Activation of peritoneal macrophages by lysophosphatidylcholine

Lysophosphatidylcholine (lyso-PC), a product of inflammation induced by infectious and other agents, is able to stimulate mouse peritoneal macrophages to ingest target cells coated with IgG but not IgM regardless of the presence of complement. In vitro treatment of mouse resident peritoneal macrophages (adherent cells) alone with lyso-PC stimulated spreading activity but did not enhance ingestion activity of macrophages. However, when mixed cultures of adherent and nonadherent (lymphocytes) cells were treated with lyso-PC, macrophage ingestion activity of IgG-coated target cells (i.e., via Fc-mediated ingestion) was markedly enhanced. Analysis of lyso-PC activation process of macrophages for ingestion activity suggests that nonadherent (lymphocytes) cells are required for the induction of the manifestation of ingestion capacity. This requirement was also met by addition of untreated nonadherent cells to treated adherent cells. Thus, the activation mechanism of macrophages by lyso-PC for ingestion requires contribution of lymphocytes to promote enhanced ingestion activity. Since lyso-PC is a metabolite of a representative membrane phospholipid, we propose that lyso-PC and other lysophospholipids are mediators for activation of macrophages regardless of the type of inflammation-causative agent.     

Effects of inflammation products on immune systems

Summary Microbial infection causes inflammation which stimulates macrophage functions. One of the inflammatory products, lysophosphatidylcholine (lyso-Pc), can stimulate macrophage activities. Treatment of mice with lyso-Pc enhanced spreading and ingestion activities of peritoneal macrophages. In vitro treatment of macrophages with lyso-Pc greatly enhanced spreading but not ingestion activities. However, incubation of a mixture of adherent and nonadherent cells with lyso-Pc produced a markedly enhanced ingestion activity of macrophages, implying the contribution of nonadherent cells to the stimulation of macrophages. Time course studies of the stimulation of these macrophages showed that spreading activity is stimulated immediately, even 30 min, after their contact with lyso-Pc while induction of ingestion activity requires a latent period of about 5 h. When the specificity of the macrophage receptors for ingestion was analyzed using defined immunoglobulins (i.e., IgG and IgM) with or without complement, lyso-Pc-activated macrophages efficiently ingested IgG-coated sheep erythrocytes independent of complement. However, macrophages of the same lyso-Pc-treated mice did not ingest erythrocytes coated with IgM and complement. These observations suggest that lyso-Pc-stimulated macrophages ingest the targets via Fc-receptors but not C3b receptors.     

Identification of the serum factor required for in vitro activation of macrophages. Role of vitamin D3-binding protein (group specific component, Gc) in lysophospholipid activation of mouse peritoneal macrophages

In vitro treatment of mouse peritoneal cells (mixture of adherent and nonadherent cells) with lysophosphatidylcholine (lyso-Pc) in 10% FCS supplemented medium RPMI 1640 results in a greatly enhanced FcR-mediated phagocytic activity of macrophages. This macrophage-activation process requires a serum factor. Fractionation studies with starch block electrophoresis of fetal calf and human sera revealed that alpha 2-globulin fraction contains a serum factor essential for macrophage activation. To identify the serum factor, human serum was precipitated with 50% saturated ammonium sulfate and fractionated on a Sephadex G-100 column. A protein fraction with a lower m.w. than albumin had the capacity to support activation of macrophages. The active serum factor in this protein fraction was analyzed by immunoabsorption by using rabbit antisera against three major proteins of human alpha 2-globulin. This active serum factor was shown to be a vitamin D3-binding protein (group specific component, Gc). By using a monoclonal anti-Gc-absorbed active column fraction of human serum, we observed no enhanced macrophage activation over the results with serum fraction-free cultivation of lyso-Pc-treated peritoneal cells. Cultivation of lyso-Pc-treated peritoneal cells in a medium containing a low concentration of purified human Gc protein (0.1 to 2.6 ng/ml) produced a greatly enhanced phagocytic activity of macrophages. When purified human Gc protein was used in a serum-free medium for stepwise cultivation of lyso-Pc-treated nonadherent cell types, a macrophage-activating factor was efficiently generated. Therefore, it is concluded that the vitamin D3-binding protein is the essential serum factor for the lyso-Pc-primed activation of macrophages.     

Enhancement of antibody production by lysophosphatidylcholine and alkylglycerol

Inflammation products of normal and cancerous tissues, lysophosphatidylcholine and dodecylglycerol, were tested for their adjuvant effect on the antibody response. Mice treated with these agents and immunized with sheep erythrocytes simultaneously or at 3 days posttreatment developed a greatly enhanced antibody production as demonstrated by the Jerne plaque assay. Mice immunized at 3 days postadministration of agents did not significantly produce enhanced antibody-secreting cells as compared with those of mice simultaneously immunized. Since the mechanism of macrophage activation by lysophospholipids requires contribution of B and T cells, BALB/c-nu/nu mice treated with these agents and subsequently immunized with sheep erythrocytes did not produce antibodies. However, conditioned medium of in vitro-treated BALB/c-nu/nu B cells efficiently transmitted a signal to untreated BALB/c +/+ T cells for enhanced macrophage ingestion activity. This observation suggests that lysophospholipid-activated macrophages and T cells efficiently transmitted antigenic signal to the antibody-producing B cell population. Therefore, we conclude that these lipid metabolites have dual beneficial effects for the host by enhancing phagocytosis and antibody production. Thus, lysophosphatidylcholine and dodecylglycerol have potential practical application as adjuvants that could be administered separately or in combination with antigens.     

Activation of macrophages by ether analogues of lysophospholipids

Summary Inflammation processes cause activation of phospholipase A in plasma membranes resulting in the production of various lysophospholipids. Treatment of mice with L--lysophosphatidyl-DL-glycerol (lyso-Pg) resulted in an enhanced ingestion activity of peritoneal macrophages as did other lysophospholipids. However, lyso-Pg is rather toxic as indicated by a rapid decrease in macrophage activity 3 days after treatment while macrophage activity of lysophosphatidylcholine-treated mice continued to increase at least up to the 6th day after treatment. Alkyl-lysophospholipid derivatives, racemic 1-0-octadecyl-2-methylglycero-3-phosphocholine and -phosphoethanolamine stimulated mouse macrophages for Fc-mediated ingestion. Decomposed products of alkyl-lysophospholipids, alkylglycerols, were also found to be excellent activators of macrophages not only for ingestion of IgG-coated target cells but also antibody-mediated tumoricidal activity. Macrophages from mice treated with alkylglycerols developed superoxide generating capacity. Furthermore, alkylglycerols were found to be tumoricidal by direct contact with retinoblastoma cells. Therefore, the advantage of the potential application of alkylglycerols as chemotherapeutic agents is that they have dual beneficial effects: potentiation of macrophage activity and cytotoxicity to malignant cells.     

In vivo and in vitro activation of macrophages with a cyanine photosensitizing dye,platonin

A cyanine photosensitizing dye, platonin, is a potent macrophage-activating agent. Four days after the administration to mice of small amounts of platonin (20–40 ng/mouse), peritoneal macrophages exhibited greatly enhanced Fc-receptor-mediated phagocytic and superoxide-generating capacities. Much higher doses (more than 3000 ng/mouse) did not have this effect. Photodynamic experiments for macrophage activation were performed by exposing mouse peritoneal cells (mixture of macrophages and B and T lymphocytes) to white fluorenscent light (3 J m^–2s^–1) in media containing various low concentrations of platonin. A short exposure to white fluorescent light (5 s, 15 J m^–2) of peritoneal cells in a medium containing 3 ng platonin/ml produced a maximal level of phagocytic capacity of macrophages. Although platonin absorbs light poorly at wavelengths longer than 630 nm, the region of the spectrum in which the tissues are transparent allows reasonable penetration of light. Thus, we designed experiments in which peritoneal cells were exposed to a red fluorescent light (0.5 J m^–2s^–1). In a medium containing 10 ng platonin/ml with 15 J m^–2 red light, a markedly enhanced ingestion activity of macrophages was observed. Photodynamic treatment of peritoneal macrophages alone did not activate macrophages. Thus, participation of nonadherent cells is required for photodynamic activation of macrophages, implying that a macrophage-activating factor is generated within the nonadherent cells and transmitted to macrophages.     

Activation of peritoneal macrophages by orally administered ether analogues of lysophospholipids.

Oral administration of dodecylglycerol, inflammatory product of cancerous tissues, and the alkyl lysophospholipid derivative, 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3-choline), greatly activated mouse peritoneal macrophages. The activation was dose related and was assessed as increased Fc-mediated ingestion of red blood cells, superoxide production, chemiluminescence activity, and incorporation of radioactive thymidine and leucine. Furthermore, the data show that dodecylglycerol or ET-18-OCH3-choline was capable of inducing equally high levels of macrophage activation and cytotoxic action on tumor cells, just as occurs with intraperitoneal administration. Dodecylglycerol appeared to activate the macrophages at a relatively lower dose (5 micrograms/mouse) than ET-18-OCH3-choline (15 micrograms/mouse). The optimal oral doses required to activate macrophages for ingestion and cytotoxic activities were relatively higher than previously observed when these agents were administered intraperitoneally. Thus, the dose difference provided crucial information for correlating oral dosages with in vivo concentration of these agents as bioassayed by macrophage activation. These observations have extended and further support our earlier findings that these agents are effective immunopotentiators and thus could therapeutically be used to activate macrophages for cytotoxic effects on tumor cells via the oral route.     

The Trypanosoma cruzi protease cruzain mediates immune evasion

Trypanosoma cruzi is the causative agent of Chagas' disease. Novel chemotherapy with the drug K11777 targets the major cysteine protease cruzain and disrupts amastigote intracellular development. Nevertheless, the biological role of the protease in infection and pathogenesis remains unclear as cruzain gene knockout failed due to genetic redundancy. A role for the T. cruzi cysteine protease cruzain in immune evasion was elucidated in a comparative study of parental wild type- and cruzain-deficient parasites. Wild type T. cruzi did not activate host macrophages during early infection (<60 min) and no increase in ～P iκB was detected. The signaling factor NF-κB P65 colocalized with cruzain on the cell surface of intracellular wild type parasites, and was proteolytically cleaved. No significant IL-12 expression occurred in macrophages infected with wild type T. cruzi and treated with LPS and BFA, confirming impairment of macrophage activation pathways. In contrast, cruzain-deficient parasites induced macrophage activation, detectable iκB phosphorylation, and nuclear NF-κB P65 localization. These parasites were unable to develop intracellularly and survive within macrophages. IL 12 expression levels in macrophages infected with cruzain-deficient T. cruzi were comparable to LPS activated controls. Thus cruzain hinders macrophage activation during the early (<60 min) stages of infection, by interruption of the NF-κB P65 mediated signaling pathway. These early events allow T. cruzi survival and replication, and may lead to the spread of infection in acute Chagas' disease.     

Effect of lipopolysaccharide on the stimulation of macrophage Fc-dependent phagocytosis by splenic B lymphocytes

Macrophage phagocytic activity is regulated by a variety of products derived from activated lymphocytes. It has been reported that nonactivated splenic B and T lymphocytes enhance macrophage glucose metabolism. In addition, the enhancement of macrophage glucose metabolism was further increased by direct effects of bacterial lipopolysaccharide (LPS) on B, but not T, lymphocytes. In the present study, the effect of purified murine splenic B and T lymphocytes on Fc-dependent phagocytosis by thioglycollate-elicited peritoneal macrophages in the presence or absence of LPS has been investigated. Fc-dependent phagocytosis was assayed by measuring the ingestion of 51Cr-tagged sheep erythrocytes. After 3 or 4 days in culture, nonadherent spleen cells (NASC) and B and T lymphocytes from C3H/HeN (LPS-responder) mice produced 92 +/- 27%, 83 +/- 13%, and 147 +/- 33% increases in C3H/HeJ (LPS-hyporesponder) macrophage phagocytic activity, respectively. A similar effect was observed in Balb/c mice. Cell-free supernatant from NASC and B lymphocytes precultured for 2 or 4 days produced a 74 +/- 20% and 157 +/- 42% increase in phagocytosis respectively. At concentrations which have been previously shown to markedly enhance the ability of splenic B lymphocytes to stimulate macrophage glucose metabolism, Escherichia coli K235 LPS (10 micrograms/ml) did not alter the stimulatory effects of any of the splenic lymphocyte populations on macrophage Fc-dependent phagocytosis. These data suggest that B lymphocytes produce a soluble factor(s) which stimulates macrophage phagocytosis. In addition, LPS has different effects on the regulation of macrophage phagocytic activity and metabolism by B lymphocytes.     

Schizophyllan (SPG)-treated macrophages and anti-tumor activities against syngeneic and allogeneic tumor cells

We tested anti-tumor activities of macrophages treated with a neutral polysaccharide, schizophyllan (SPG), against syngeneic and allogeneic tumor cell lines. SPG was a macrophage stimulant which was not mitogenic to lymphocytes. That made a sharp contrast with the data that Corynebacterium parvum, BCG, and muramyl dipeptide (MDF) were macrophage stimulants which had lymphocyte-activating properties. Treatment of SPG-treated PEC with Thy12 monoclonal antibody and guinea pig complement did not affect the capabilities of tumor-cell-growth suppression by the treated PEC. Thus, the effector cells were peritoneal adherent cells (macrophages morphologically) and effector-to-target contact seemed to be necessary for effective tumor-cell-growth inhibition, although contradictory data exist for this. Murine peritoneal adherent cells harvested 4 days after a single IP injection of SPG at a dose of 100 mg/kg body weight of mouse showed the most prominent cytostatic and cytotoxic activities against syngeneic and allogeneic tumor cells. The distribution of anti-tumor activity in macrophages of various sizes followed the same pattern as macrophages treated with C. Parvum, i.e., larger macrophages showed more remarkable anti-tumor activity. Crude nonadherent peritoneal cells incubated with SPG at a concentration of 10 micrograms/ml, 100 micrograms/ml, or 1 mg/ml did not secrete lymphokine that rendered macrophages cytotoxic, while ConA-treated nonadherent cells did so. Furthermore, spleen cells treated with SPG in vivo did not secrete macrophage-activating lymphokine in the presence of SPG. On the other hand, addition of 1 mg/ml of SPG-treated peritoneal adherent cells and bone-marrow-derived macrophages in vitro rendered them cytotoxic to a moderate degree. This implies that SPG may activate macrophages directly, allowing them to become cytotoxic in the peritoneal cavity. Lastly, SPG could induce production of II-1-like factor to a moderate degree. SPG, whose molecular structure is well elucidated, will provide us with a strong tool to analyze the mechanism of macrophage activation both in vitro and in vivo.     

Cutting edge: IFN-gamma signaling to macrophages is required for optimal Valpha14i NK T/NK cell cross-talk

Activated NK T cells are known to rapidly stimulate NK cells and, subsequently, CD8(+) T cells and B cells. In this report, we first demonstrate that the downstream effects induced by alpha-galactosylceramide activated NK T cells on NK cells are mainly dependent on IFN-gamma. We found that NK T cell activation of NK cells requires a functional IFN-gamma signaling in macrophages and dendritic cells but not in B cells, NK cells, or NK T cells. NK T cell activation is dendritic cell-dependent whereas NK T cell activation of NK cells is indirect and in part mediated by macrophages. Interestingly, in this context, macrophage participation in the CD1d Ag presentation of alpha-galactosylceramide to NK T cells is not necessary. These data indicate that NK T cell-dependent activation of macrophages is required for optimal NK T cell-induced stimulation of NK cells.     

Tumor cell apoptosis polarizes macrophages role of sphingosine-1-phosphate

Macrophage polarization contributes to a number of human pathologies. This is exemplified for tumor-associated macrophages (TAMs), which display a polarized M2 phenotype, closely associated with promotion of angiogenesis and suppression of innate immune responses. We present evidence that induction of apoptosis in tumor cells and subsequent recognition of apoptotic debris by macrophages participates in the macrophage phenotype shift. During coculture of human primary macrophages with human breast cancer carcinoma cells (MCF-7) the latter ones were killed, while macrophages acquired an alternatively activated phenotype. This was characterized by decreased tumor necrosis factor (TNF)-alpha and interleukin (IL) 12-p70 production, but increased formation of IL-8 and -10. Alternative macrophage activation required tumor cell death because a coculture with apoptosis-resistant colon carcinoma cells (RKO) or Bcl-2-overexpressing MCF-7 cells failed to induce phenotype alterations. Interestingly, phenotype alterations were achieved with conditioned media from apoptotic tumor cells, arguing for a soluble factor. Knockdown of sphingosine kinase (Sphk) 2, but not Sphk1, to attenuate S1P formation in MCF-7 cells, restored classical macrophage responses during coculture. Furthermore, macrophage polarization achieved by tumor cell apoptosis or substitution of authentic S1P suppressed nuclear factor (NF)-kappaB signaling. These findings suggest that tumor cell apoptosis-derived S1P contributes to macrophage polarization.     

Potent effects of,and mechanisms for,modification of crosstalk between macrophages and adipocytes by lactobacilli

The murine macrophage‐like cell line J774.1 was treated with heat‐killed cells of Lactobacillus GG (LGG) and L. gasseri TMC0356 (TMC 0356). Interleukin (IL)‐6, IL‐12, and tumor necrosis factor‐α were profiled from the J774.1 cells using enzyme‐linked immunosorbent assay methods. The conditioned medium from cultured J774.1 cells was transferred to the preadipocyte cell line 3T3‐L1 (which is a mouse embryonic fibroblast‐adipose‐like cell line). Growth and differentiation of 3T3‐L1 cells were monitored by analyzing lipid accumulation and expression of peroxisome proliferator‐activated receptor (PPAR)‐γ mRNA. The medium conditioned by 3T3‐L1 cells was added to J774.1 cells and the cytokines in the supernatant analyzed. Compared with that of cells exposed to a PBS‐conditioned medium, lipid accumulation in 3T3‐L1 cells was significantly suppressed in a dose‐dependent manner by each medium that had been conditioned with LGG and TMC0356. PPAR‐γ mRNA expression in 3T3‐L1 cells was also significantly downregulated (P < 0.01, P < 0.05, respectively). The conditioned medium of 3T3‐L1 adipose phenotype significantly stimulated production of IL‐6 and IL‐12 in J774.1 cells treated with LGG and TMC0356. These results suggest that lactobacilli may suppress differentiation of preadipocytes through macrophage activation and alter the immune responses of macrophages to adipose cells.     

Adipocyte-derived lipoprotein lipase induces macrophage activation and monocyte adhesion: role of fatty acids

Objective: We evaluated the effect of adipocyte‐derived lipoprotein lipase (LPL) on macrophage activation and monocyte adhesion and the role of fatty acids in these effects. Research Methods and Procedures: 3T3‐L1 adipocytes were incubated with heparin or insulin to induce LPL secretion; then adipocyte conditioned media (CM) were added to cultured J774 macrophages or human aortic endothelial cells (HAECs). Macrophage cytokine production and monocyte adhesion to HAECs were determined. Results: Incubation of macrophages with heparin‐ or insulin‐treated adipocyte CM increased tumor necrosis factor α, interleukin‐6, and nitric oxide production by these cells. LPL neutralization and heparinase treatment prevented these effects. Addition of active LPL or palmitate to cultured macrophages replicated these effects. Blockade of leptin also reduced the effect of insulin‐treated adipocyte CM on macrophage inflammatory changes. Induction of macrophage cytokine secretion by leptin was prevented by LPL immunoneutralization. Finally, addition of CM of heparin‐ or insulin‐treated adipocytes to HAECs stimulated monocyte adhesion to these cells, an effect that was abrogated by an anti‐LPL antibody. This effect was reproduced by treating HAECs with active LPL or palmitate. Discussion: These results point to an effect of LPL‐mediated lipolysis in macrophage activation and monocyte adhesion.     

Generation of alloreactive cytotoxic T lymphocytes: production of T cell and macrophage helper factors in addition to IL 1 and IL 2 by peritoneal cells from mice immunized to Listeria monocytogenes

We investigate the production and biological activity of soluble helper factors produced by peritoneal T cells and macrophage derived from mice primed in vivo with Listeria monocytogenes. Supernatant fluids from co-cultures of these immune T cells and activated macrophages contained Interleukin 1 (IL 1) and Interleukin 2 (IL 2), and had the ability to assist the generation of cytotoxic T lymphocytes (CTL) from a population of nylon wool nonadherent spleen cells sensitized to allogeneic heat-treated thymocytes. The ability to assist CTL development involved T cell and macrophage factors in addition to IL 1 and IL 2. Immune T cells cultured alone produced a factor, devoid of significant IL 2 activity, that assisted CTL development only if adherent cells were present in the responding population. Activated macrophage produced a 38,000 dalton component, distinct from IL 1 on the basis of m.w., that assisted the development of CTL from nylon wool nonadherent splenic cells. Supernatants fluids from co-cultures of immune T cells and allogeneic, nonactivated macrophage contained a CTL helper factor but did not contain IL 1 or IL 2 activities. In contrast, supernatant fluids from co-cultures of immune T cells and syngeneic, nonactivated macrophage contained all 3 activities. This suggests a genetic restriction for the production of IL 1 and IL 2 that does not restrict the production of a CTL helper factor. These results demonstrate that T cell- and macrophage-derived helper factors distinct from IL 1 and IL 2 participate in the development of CTL.     

Blockade of CD38 diminishes lipopolysaccharide-induced macrophage classical activation and acute kidney injury involving NF-κB signaling suppression

The CD38, possessing ADP-ribosyl cyclase (ADPR-cyclase) and cyclic ADP-ribose hydrolase (cADPR-hydrolase), is able to regulate a variety of cellular activities. However, the role and mechanisms for CD38 in macrophage activation and sepsis-induced acute kidney injury (AKI) remain to be determined. Here we report that in cultured macrophages, Lipopolysaccharide (LPS) could upregulate CD38 expression in time and dose dependent manner. Knocking down or blockade of CD38 in macrophages could inhibit LPS-induced macrophage M1 polarization accompanied by diminished NF-κB signaling activation. In mouse model with LPS-induced acute kidney injury, blocking CD38 with quercetin could significantly relieve kidney dysfunction, kidney pathological changes as well as inflammatory cell accumulation. Similar to those in the cultured cells, quercetin could inhibit macrophage M1 polarization and NF-κB signaling activation in macrophages from kidneys and spleens in mice after LPS injection. Together, these results demonstrate that CD38 mediates LPS-induced macrophage activation and AKI, which may be treated as a therapeutic target for sepsis-induced AKI in patients.     

Fc- and complement-receptor activation stimulates cell cycle progression of macrophage cells from G1 to S

Phagocytosis of microorganisms by macrophages is an important host defense mechanism. While studying the phagocytosis of the human pathogenic fungus Cryptococcus neoformans, we noted that macrophage-like J774 cells with ingested fungal cells had frequent mitotic figures. By analyzing the relative proportion of phagocytic cells as a function of cell cycle phase, we observed an increase in S phase cells after Fc-mediated phagocytosis of polystyrene beads, live or heat-killed C. neoformans. This result was confirmed by increased nuclear BrdU incorporation after Fc-mediated phagocytosis. The induced progression to S phase was observed after both Fc- and complement-mediated phagocytosis of live yeasts. Fc-mediated stimulation of cell division did not require ingestion, because it could be triggered by incubating cells in IgG1-coated plates. Phagocytosis-mediated stimulation of replication was confirmed in vitro using primary bone marrow macrophages and in vivo for peritoneal macrophages. We conclude that phagocytosis of microbes or inert particles can stimulate macrophages to enter S phase and commence cell division. This observation suggests a potential mechanism for increasing the number of effector cells after microbial ingestion, but can also promote the spread of infection.     

Cytokine-stimulated T cells induce macrophage IL-10 production dependent on phosphatidylinositol 3-kinase and p70S6K: implications for rheumatoid arthritis

IL-10 is an anti-inflammatory cytokine produced in the joint in rheumatoid arthritis by macrophages and infiltrating blood lymphocytes. Regulation of its expression is poorly understood, but previous findings have suggested that physical interactions with T cells may play a role. This report investigates signalling mechanisms involved in the production of macrophage IL-10 upon interaction with fixed, cytokine-stimulated T cells (Tck). Elutriated monocytes were differentiated to macrophages by macrophage-colony-stimulating factor (M-CSF) and co-cultured with fixed T cells chronically stimulated in a cytokine cocktail of IL-2/IL-6/tumour necrosis factor (TNF)-α in the presence or absence of wortmannin and LY294002, inhibitors of phosphatidylinositol 3-kinase (PI3K), or of rapamycin, an inhibitor of p70 S6-kinase (p70S6K). Spontaneous IL-10 production by rheumatoid arthritis synovial-membrane mononuclear cells (RA-SMCs) and co-cultures of rheumatoid arthritis T cells (RA-Ts) and macrophages was also assessed. RA-T and Tck induction of macrophage IL-10 production was suppressed by cell separation and inhibition of PI3K and p70S6K. PI3K involvement was also shown by phosphorylation of the downstream effector protein kinase B. Spontaneous IL-10 production by RA-SMCs was also inhibited by LY294002 and depletion of the nonadherent (T-cell-enriched) fraction of the cell population. IL-10 production in RA-SMCs and M-CSF-primed macrophages, activated by interaction with Tck, is PI3K- and p70S6K-dependent.     

Cytokine-stimulated T cells induce macrophage IL-10 production dependent on phosphatidylinositol 3-kinase and p70S6K: implications for rheumatoid arthritis

IL-10 is an anti-inflammatory cytokine produced in the joint in rheumatoid arthritis by macrophages and infiltrating blood lymphocytes. Regulation of its expression is poorly understood, but previous findings have suggested that physical interactions with T cells may play a role. This report investigates signalling mechanisms involved in the production of macrophage IL-10 upon interaction with fixed, cytokine-stimulated T cells (Tck). Elutriated monocytes were differentiated to macrophages by macrophage-colony-stimulating factor (M-CSF) and co-cultured with fixed T cells chronically stimulated in a cytokine cocktail of IL-2/IL-6/tumour necrosis factor (TNF)-alpha in the presence or absence of wortmannin and LY294002, inhibitors of phosphatidylinositol 3-kinase (PI3K), or of rapamycin, an inhibitor of p70 S6-kinase (p70S6K). Spontaneous IL-10 production by rheumatoid arthritis synovial-membrane mononuclear cells (RA-SMCs) and co-cultures of rheumatoid arthritis T cells (RA-Ts) and macrophages was also assessed. RA-T and Tck induction of macrophage IL-10 production was suppressed by cell separation and inhibition of PI3K and p70S6K. PI3K involvement was also shown by phosphorylation of the downstream effector protein kinase B. Spontaneous IL-10 production by RA-SMCs was also inhibited by LY294002 and depletion of the nonadherent (T-cell-enriched) fraction of the cell population. IL-10 production in RA-SMCs and M-CSF-primed macrophages, activated by interaction with Tck, is PI3K- and p70S6K-dependent.     

Control of suppressor cell activation via endogenous prostaglandin synthesis: The role of T cells and macrophages

Splenic macrophages were cultured in the inner chamber of a Marbrook flask and their ability to induce suppressor activity in a glass-wool-adherent T-cell population was determined. Nontreated, lipopolysaccharide-, or phytohemagglutinin-treated macrophages, all induced comparable levels of suppressor activity in glass-wool-adherent T cells. Suppression induced by untreated macrophages was reversible by the addition of a prostaglandin synthetase inhibitor. In addition, macrophages enhanced non-glass-adherent T-cell activation of glass-wool-adherent cells. The results indicate that macrophage in concert with T cells may activate T suppressor cells via a prostaglandin-dependent pathway.