Activation of the production of cytotoxic factors by mouse spleen cells exposed to the combined action of lipopolysaccharide and muramyl dipeptide in vitro

Lipopolysaccharide (LPS) and muramyl dipeptide (MDP) stimulated murine splenocytes in vitro to produce cytotoxic factors (CF) that killed target cells L-929. This effect was synergic at LPS dose of 10 ng/ml and MDP dose of 10 micrograms/ml. CF production started 2 hours after spleen cell activation and was maximum in 6 hours. CF were produced by macrophages as well as by lymphocytes stimulated by LPS, MDP or their combination. However, synergic effect of immunomodulators was registered only if nonfractionated spleen cells were stimulated during 24 hours. Lymphocytes depleted on T cells did not lack the ability to generate CF upon activation. In addition, LPS and MDP activated synergically the production of interleukin-I by spleen cells in vitro.     

Regional Dissociation of Cyclic AMP and Inositol Phosphate Formation in Response to Thyrotropin-Releasing Hormone in the Rat Brain

The present study was undertaken to define effects of thyrotropin-releasing hormone (TRH) on formation of cyclic AMP (cAMP) and inositol phosphates (IPs) in rat brain regions. The brain of male Wistar rats was dissected into seven discrete regions, and each region was sliced. The slices were incubated in Krebs-Henseleit glucose buffer containing varying doses of TRH. TRH caused a significant and consistent increase in cAMP level, but not in formation of IPs, in the hypothalamus, striatum, and midbrain. TRH stimulated formation of IPs in the cerebellum, where the tripeptide did not change the cAMP level. In contrast, formation of neither cAMP nor IPs was affected by TRH in the cortex, hippocampus, or pons-medulla. These data suggest that TRH possesses two distinct types of brain intracellular signaling systems, which vary with brain regions.     

Macrophage Activation by Muramyl Dipeptide (MDP) without Lymphocyte Participation

An increase in the numbers of spread macrophages caused by macrophage stimulants was found to be a very sensitive measure for macrophage activation. Muramyl dipeptide (MDP), bacterial lipopolysaccharide (LPS), and lymphokines were found to activate macrophages dose-dependently as measured by this parameter. Macrophage activation by MDP was strictly dependent on its adjuvant-active stereochemically specific structures. Macrophage activation by MDP and LPS occurred without lymphocyte participation. It is suggested that LPS also activates macrophages via lymphocytes.     

Differential effects of liposome-incorporation on liver macrophage activating potencies of rough lipopolysaccharide, lipid A, and muramyl dipeptide. Differences in susceptibility to lysosomal enzymes

We investigated the in vitro activation of rat liver macrophages to a tumor-cytotoxic state with muramyl dipeptide (MDP), rough LPS (Re-LPS) and lipid A in both a free and liposome-encapsulated form. The tumor cytotoxic state of the liver macrophages was determined with a [methyl-3H]thymidine release assay using C26 colon adenocarcinoma cells as target cells. As was shown previously, the encapsulation of MDP within multi-lamellar phospholipid vesicles greatly enhanced the activating potency of the drug; by contrast, encapsulation of Re-LPS or lipid A significantly reduced the activation of macrophages as compared to the free form of these agents. At a dose of 1 ng of free Re-LPS per ml a significant induction of tumor cell lysis was observed whereas a maximal level was obtained at a concentration of approximately 10 ng/ml. By encapsulation of Re-LPS in liposomes the activating potency diminished 20- to 100-fold. The minimal concentration required to induce detectable macrophage activation with free lipid A was 10 ng/ml, while liposome-encapsulated lipid A did not induce any detectable tumor cell lysis up to a concentration of 200 ng/ml. After a 1-h pre-incubation with a lysosomal fraction from rat liver at pH 4.8, the macrophage-activating potency of Re-LPS and lipid A was diminished by up to 95% whereas MDP remained fully active under these conditions. We conclude that, due to endocytic uptake of liposome-incorporated Re-LPS and lipid A and subsequent intralysosomal degradation, these immunomodulators are inactivated with respect to their potency to activate liver macrophages to tumor cytotoxicity.     

Interleukin 1 and prostaglandin production by cells of the mononuclear phagocyte system isolated from mycobacterial granulomas

A study has been made of the activity of interleukin 1 (IL-1) and prostaglandins (PGs) in the culture supernatants from unstimulated and lipopolysaccharide (LPS)-stimulated mycobacteria-induced granuloma cells. Both epithelioid cells from bacillus Calmette-Guerin (BCG)-induced granulomas and macrophages from Mycobacterium leprae-induced granulomas, separated on a fluorescence-activated cell sorter using monoclonal antibody specific to guinea pig macrophages, spontaneously secreted low levels of IL-1 (assayed by thymocyte comitogenic and fibroblast mitogenic activities) into culture supernatants. However, culture supernatants from LPS-stimulated epithelioid cells showed significantly higher IL-1 activity than those from unstimulated cells. In contrast, LPS stimulation of M. leprae granuloma macrophages failed to enhance IL-1 production. Nevertheless, IL-1 activity in the culture supernatants from stimulated mycobacterial granuloma cells of both types was much lower than that from LPS-stimulated peritoneal exudate macrophage culture supernatants. There was no detectable amount of prostaglandin E2 (PGE2) in the culture supernatants from both unstimulated and LPS-stimulated BCG- and M. leprae-induced granuloma cells in comparison to much higher levels of PGE2 produced by unstimulated (0.28-6.2 ng/ml) or LPS-stimulated (greater than 15 ng/ml) peritoneal exudate macrophages. However, BCG granuloma cells either secreted prostaglandin F2 alpha (PGF2 alpha) spontaneously or produced comparable levels of PGF2 alpha to those from peritoneal exudate macrophages on stimulation, while M. leprae granuloma macrophages produced much lower levels of PGF2 alpha.     

IgG-coated erythrocytes augment LPS-stimulated TNF-alpha secretion, TNF-alpha mRNA levels, and TNF-alpha mRNA stability in macrophages

Previous studies have shown that IgG-coated erythrocytes (EIgG) augment the LPS-stimulated increase in serum TNF-alpha levels in animals and the LPS-stimulated secretion of TNF-alpha by isolated macrophages. The present study evaluated the mechanism for the effect of EIgG on LPS-stimulated TNF-alpha secretion in the murine macrophage cell line, RAW 264.7. Incubation of the macrophages with EIgG or IgG-coated glass beads caused a dose-dependent augmentation of LPS-stimulated TNF-alpha secretion. The addition of EIgG increased the rate of LPS-stimulated TNF-alpha protein secretion between 2 and 4 hr after LPS. Accordingly, EIgG increased the levels of TNF-alpha mRNA at 2 and 3 hr after LPS. The increase in the LPS-stimulated TNF-alpha mRNA levels caused by EIgG was associated with an increase in TNF-alpha mRNA stability. Thus, the augmentation of LPS-stimulated TNF-alpha secretion by EIgG was associated with an increase in TNF-alpha mRNA levels which at least partly resulted from an increase in the stability of TNF-alpha mRNA.     

The role of inositol lipids in the activation of monocytes by interleukin-1 and bacterial endotoxin

Abstract The effect of interleukin-1 (IL-1) and bacterial endotoxin (lipopolysaccharide, LPS) on the activation of phosphoinositidase C (PIC) and on prostaglandin E₂ release was studied in monocytes (Mø). Both IL-1α and IL-1β increased the release of PGE₂ in a concentration-dependent manner, with EC₅₀s of 0.48 nM and 0.12 nM, respectively. Intact Mø were prelabelled with [³H]inositol and the formation of inositol phosphates (IPs) was estimated by ion exchange chromatography. PIC activity was estimated directly by measuring the conversion of [³H]phosphatidylinositol-4,5,-bisphosphate to aqueous soluble radioactivity by Mø homogenates. IL-1α (5.8 nM) increased the accumulation of IPs within 1–4 minutes and increases in IP₃ and IP₄ occured before the increase in IP₁₊₂ whereas LPS only increased the IPs level after at least 30 min. IL-1α increased PIC activity in Mø homogenates within 15 min with an EC₅₀ of 0.58 nM and IL-1β (0.1 nM) also increased activity. Neither IL-1α nor IL-1β affected the PIC activity of membrane or cytosolic fractions. LPS decreased activity in all fractions. These data indicate that IL-1, but not LPS, can directly lead to an increased activity of PIC which may be involved in eicosanoid formation in Mø.     

Lysophosphatidylserine stimulates leukemic cells but not normal leukocytes

In this study, we observed that lysophosphatidylserine (LPS) stimulated intracellular calcium ([Ca(2+)](i)) increase in leukemic cells but not in normal human peripheral blood mononuclear cells. LPS also stimulated [Ca(2+)](i) increase in human leukemic THP-1 cells. LPS-stimulated [Ca(2+)](i) increase was inhibited by U-73122 but not by U-73343. LPS also stimulated inositol phosphates formation in THP-1 cells, suggesting that LPS stimulates calcium signaling via phospholipase C activation. Moreover, pertussis toxin (PTX) completely inhibited [Ca(2+)](i) increase by LPS, indicating the activation of PTX-sensitive G-proteins. We also found that LPS-induced [Ca(2+)](i) increase was completely inhibited by suramin, suggesting G-protein coupled receptor activation. Since LPS specifically stimulates PTX-sensitive G-proteins, phospholipase C-dependent [Ca(2+)](i) increase in leukemic cells but not normal peripheral blood leukocytes, LPS receptor may be associated with leukemia.     

Stimulation of polyphosphoinositide hydrolysis in Swiss 3T3 cells by recombinant fibroblast growth factors

Mitogenic concentrations of recombinant acidic or basic fibroblast growth factor (FGF) stimulated the accumulation of [3H]inositol phosphates ([3H]IPs) in Swiss 3T3 cells pre-labelled for 48 h with [3H]inositol. Maximal effects were obtained at 0.3 ng/ml and 3 ng/ml for basic and acidic FGF, respectively. Higher doses of either factor led to a diminished stimulation. FGF also stimulated 45Ca2+ release from cells pre-labelled with the isotope. However, FGF-stimulated production of [3H]IPs and release of 45Ca2+ exhibited marked differences when compared with the responses to the peptide mitogen bombesin; the FGF responses were markedly slower and were not inhibited by phorbol esters.     

Augmentation of the proliferative response of thymocytes to phytohemagglutinin by the muramyl dipeptide

A synthetic N-acetylmuramyl-l-alanyl-d-isoglutamine or muramyl dipeptide (MDP) and adjuvant-active analogs, but not lipopolysaccharide (LPS), exhibited the augmenting effect on the proliferative response of thymocytes to phytohemagglutinin (PHA). MDP also had a comitogenic effect on PHA-stimulated T lymphocytes. It was shown that the thymocyte-stimulating effect of MDP is not through the production of the monokines by MDP-stimulated macrophages and that MDP has a direct action on lymphocytes.     

Effect of cisplatin, lipopolysaccharide, muramyl dipeptide and recombinant interferon-gamma on murine macrophages in vitro. I. Macrophage-mediated tumor cell lysis

Murine macrophage monolayers treated with cisplatin, lipopolysaccharide (LPS), muramyl dipeptide (MDP) or recombinant interferon-gamma (rIFN gamma) were observed to have significantly increased tumoricidal activity. rIFN gamma had synergistic effects with cisplatin, LPS or MDP in activating macrophages. However, MDP showed much more pronounced synergism with cisplatin and LPS than with rIFN gamma. Supernatants collected from these activated macrophage monolayers also showed increased tumoricidal activity. Tumor cell lysis mediated by cisplatin-treated macrophages did not require priming with rIFN gamma though it may be necessary as a first signal for the increased macrophage activation with LPS and MDP.     

Inositol lipids and phosphates in the regulation of the growth and differentiation of haemopoietic and other cells

Stimulation of phosphatidylinositol 4,5-bisphosphate hydrolysis is an important signalling reaction involved in the responses of cells to some, but not all, stimuli that promote cell proliferation. Active agents in this regard include antigens activating T and B lymphocytes, angiotensin (employing a receptor encoded by the mas oncogene), bombesin and platelet-derived growth factor PDGF). However, accumulating evidence suggests that inositol lipids and phosphates also have other roles in the regulation of cell growth and differentiation. Growth factor receptors that encode tyrosine kinases (such as that for PDGF) activate a kinase that synthesises phosphatidylinositol 3-phosphate, a novel lipid, and loss of this kinase-activating function abolishes growth-promoting activity. Human interleukin-4, a lymphokine that activates B lymphocytes, appears to employ phosphatidylinositol 4,5-bisphosphate hydrolysis as a brief initial signal that is followed by a sustained rise in cyclic adenosine monophosphate (cAMP): both signals are needed for the successful induction of the surface antigen CD23. Moreover, the same inositol lipid signalling pathway as is employed by antigen-stimulated mature T lymphocytes to provoke proliferation may be redeployed in immature T cells to trigger their elimination when they encounter self-antigens. Finally, studies of HL60 promyelocytic cells have shown that these cells contain high concentrations of inositol 3,4,5,6-tetrakisphosphate, 1,3,4,5,6-pentakisphosphate and hexakisphosphate, three inositol polyphosphates that are probably formed independently of inositol lipid metabolism. When these cells are induced to differentiate either towards neutrophils (in the presence of dimethylsulphoxide) or macrophages (in phorbol myristate acetate), cessation of growth and acquisition of differentiated characteristics are accompanied by large and different changes in the concentrations of these inositol phosphates that may be characteristic of these two pathways of differentiation.     

Hydrolysis of phosphatidylinositol 4,5-bisphosphate and increase in cytosolic free Ca2+-concentration induced in a human T cell leukemia line, JURKAT, by monoclonal antibodies against the T3 complex

The monoclonal antibodies against the T3 complex on human T lymphocytes, anti-Leu-4, OKT3, and T3, induced an accumulation of inositol phosphates in a human T cell leukemia line, JURKAT, in the presence of LiCl. The monoclonal antibodies also induced an increase in the cytosolic free Ca2+-concentration ([Ca2+]i) in JURKAT. The accumulation of inositol phosphates and the increase in [Ca2+]i were specifically induced by the monoclonal antibodies against the T3 complex. Other monoclonal antibodies against differentiation antigens on human T lymphocytes were not active in inducing these responses in JURKAT. Stimulation of JURKAT by anti-Leu-4 induced a rapid and immediate decrease in phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] and an increase in the 32P-labeling of phosphatidic acid, which occurred after a short lag period. An analysis of inositol phosphates formed in the anti-Leu-4-stimulated JURKAT indicated the formation of inositol trisphosphate. These results strongly suggested that the T3 complex or T3/antigen receptor (Ti) complex functions as a receptor which transduces antigen signal, presented by either antigen-presenting cells or target cells, into the hydrolysis of PtdIns(4,5)P2. Fetal bovine serum at a dose of 1-20 microliters/ml induced a marked and transient [Ca2+]i increase in JURKAT immediately after addition. However, the level of formation of inositol phosphates was very small in cells stimulated by fetal bovine serum. Fetal bovine serum induced an immediate increase in the 32P-labeling of phosphatidic acid in JURKAT. These and other results suggested that serum increased [Ca2+]i in JURKAT by a mechanism different from that for the anti-Leu-4-induced [Ca2+]i response.     

Relationship between stimulated phosphatidic acid production and inositol lipid hydrolysis in intestinal longitudinal smooth muscle from guinea pig.

Accumulation of [32P]phosphatidic acid (PA) and total [3H]inositol phosphates (IPs) was measured in the longitudinal smooth-muscle layer from guinea-pig small intestine. Stimulation with carbachol, histamine and substance P produced increases in accumulation of both [3H]IPs and [32P]PA over the same concentration range. The increase in [32P]PA accumulation in response to carbachol (1 microM-0.1 mM) was inhibited in the presence of atropine (0.5 microM). Buffering the external free [Ca2+] to 10 nM did not prevent the carbachol-stimulated increase in [32P]PA accumulation. Carbachol and Ca2+ appear to act synergistically to increase accumulation of [32P]PA. In contrast, although incubation with noradrenaline also increased accumulation of [3H]IPs, no increase in accumulation of [32P]PA could be detected. These results suggest that an increase in formation of IPs is not necessarily accompanied by an increase in PA formation, and imply the existence of receptor-modulated pathways regulating PA concentrations other than by phospholipase-C-catalysed inositol phospholipid hydrolysis.     

A differential role for the mitogen-activated protein kinases in lipopolysaccharide signaling: the MEK/ERK pathway is not essential for nitric oxide and interleukin 1beta production.

Endotoxin (lipopolysaccharide, LPS) is a component of the outer membrane of Gram-negative bacteria and promotes the activation of macrophages and microglia. Although these cells are highly LPS-responsive, they serve unique tissue-specific functions and exhibit different LPS sensitivities. Accordingly, it was of interest to evaluate whether these biological differences reside in variations within LPS signaling pathways between these two cell types. Because the mitogen-activated protein kinases ERK-1 and ERK-2 have been implicated in the control of many immune responses, we tested the concept that they are a key indicator for differences in cellular LPS sensitivity. We observed that murine RAW 264.7 macrophages and murine BV-2 microglial cells both respond to LPS by exhibiting increased IkappaBalpha degradation, enhanced NF-kappaB DNA binding activity, and elevated nitric oxide and interleukin-1beta production. Although LPS potently stimulates ERK activation in RAW 264.7 macrophages, it does not activate ERK-1/-2 in BV-2 microglia. Moreover, antagonism of the MEK/ERK pathway potentiates LPS-stimulated nitric oxide production, suggesting that LPS-stimulated ERK activation can exert inhibitory effects in macrophage-like cells. These data support the idea that ERK activation is not a required function of LPS-mediated signaling events and illustrate that alternative/additional pathways for LPS action exist in these cell types.     

Effects of disodium ethane-1-hydroxy-1,1-diphosphonate (EHDP) on interleukin 1 production by macrophages

The effects of disodium ethane-1-hydroxy-1,1-diphosphonate (EHDP) on the in vitro functions of guinea pig macrophages were studied. A high dose (1 mg/ml) of EHDP inhibited interleukin 1 (IL 1) production by oil-induced peritoneal macrophages stimulated with muramyl dipeptide (MDP), lipopolysaccharide (LPS), phorbol myristic acetate (PMA), heat-aggregated IgG2 or calcium ionophore A23187. On the other hand, low doses (less than 0.125 mg/ml) of EHDP augmented the MDP induced IL 1 production by macrophages. This biphasic effect was also observed when macrophages were exposed to EHDP at 37 C for 24 hr and then stimulated with IL 1 inducers. Superoxide anion generation induced by formyl peptide or PMA was not affected by preincubation of the macrophages with doses of EHDP up to 1 mg/ml. Adherence and spreading of macrophages was inhibited by EHDP in a dose dependent manner without affecting cell viability. These results demonstrated that EHDP acted on macrophages directly and modulated IL 1 production in vitro.     

Effects of bacterial products on enterocyte-macrophage interactions in vitro

We describe a coculture model of a human intestinal epithelial cell line and human peripheral blood monocytes in which monocytes differentiate into cells with features of resident intestinal macrophages. Caco-2 cells are grown on the lower surface of a semipermeable filter with pore size of 3 μm (Transwells®) until they differentiate into enterocytes. Peripheral-blood monocytes are added and the co-culture incubated for two days. Monocytes migrate through the pores of the membrane, come into direct contact with the basolateral surfaces of the epithelial cell monolayer, and develop characteristics of resident intestinal macrophages including downregulation of CD14 expression and reduced pro-inflammatory cytokine responses (IL-8, TNF and IL-1β) to bacterial products. The apical application of lipopolysaccharide (LPS) and muramyl dipeptide (MDP) resulted in an increased number of integrated monocytes, but abrogated the downregulation of CD14 expression and the diminished cytokine responses. MDP also reduced tight-junctional integrity, whilst LPS had no effect. These data indicate that LPS and MDP have significant pathophysiological effects on enterocyte–monocyte interactions, and confirm other studies that demonstrate that enterocytes and their products influence monocyte differentiation. This model may be useful in providing insights into the interaction between monocytes, epithelial cells and intestinal bacteria in health and disease.     

Thrombin and IgE antigen induce formation of inositol phosphates by mouse E-mast cells

Stimulation of murine chondroitin sulfate E containing mast cells (E-MC) in vitro either by thrombin or immunologically resulted in a rapid formation of inositol phosphates (IPs). Increase in all of the three IPs (IP1, IP2 and IP3) could be detected 20 s after stimulation. The depletion of Ca2+ from the medium resulted in more than 80% reduction in beta-hexosaminidase release from either thrombin or IgE antigen stimulated cells. However, both thrombin and IgE antigen increased the formation of IP3 under these conditions independent of the presence of extracellular Ca2+.