Regulation of the RON receptor tyrosine kinase expression in macrophages: blocking the RON gene transcription by endotoxin-induced nitric oxide

Previous studies have shown that activation of the RON receptor tyrosine kinase inhibits inducible NO production in murine peritoneal macrophages. The purpose of this study is to determine whether inflammatory mediators such as LPS, IFN-gamma, and TNF-alpha regulate RON expression. Western blot analysis showed that RON expression is reduced in peritoneal macrophages collected from mice injected with a low dose of LPS. The inhibition was seen as early as 8 h after LPS challenge. Experiments in vitro also demonstrated that the levels of the RON mRNA and protein are diminished in cultured peritoneal macrophages following LPS stimulation. TNF-alpha plus IFN-gamma abrogated macrophage RON expression, although individual cytokines had no significant effect. Because LPS and TNF-alpha plus IFN-gamma induce NO production, we reasoned that NO might be involved in the RON inhibition. Two NO donors, S-nitroglutathione (GSNO) and (+/-)-S-nitroso-N-acetylpenicillamine (SNAP), directly inhibited macrophage RON expression when added to the cell cultures. Blocking NO production by NO inhibitors like TGF-beta prevented the LPS-mediated inhibitory effect. In Raw264.7 cells transiently transfected with a report vector, GSNO or SNAP inhibited the luciferase activities driven by the RON gene promoter. Moreover, GSNO or SNAP inhibited the macrophage-stimulating protein-induced RON phosphorylation and macrophage migration. We concluded from these data that RON expression in macrophages is regulated during inflammation. LPS and TNF-alpha plus IFN-gamma are capable of down-regulating RON expression through induction of NO production. The inhibitory effect of NO is mediated by suppression of the RON gene promoter activities.     

Inhibition of IL-6 and IL-10 signaling and Stat activation by inflammatory and stress pathways

The development and resolution of an inflammatory process are regulated by a complex interplay among cytokines that have pro- and anti-inflammatory effects. Effective and sustained action of a proinflammatory cytokine depends on synergy with other inflammatory cytokines and antagonism of opposing cytokines that are often highly expressed at inflammatory sites. We analyzed the effects of the inflammatory and stress agents, IL-1, TNF-alpha, LPS, sorbitol, and H(2)O(2), on signaling by IL-6 and IL-10, pleiotropic cytokines that activate the Jak-Stat signaling pathway and have both pro- and anti-inflammatory actions. IL-1, TNF-alpha, and LPS blocked the activation of Stat DNA binding and tyrosine phosphorylation by IL-6 and IL-10, but not by IFN-gamma, in primary macrophages. Inhibition of Stat activation correlated with inhibition of expression of IL-6-inducible genes. The inhibition was rapid and independent of de novo gene induction and occurred when the expression of suppressor of cytokine synthesis-3 was blocked. Inhibition of IL-6 signaling was mediated by the p38 subfamily of stress-activated protein kinases. Jak1 was inhibited at the level of tyrosine phosphorylation, indicating that inhibition occurred at least in part upstream of Stats in the Jak-Stat pathway. Experiments using Stat3 mutated at serine 727 and using truncated IL-6Rs suggested that the target of inhibition is contained within the membrane-proximal region of the cytoplasmic domain of the gp130 subunit of the IL-6 receptor and is different from the SH2 domain-containing protein-tyrosine phosphatase/suppressor of cytokine synthesis-3 docking site. These results identify a new level at which IL-1 and TNF-alpha modulate signaling by pleiotropic cytokines such as IL-6 and IL-10 and provide a molecular basis for the previously described antagonism of certain IL-6 actions by IL-1.     

Production of proinflammatory cytokines by resident tissue macrophages after phagocytosis of apoptotic cells

It is generally believed that apoptosis is not associated with inflammation. However, we have found that phagocytosis of apoptotic cells by PMA-treated THP-1 cells and human monocyte-derived macrophages led to the production of proinflammatory cytokines, notably IL-8. These macrophages were obtained either by PMA treatment or by M-CSF treatment, possibly affecting the cytokine production after phagocytosis of apoptotic cells. In order to exclude the possibility, we employed resident tissue macrophages such as Kupffer cells and alveolar macrophages in this study and examined the production of cytokines after phagocytosis of apoptotic cells. Kupffer cells produced proinflammatory cytokines MIP-2 and TNF-alpha at the mRNA level. The MIP-2 protein was also detected by means of ELISA. Alveolar macrophages also produced the MIP-2 protein after phagocytosis of apoptotic cells. Furthermore, apoptotic thymocytes induced a similar response by these macrophages. These findings do support the notion that macrophages are apt to produce proinflammatory cytokines after phagocytosis of apoptotic cells.     

Paradoxical anti-inflammatory actions of TNF-alpha: inhibition of IL-12 and IL-23 via TNF receptor 1 in macrophages and dendritic cells

IL-12 and TNF-alpha are central proinflammatory cytokines produced by macrophages and dendritic cells. Disregulation of TNF-alpha is associated with sepsis and autoimmune diseases such as rheumatoid arthritis. However, new evidence suggests an anti-inflammatory role for TNF-alpha. TNF-alpha-treated murine macrophages produced less IL-12p70 and IL-23, after stimulation with IFN-gamma and LPS. Frequency of IL-12p40-producing macrophages correspondingly decreased as measured by intracellular cytokine staining. IL-12p40 production was also inhibited in dendritic cells. TNFR1 was established as the main receptor involved in IL-12p40 regulation, because IL-12p40 levels were not affected by TNF-alpha in TNFR1(-/-)-derived macrophages. Macrophages activated during Listeria monocytogenes infection were more susceptible to inhibition by TNF-alpha than cells from naive animals, which suggests a regulatory role for TNF-alpha in later stages of infection. This nonapoptotic anti-inflammatory regulation of IL-12 and IL-23 is an important addition to the multitude of TNF-alpha-induced responses determined by cell-specific receptor signaling.     

IL-10-independent STAT3 activation by Toxoplasma gondii mediates suppression of IL-12 and TNF-alpha in host macrophages

Infection of mouse macrophages by Toxoplasma gondii renders the cells resistant to proinflammatory effects of LPS triggering. In this study, we show that cell invasion is accompanied by rapid and sustained activation of host STAT3. Activation of STAT3 did not occur with soluble T. gondii extracts or heat-killed tachyzoites, demonstrating a requirement for live parasites. Parasite-induced STAT3 phosphorylation and suppression of LPS-triggered TNF-alpha and IL-12 was intact in IL-10-deficient macrophages, ruling out a role for this anti-inflammatory cytokine in the suppressive effects of T. gondii. Most importantly, Toxoplasma could not effectively suppress LPS-triggered TNF-alpha and IL-12 synthesis in STAT3-deficient macrophages. These results demonstrate that T. gondii exploits host STAT3 to prevent LPS-triggered IL-12 and TNF-alpha production, revealing for the first time a molecular mechanism underlying the parasite's suppressive effect on macrophage proinflammatory cytokine production.     

Dectin-1 expression and function are enhanced on alternatively activated and GM-CSF-treated macrophages and are negatively regulated by IL-10, dexamethasone, and lipopolysaccharide

Dectin-1 is the major macrophage receptor for beta-glucans and generates a proinflammatory response through the recognition of these carbohydrates on fungal pathogens. We have examined the effects of cytokines and other agents on the expression and functions of dectin-1 in both resident and elicited murine peritoneal macrophages (Mphi). Dectin-1 expression was found to be highly up-regulated by GM-CSF and by the cytokines that induce alternative macrophage activation, IL-4 and IL-13. In contrast, IL-10, LPS, and dexamethasone, but not IFN-gamma, down-regulated the expression of this receptor. Modulation of dectin-1 receptor levels correlated with the ability of these macrophages to bind zymosan and significantly affected the contribution of this receptor to the resultant proinflammatory response, as measured by the production of TNF-alpha, although some Mphi-specific differences were observed. These results correlate with the known effects of these cytokines and other agents on the ability of the immune system to recognize and respond to fungal pathogens.     

HMGB1 develops enhanced proinflammatory activity by binding to cytokines

High mobility group box 1 protein (HMGB1), originally characterized as a nuclear DNA-binding protein, has also been described to have an extracellular role when it is involved in cellular activation and proinflammatory responses. In this study, FLAG-tagged HMGB1 was inducibly expressed in the presence of culture media with or without added IL-1beta, IFN-gamma, or TNF-alpha. HMGB1 purified from cells grown in culture media alone only minimally increased cytokine production by MH-S macrophages and had no effect on murine neutrophils. In contrast, HMGB1 isolated from cells cultured in the presence of IL-1beta, IFN-gamma, and TNF-alpha had enhanced proinflammatory activity, resulting in increased production of MIP-2 and TNF-alpha by exposed cells. IL-1beta was bound to HMGB1 isolated from cells cultured with this cytokine, and purified HMGB1 incubated with recombinant IL-1beta acquired proinflammatory activity. Addition of anti-IL-1beta Abs or the IL-1 receptor antagonist to cell cultures blocked the proinflammatory activity of HMGB1 purified from IL-1beta-exposed cells, indicating that such activity was dependent on interaction with the IL-1 receptor. These results demonstrate that HMGB1 acquires proinflammatory activity through binding to proinflammatory mediators, such as IL-1beta.     

Surfactant strengthens the inhibitory effect of C-reactive protein on human lung macrophage cytokine release

In this study we investigated the effect of acute-phase levels of C-reactive protein (CRP) on cytokine production by pulmonary macrophages in the presence or absence of pulmonary surfactant. Both human alveolar and interstitial macrophages as well as human surfactant were obtained from multiple organ donor lungs. Precultured macrophages were stimulated with LPS alone or together with IFN-gamma in the presence or absence of CRP, surfactant, and combinations. Releases of TNF-alpha and of IL-1beta to the medium were determined. We found that CRP could modulate lung inflammation in humans by decreasing the production of proinflammatory cytokines by both alveolar and interstitial macrophages stimulated with LPS alone or together with IFN-gamma. The potential interaction between CRP and surfactant phospholipids did not overcome the effect of either CRP or surfactant on TNF-alpha and IL-1beta release by lung macrophages. On the contrary, CRP and pulmonary surfactant together had a greater inhibitory effect than either alone on the release of proinflammatory cytokines by lung macrophages.     

Lung-specific delivery of cytokines induces sustained pulmonary and systemic immunomodulation in rats

The recombinant cytokines IFN-gamma and TNF-alpha stimulate several macrophage-mediated functions important in host defense. However, systemic administration of cytokines may be limited by significant host toxicity. We investigated whether aerosolized cytokines can stimulate alveolar macrophage and blood monocyte function, and whether they induce an inflammatory response in the lungs of normal rats. We found that aerosolized murine rIFN-gamma or recombinant human TNF-alpha increased IL-1 production by both alveolar macrophages and blood monocytes for at least 5 days after administration. Furthermore, murine rIFN-gamma increased the expression of Ia Ag on alveolar macrophages and human rTNF-alpha increased alveolar macrophage- and blood monocyte-mediated tumor lysis. Sequential aerosolization of IFN-gamma and TNF-alpha significantly increased both IL-1 release and Ia expression compared to either cytokine administered alone. Aerosolized human rTNF-alpha achieved lung levels comparable to those produced by an i.v. TNF-alpha dose reported to cause diffuse organ injury and death in rats. However, plasma TNF-alpha levels were several thousand-fold lower after aerosol administration. Aerosolized cytokines did not induce lung edema or an inflammatory cell infiltrate within the airways or alveoli. Aerosolized human rTNF-alpha alone, or murine rIFN-gamma and human rTNF-alpha, induced margination of leukocytes in pulmonary blood vessels 1 day after aerosolization, and a few small foci of pulmonary hemorrhage 5 days later. We conclude that aerosol administration of IFN-gamma or TNF-alpha enhances both pulmonary and systemic monocyte function, and that the combination of IFN-gamma and TNF-alpha produce additive or synergistic effects. Aerosolized cytokines induce only a minimal pulmonary inflammatory response. Aerosolized TNF-alpha produces high cytokine levels in the lung but very low uptake into the circulation.     

Kinetics and cellular origin of cytokines in the central nervous system: insight into mechanisms of myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis induced by myelin oligodendrocyte glycoprotein (MOG) in C57BL/6 (H-2b) mice is characterized by early (day 12) acute paralysis, followed by a sustained chronic clinical course that gradually stabilizes. Extensive inflammation and demyelination coincide with clinical signs of disease. To identify the mechanisms of these processes, individual proinflammatory and anti-inflammatory cytokines and chemokines were studied. Sensitive single-cell assays were utilized to determine the cellular origin and kinetics of cytokine production in the CNS. Immunization with MOG35-55 peptide resulted in priming of both Th1 (lymphotoxin, IFN-gamma, and TNF-alpha) and Th2 (IL-4) cells in the spleen. However, only Th1 cells were apparent in the CNS. CD4 T cells that produced IFN-gamma or TNF-alpha were present in the CNS by day 7 after immunization with MOG35-55, peaked at day 20, and then waned. TNF-alpha was also produced in the CNS by Mac-1+ cells. On days 7 and 10 after immunization, the TNF-alpha-producing Mac1+ cells were predominantly microglia. By day 14, a switch occurred in that the Mac1+ TNF-alpha-producing cells had the phenotype of infiltrating macrophages. RANTES, IFN-inducible protein 10 (IP-10), and monocyte chemotactic protein 1 chemokine mRNA were detected in the CNS by day 8 after immunization. The early presence of monocyte chemotactic protein 1 (MCP-1) in the CNS provides a mechanism for the recruitment of macrophages. These data implicate TNF-alpha production by a continuum of T cells, microglia, and macrophages at various times during the course of disease. The importance of Th1 cytokines is highlighted, with little evidence for a role of Th2 cytokines.     

Macrophage-stimulating protein, the ligand for the stem cell-derived tyrosine kinase/RON receptor tyrosine kinase, inhibits IL-12 production by primary peritoneal macrophages stimulated with IFN-gamma and lipopolysaccharide

IL-12, produced by APCs during the initial stages of an immune response, plays a pivotal role in the induction of IFN-gamma by NK and gammadeltaT cells and in driving the differentiation of Th1 cells, thus providing a critical link between innate and acquired immunity. Due to the unique position occupied by IL-12 in the regulation of immunity, many mechanisms have evolved to modulate IL-12 production. We have shown previously that macrophage-stimulating protein (MSP), the ligand for the stem cell-derived tyrosine kinase/recepteur d'origine nantais (RON) receptor, inhibits NO production by macrophages in response to IFN-gamma and enhances the expression of arginase. Mice lacking RON exhibit increased inflammation in a delayed-type hypersensitivity reaction and increased susceptibility to endotoxic shock. In this study we demonstrate that pretreatment of macrophages with MSP before IFN-gamma and LPS results in the complete inhibition of IL-12 production due to suppression of p40 expression. This response is mediated by the RON receptor, and splenocytes from RON(-/-) animals produce increased levels of IFN-gamma. MSP pretreatment of macrophages resulted in decreased tyrosine phosphorylation of Stat-1 and decreased expression of IFN consensus sequence binding protein in response to inflammatory cytokines. In addition to IL-12, the expression of IL-15 and IL-18, cytokines that are also dependent on IFN consensus sequence binding protein activation, is inhibited by pretreatment with MSP before IFN-gamma and LPS. We also show that the ability of MSP to inhibit IL-12 production is independent of IL-10. Taken together, these results suggest that MSP may actively suppress cell-mediated immune responses through its ability to down-regulate IL-12 production and thus inhibit classical activation of macrophages.     

Both viable and killed Candida albicans cells induce in vitro production of TNF-alpha and IFN-gamma in murine cells through a TLR2-dependent signalling

The in vitro production of TNF-alpha and IFN-gamma in response to Candida albicans was investigated in wild type, TLR2-/- and TLR4-/- murine cells. TLR2-/- resident peritoneal macrophages showed a strong impairment of TNF-alpha production in response to viable and non-viable (heat-killed, antimycotic-treated and formaldehyde-fixed) yeasts and hyphae (germ tube-bearing cells) of the high virulence C. albicans ATCC 26555 strain, as compared with macrophages from wild-type and TLR4-/- mice. The in vitro production of IFN-gamma was investigated in murine splenocytes obtained three days after intravenous injection with the low virulence, non-germinative C. albicans PCA2 strain, and again, TLR2-/- splenocytes showed a strong impairment of the in vitro production of IFN-gamma in response to non-viable (heat-killed, antimycotic-treated and formaldehyde-fixed) C. albicans ATCC 26555 yeasts, as compared with splenocytes of TLR4-/- and wild type mice. These results indicate that the TLR2-mediated recognition of C. albicans leading to a proinflammatory Th1 host response appears to be well conserved in killed C. albicans cells, regardless of the inactivating treatment employed.     

Raf1 plays a pivotal role in lipopolysaccharide-induced activation of dendritic cells

Activation of extracellular-regulated kinases 1/2 (ERK) is involved in lipopolysaccharide (LPS)-induced cellular responses such as the increased production of proinflammatory cytokines. However, mitogen-activated protein kinases (MAPKs) such as p38 are also activated by LPS and have been postulated to be important in the control of these end points. Therefore, establishing the relative contribution of MAPKs in each cell type is important, as is elucidating the molecular mechanisms by which these MAPKs are activated in LPS-induced signaling cascades. We demonstrated in DC2.4 dendritic cells that ERK regulates tyrosine phosphorylation of phosphatidyl-inositol-3-kinase (PI3-K) and the production of TNF-alpha. We also demonstrated that Raf1 is phosphorylated and involved in the production of TNF-alpha and tyrosine phosphorylation of PI3-K via ERK. Raf1 also regulates the activation of NF-kappaB. We propose that Raf1 plays a pivotal role in LPS-induced activation of the dendritic cells.     

Epigallocatechin gallate modulates cytokine production by bone marrow-derived dendritic cells stimulated with lipopolysaccharide or muramyldipeptide, or infected with Legionella pneumophila

The primary polyphenol in green tea extract is the catechin epigallocatechin gallate (EGCG). Various studies have shown significant suppressive effects of catechin on mammalian cells, either tumor or normal cells, including lymphoid cells. Previous studies from this laboratory reported that EGCG has marked suppressive activity on murine macrophages infected with the intracellular bacterium Legionella pneumophila (Lp), an effect mediated by enhanced production of both tumor necrosis factor-alpha (TNF-alpha) and gamma-interferon (IFN-gamma). In the present study, primary murine bone marrow (BM)-derived dendritic cells (DCs), a phagocytic monocytic cell essential for innate immunity to intracellular microorganisms, such as Lp, were stimulated in vitro with the microbial stimulant lipopolysaccharide (LPS) from gram-negative bacteria, the cell wall component from gram-positive bacteria muramyldipeptide (MDP) or infected with Lp. Production of the T helper cell (Th1)-activating cytokine, interleukin-12 (IL-12) and the proinflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha),produced mainly by phagocytic cells and important for antimicrobial immunity, was determined in cell culture supernatants by enzyme-linked immunosorbent assay (ELISA). Treatment of the cells with EGCG inhibited, in a dose-dependent manner, production of IL-12. In contrast, enhanced production of TNF-alpha occurred in a dose-dependent manner in the DC cultures stimulated with either soluble bacterial product or infected with Lp. Thus, the results of this study show that the EGCG catechin has a marked effect in modulating production of these immunoregulatory cytokines in stimulated DCs, which are important for antimicrobial immunity, especially innate immunity. Further studies are necessary to characterize the physiologic function of the effect of EGCG on TNF-alpha and IL-12 during Lp infection, and the mechanisms involved.     

Essential requirement of antigen presentation by monocyte lineage cells for the activation of primary human gamma delta T cells by aminobisphosphonate antigen

Human gammadelta T cells respond to nonpeptide Ags such as pyrophosphomonoesters and alkylamines in a gammadelta TCR-dependent manner in the absence of other APCS: Recently, aminobisphosphonates such as pamidronate have also been shown to activate human gammadelta T cells. In the present study, we indicate that activation of primary gammadelta T cells by pamidronate strictly depends on the presence of monocyte-lineage cells, unlike that by pyrophosphomonoesters. Thus, although pamidronate induced cell clustering, proliferation, and IFN-gamma production of gammadelta T cells in the culture of PBMC, it failed to induce any of these activities in the culture of purified primary gammadelta T cells. By adding back the purified monocytes, however, both cell clustering and IFN-gamma production of gammadelta T cells by pamidronate could be restored. The pamidronate-pulsed, but not untreated, myelomonocytic line, THP-1, was capable of activating the purified gammadelta T cells to produce IFN-gamma, which was associated with the down-regulation of gammadelta TCR. Furthermore, pamidronate-pulsed THP-1 cells were significantly more susceptible to gammadelta T cell-mediated cytotoxicity than untreated THP-1. Also, TCR-defective Jurkat T cells transfected with gammadelta TCR genes produced a significant level of IL-2 in response to the pamidronate-pulsed THP-1 cells. These results have suggested strongly that human gammadelta T cells are functionally activated via gammadelta TCR by aminobisphosphonate Ag presented on the surface of monocyte lineage cells rather than directly by its free form.