood monocytes. IL-1 beta production was demonstrated biologically as the stimulation of proliferation of the cloned IL-1-dependent murine T cell line D10.G4.1 in the presence of a submitogenic concentration of PHA. In a representative experiment, 3H-thymidine uptake increased from 57826 cpm in the presence of supernatants obtained from unstimulated cells to 200774 cpm with supernatants from monocytes stimulated by IgE/alpha IgE immune complexes. By ELISA, IgE complexes increased IL-1 beta production from 0.54 +/- 0.06 ng (per 10(6) monocytes) to 2.60 +/- 0.62 ng (p less than 0.01; mean of eight experiments) and TNF-alpha production from 0.17 +/- 0.10 ng to 3.00 +/- 0.54 ng (p less than 0.01; mean of four experiments). No IL-1 alpha secretion was observed. RNA hybridization analysis demonstrated that IL-1 beta production represented de novo synthesis of the cytokine. Stimulated RNA production was observed after a minimal 1/2-h incubation and was maximal at 2 h. The IgE-dependent secretion of these pro-inflammatory cytokines by mononuclear phagocytic cells may contribute to the inflammation characteristic of allergic responses.     

Relaxin modulates proinflammatory cytokine secretion from human decidual macrophages

Relaxin (RLN) is a systemic hormone from the corpus luteum, and its levels remain low during normal human gestation. Indeed, elevation of circulating RLN has long been associated with preterm birth, for which there has been no physiological explanation. Recent studies have shown that RLN suppresses endotoxin-induced cytokine secretion from THP-1 monocytic cells by acting on the glucocorticoid receptor (GR), but its effects on primary macrophages are unknown. Therefore, in the present study, we examined the effects of RLN on cytokine secretion from primary decidual macrophages (DMs) obtained at term before labor. Unlike THP-1 cells, RLN had no effects on the cytokine responses induced by either lipopolysaccharide (LPS) or interleukin (IL) 1B, mimicking infection-induced or sterile inflammation, respectively. However, RLN alone for 4 h significantly decreased (P < 0.05) colony-stimulating factor 2 (CSF2; also known as granulocyte-macrophage colony-stimulating factor) and IL8 but for 24 h significantly increased IL6 (P < 0.01). We show that DMs express both the RLN receptor (RXFP1) and the GR. RLN suppression of CSF2 and IL8 was sensitive to the GR-antagonist mifepristone (RU-486). However, RLN activation of RXFP1 induced a dose-dependent cAMP response, which when mimicked by forskolin also caused significantly increased (P < 0.05) secretion of IL6. Thus, RLN may be anti-inflammatory in DMs via activation of the GR but proinflammatory via activation of RXFP1 and cAMP. In summary, we have shown that RLN targeting DMs may modulate proinflammatory cytokine secretion at the maternal-fetal interface and contribute to the localized inflammatory response associated with parturition in women.     

Mannose-specific oligosaccharide recognition by mononuclear phagocytes

Glycoproteins terminating in mannose are recognized by receptors on macrophages. The mannose receptor is expressed by a variety of macrophages but expression is closely regulated. Activated macrophages, for example, express little mannose receptor activity. Kinetic and fractionation experiments suggest that cell surface mannose receptors recycle to and from an acidic, pre-lysosomal compartment. Preliminary evidence suggests that the mannose receptor is a large polypeptide and that it is structurally related to the mannose binding protein found in serum. The mannose receptor may, among other possibilities, regulate the extracellular levels of lysosomal hydrolases.     

Formation of endogenous pyrogen by mononuclear phagocytes

Production of endogenous pyrogen by human and rabbit blood monocytes in response to stimulation with agents of different origin was studied by inhibitory analysis under comparable conditions. Actinomycin D and cytochalasin B were applied. New evidence was obtained about an important role in the mechanism of activation of mononuclear phagocytes of initial interaction between a stimulating agent and the leukocyte membrane and of the biphasic process of endogenous pyrogen production.     

Cysteine proteinase inhibitors produced by mononuclear phagocytes

Summary Monocytes were separated from human peripheral blood and allowed to attach to culture flasks, after which the content and production of a number of cysteine proteinase inhibitors was assayed. These were: a low molecular weight (MW 12000) acid cysteine proteinase inhibitor (ACPI); a low-molecular weight inhibitor of the same size with neutral pH (NCPI), and -cysteine proteinase inhibitor with a molecular weight around 90000 (-CPI). Only NCPI was detectable in the cultures at the beginning of the incubation, and it was synthesized and released into the incubation mixture during the incubation, especially if the cells were stimulated with silica. The amount of NCPI contained in and released from the cells was drastically decreased by puromycin. Immunoblots after cell electrophoresis in polyacrylamide gel revealed only one molecular form of NCPI with a molecular weight of 12000 both in the cells and in the culture medium. No ACPI or -CPI could be detected.     

Regulation of enzyme secretion by mononuclear phagocytes: studies with macrophage plasminogen activator and lysozyme.

Lysozyme and plasminogen activator (PA) are independently regulated secretion products of the macrophage. Lysozyme is released constitutively by all types of macrophage, whereas PA is induced during macrophage activation by nonspecific stimuli or by an immunologically specific pathway under control of sensitized T lymphocytes and antigen. Production of PA is closely related to the ability of the macrophage to proliferate in the presence of colony stimulating factor (CSF). Production of lysozyme is often extinguished after hybridization of macrophages with other cells, but may persist and serve as a useful marker for expression of the differentiated macrophage phenotype in somatic cell hybrids.     

Onset of apoprotein E secretion during differentiation of mouse bone marrow-derived mononuclear phagocytes

A number of macrophage functions were sequentially expressed when the bone marrow precursors of mononuclear phagocytes differentiated in culture in the presence of a specific growth factor, colony-stimulating factor-1. We have defined the expression of apoprotein E (ApoE), a major secreted protein of resident peritoneal macrophages, during maturation of adherent bone marrow-derived mononuclear phagocytes into macrophages. By 5 d the bone marrow macrophages were active secretory cells, but few cells contained intracellular immunoreactive ApoE, and little, if any, ApoE was secreted. ApoE secretion was initiated at 9 d, and this correlated with an increase in the percentage of macrophages containing intracellular ApoE. The onset of ApoE secretion was selective, and little change occurred in the other major secreted proteins detected by [35S]methionine incorporation. In parallel, the high rate of plasminogen activator secretion, which peaked at 7 d, decreased markedly. ApoE secretion was not associated with altered expression of the macrophage surface antigen, Ia, or with secretion of fibronectin. Virtually all cells in independent colonies of bone marrow- derived macrophages eventually expressed ApoE. The proliferating monocyte/macrophage-like cell lines P388D1, J774.2, WEHI-3, RAW 264.1, and MGI.D+ secreted little or no ApoE. These data establish that ApoE secretion is developmentally regulated.     

VLDL modulates the cytokine secretion profile to a proinflammatory pattern.

Human very-low-density lipoprotein (VLDL) inhibits DNA synthesis in lymphocytes activated by the nonspecific mitogen concanavalin A (Con A). We studied the effects of VLDL on lymphocyte activation (IL-2 receptor expression), cell cycle progression, and production of IL-2 and of IL-4 (a proinflammatory and an anti-inflammatory interleukin, respectively) to understand why an atherogenic lipoprotein inhibits cell proliferation. After 48 h of stimulation with the mitogen, VLDL decreased the population of cells bearing IL-2 receptor and the population of T-cells that progress through the cell cycle, increasing the population of T-cells in G(0)/G(1). Cells cultured in the presence of Con A and VLDL produced higher levels of IL-2 and lower levels of IL-4 than cells cultured without VLDL. These results suggest that VLDL inhibits lymphocyte proliferation by reducing IL-2 receptor and enhancing the levels of IL-2. Probably, one atherogenic effect of VLDL is to modulate the cytokine secretion profile of lymphocytes to a predominantly proinflammatory response.     

CD45 regulates TLR-induced proinflammatory cytokine and IFN-beta secretion in dendritic cells

CD45 is a leukocyte-specific protein tyrosine phosphatase and an important regulator of AgR signaling in lymphocytes. However, its function in other leukocytes is not well-understood. In this study, we examine the function of CD45 in dendritic cells (DCs). Analysis of DCs from CD45-positive and CD45-null mice revealed that CD45 is not required for the development of DCs but does influence DC maturation induced by TLR agonists. CD45 affected the phosphorylation state of Lyn, Hck, and Fyn in bone marrow-derived DCs and dysregulated LPS-induced Lyn activation. CD45 affected TLR4-induced proinflammatory cytokine and IFN-beta secretion and TLR4-activated CD45-null DCs had a reduced ability to activate NK and Th1 cells to produce IFN-gamma. Interestingly, the effect of CD45 on TLR-induced cytokine secretion depended on the TLR activated. Analysis of CD45-negative DCs indicated a negative effect of CD45 on TLR2 and 9, MyD88-dependent cytokine production, and a positive effect on TLR3 and 4, MyD88-independent IFN-beta secretion. This indicates a new role for CD45 in regulating TLR-induced responses in DCs and implicates CD45 in a wider regulatory role in innate and adaptive immunity.     

Lysophospholipid regulation of mononuclear phagocytes

Blood monocytes and tissue macrophages derived from monocyte differentiation in tissues are central elements of innate immunity in host defense against numerous pathogens and other challenges. These mononuclear phagocytes also participate in wound healing and normal tissue remodeling in development and growth. Pathological perversion of their physiological roles leads to participation of mononuclear phagocytes in fibrosing diseases including granulomatous disorders, chronic inflammation typical of arthritis, and atherosclerosis. Lysophospholipids, including lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P), are platelet-derived lipid growth factors considered to participate in leukocyte differentiation and activation. This section summarizes our recent observations of the effects of lysophospholipids on mononuclear phagocytes.     

Endogenous peroxidase activity in mononuclear phagocytes

The diaminobenzidine (DAB) technique has been used to visualize the subcellular localization of peroxidatic enzymes in mononuclear phagocytes. The latter cells are part of the mononuclear phagocyte system (MPS), which includes the monocytes in the bone marrow and blood, their precursors in the bone marrow, and the resident macrophages in the tissues. The DAB cytochemistry has revealed distinct subcellular distribution patterns of peroxidase in the mononuclear phagocytes. Thus the technique facilitates the identification of the various phagocyte types: Promonocytes contain peroxidase reaction in the nuclear envelope, endoplasmic reticulum, Golgi apparatus, and cytoplasmic granules. Monocytes exhibit the reaction product only in cytoplasmic granules. Most resident macrophages show the activity only in the nuclear envelope and endoplasmic reticulum. Furthermore, new phagocyte types have been detected based on the peroxidase cytochemistry. Intermediate cells between monocytes and resident macrophages contain reaction product in the nuclear envelope, endoplasmic reticulum and cytoplasmic granules. The resident macrophages can be divided into two subtypes. Most of them exhibit the pattern noted above. Some, however, are totally devoid of peroxidase reaction. Most studies on peroxidase cytochemistry of monocytes and macrophages agree that the peroxidase patterns reflect differentiation or maturation stages of one cell line. Some authors, however, still interpret the patterns as invariable characteristics of separate cell lines. As to the function of the peroxidase in phagocytes, the cytochemical findings imply that two different peroxidatic enzymes exist in the latter cells: one peroxidase is synthesized in the endoplasmic reticulum of promonocytes and transported to granules via the Golgi apparatus. The synthesis ceases when the promonocyte matures to the monocyte. Upon phagocytosis the peroxidase is discharged into the phagosomes. Biochemical and functional studies have indicated that this peroxidase (myeloperoxidase) is part of a microbicidal system operating in host defence mechanisms. The other enzyme with peroxidatic activity is confined to the nuclear envelope and endoplasmic reticulum of resident macrophages in-situ and of monocytes at early stages in culture. As suggested by the subcellular distribution, the inhibition by peroxidase blockers, and the localization during phagocytosis studies, the latter peroxidase is functionally different from the myeloperoxidase.(ABSTRACT TRUNCATED AT 400 WORDS)     

Modulation of cytokine secretion by pentacyclic triterpenes from olive pomace oil in human mononuclear cells

Olive pomace oil, also known as "orujo" olive oil, is a blend of refined-pomace oil and virgin olive oil, fit for human consumption. Maslinic acid, oleanolic acid, erythrodiol, and uvaol are pentacyclic triterpenes, found in the non-glyceride fraction of orujo oil, which have previously been reported to have anti-inflammatory properties. In the present work, we investigated the effect of these minor components on pro-inflammatory cytokine production by human peripheral blood mononuclear cells in six different samples. Uvaol, erythrodiol, and oleanolic acid significantly decreased IL-1beta and IL-6 production in a dose-dependent manner. All three compounds significantly reduced TNF-alpha production at 100microM; however, at 10microM, uvaol and oleanolic acid enhanced the generation of TNF-alpha. In contrast, maslinic acid did not significantly alter the concentration of those cytokines, with the exception of a slight inhibitory effect at 100microM. All four triterpenes inhibited production of I-309, at 50microM and 100microM. However, uvaol enhanced I-309 production at 10microM. The triterpenic dialcohols had a similar effect on MIG production. In conclusion, this study demonstrates that pentacyclic triterpenes in orujo oil exhibit pro- and anti-inflammatory properties depending on chemical structure and dose, and may be useful in modulating the immune response.     

Suppression of endothelial or lipoprotein lipase in THP-1 macrophages attenuates proinflammatory cytokine secretion

LPL and endothelial lipase (EL) are associated with macrophages in human atherosclerotic lesions, and overexpression of LPL in mouse macrophages is associated with a greater extent of atherosclerosis. To investigate potential mechanisms by which macrophage-derived lipase expression may mediate proatherogenic effects, we used lentivirus-mediated RNA interference to suppress the expression of either LPL or EL within THP-1 macrophages. After suppression of either LPL or EL, significant decreases in the concentration of interleukin-1beta, interleukin-6, monocyte chemoattractant protein-1, and tumor necrosis factor-alpha were observed. Incubation of THP-1 macrophages with either mildly or extensively oxidized LDL consistently decreased cytokine expression, which was additive to that contributed by lipase suppression. Decreased lipase expression was also associated with an altered lipid composition, with reduced percentages of cholesterol (unesterified and esterified), triglycerides, and lysophosphatidylcholine. Microarray data indicated a decreased expression of proinflammatory genes, growth factors, and antiapoptotic genes. By contrast, there was an increased expression of lipoprotein receptors (scavenger receptor 1, low density lipoprotein receptor, scavenger receptor class B type I, and CD36). Thus, we conclude that the suppression of either LPL or EL decreases proinflammatory cytokine expression and influences the lipid composition of THP-1 macrophages. These results provide further insight into the specific metabolic and potential pathological roles of LPL and EL in human macrophages.