Protein kinase C independent activation of inducible nitric oxide synthase by tumor necrosis factor-alpha in TM4 Sertoli cells

To investigate the nitric oxide (NO) production and its signalling mechanism in TM4 Sertoli cells, the cells were treated with recombinant tumor necrosis factor-alpha (rTNF-alpha), recombinant interleukin-1 alpha (rIL-1alpha), or lipopolysaccharide (LPS), either alone or in combination with recombinant interferon-gamma (rIFN-gamma), and NO production was measured by using the Griess method. TM4 Sertoli cells produced a small amount of NO upon treatment with rIFN-gamma. The effect of rIFN-gamma was drastically increased by cotreatment with rTNF-alpha in a dose-dependent manner. However, combination of rIL-1alpha or LPS with rIFN-gamma did not synergize to activate cells. RIFN-gamma in combination with rTNF-alpha showed marked increase of the expression of iNOS protein. Protein kinase C inhibitors did not inhibit the production of NO induced by rIFN-gamma plus rTNF-alpha. These results suggest that the role of TNF-alpha is to provide TM4 Sertoli cells with the active cofactor for NO production and TNF-alpha-induced signaling for induction of NO synthesis is not dependent on protein kinase C activation.     

Phagocytosis enhances murine macrophage activation by interferon-gamma and tumor necrosis factor-alpha

Previously, we reported that exposure of bone marrow-derived macrophages (M phi) to a phagocytic stimulus in the simultaneous presence of interferon-gamma (IFN-gamma) induced these cells to generate nitrite (NO2-). This effect was achieved using both living (i.e. promastigotes of the protozoan parasite Leishmania enriettii) and inert (latex beads) particles. When the phagocytic stimulus was Leishmania, enhanced intracellular killing accompanied elevated NO2- secretion. As shown here, the capacity of phagocytosis to elicit NO2- production by IFN-gamma-treated M phi was inhibited by antibody to murine recombinant tumor necrosis factor-alpha (rTNF-alpha), suggesting that phagocytosis enabled IFN-gamma to activate M phi via the induction of TNF-alpha as an autocrine second signal. M phi NO2- production in response to rIFN-gamma and either exogenous TNF-alpha or Leishmania was strongly enhanced by prostaglandin E2, consistent with such a mechanism. However, addition of either Leishmania promastigotes or latex beads to M phi cultures simultaneously exposed to both IFN-gamma and exogenous murine or human rTNF-alpha further potentiated activation as measured by NO2- release. Furthermore, anti-TNF antibody failed to inhibit M phi responses to rIFN-gamma and bacterial lipopolysaccharide (LPS) in the presence or absence of Leishmania; also exogenous rTNF-alpha did not significantly affect NO2- production by IFN-gamma/LPS cultures despite a strong enhancement by Leishmania. These results suggest that phagocytosis enhances M phi responses by a process more complex than the sole induction of TNF-alpha. Phagocytosis also increased M phi NO2- production elicited by IFN-gamma plus TNF-alpha in L-arginine-deficient media. These results indicate that phagocytosis may be an important mechanism of up-regulating M phi microbicidal activity, and could be particularly relevant upon arginine depletion which occurs during an inflammatory response.     

Scrophularia buergeriana regulates cytokine production in vitro

The Scrophularia buergeriana (SB) has long been used to treat various diseases an account of its antimicrobial and anti-virus activity. However, it is unclear how SB regulates the immune responses. This study investigated the effect of SB on the production of cytokines in a human T-cell line, MOLT-4 cells, and mouse peritoneal macrophages. The MOLT-4 cells were cultured for 24 h in the presence or absence of SB plus concanavalin (con) A. SB plus con A significantly increased the level of interleukin (IL)-2, IL-4 and interferon (IFN)-gamma production compared with that of con A alone (approximately 1.79-fold for IL-2, 2-fold for IL-4, and 1.85-fold for IFN-gamma, p < 0.05). SB plus recombinant IFN-gamma (rIFN-gamma) increased the level of IL-12 and NO production compared with rIFN-gamma alone. In addition, SB plus rIFN-gamma increased the level the iNOS expression on mouse peritoneal macrophages. Overall, SB may have an immune-enhancement effect through cytokine production.     

Inhibition of the induction of the inducible nitric oxide synthase in murine brain microglial cells by sodium salicylate.

The induction of the inducible nitric oxide synthase (iNOS) has been proposed to play a role in a variety of inflammatory diseases. Sodium salicylate (NaSal) is the most commonly used anti-inflammatory agent. We investigated whether NaSal can diminish the induction of iNOS in murine brain microglial cells. In primary cultures, interferon-gamma (IFN-gamma) or lipopolysaccharide (LPS) separately did not stimulate nitric oxide (NO) production, whereas IFN-gamma combined with LPS synergistically induced iNOS. NaSal inhibited both the production of NO and expression of iNOS in microglial cells. Synergy between IFN-gamma and LPS was mainly dependent on tumour necrosis factor-alpha (TNF-alpha) secretion as the increase of the induction of the iNOS by IFN-gamma plus LPS was associated with the increase of TNF-alpha secretion and IFN-gamma plus LPS-induced TNF-alpha secretion by microglial cells was decreased by the treatment with NaSal. These results suggest a possible use of NaSal in managing inflammation of the central nervous system through inhibition of the iNOS induction.     

Inhibition of caspase 3 abrogates lipopolysaccharide-induced nitric oxide production by preventing activation of NF-kappaB and c-Jun NH2-terminal kinase/stress-activated protein kinase in RAW 264.7 murine macrophage cells

The effect of caspase inhibitors on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 267.4 murine macrophage cells was investigated. Pretreatment of RAW cells with a broad caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-FMK), resulted in a striking reduction in LPS-induced NO production. Z-VAD-FMK inhibited LPS-induced NF-kappaB activation. Furthermore, it blocked phosphorylation of c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) but not that of extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinases. Similarly, a caspase 3-specific inhibitor, Z-Asp-Glu-Val-Asp-fluoromethylketone, inhibited NO production, NF-kappaB activation, and JNK/SAPK phosphorylation in LPS-stimulated RAW cells. The attenuated NO production was due to inhibition of the expression of an inducible-type NO synthase (iNOS). The overexpression of the dominant negative mutant of JNK/SAPK and the addition of a JNK/SAPK inhibitor blocked iNOS expression but did not block LPS-induced caspase 3 activation. It was therefore suggested that the inhibition of caspase 3 might abrogate LPS-induced NO production by preventing the activation of NF-kappaB and JNK/SAPK. The caspase family, especially caspase 3, is likely to play an important role in the signal transduction for iNOS-mediated NO production in LPS-stimulated mouse macrophages.     

Endogenous tumor necrosis factor alpha is required for enhanced antimicrobial activity against Toxoplasma gondii and Listeria monocytogenes in recombinant gamma interferon-treated mice.

In vitro studies have shown that macrophages stimulated with recombinant gamma interferon (rIFN-gamma) produce tumor necrosis factor alpha (TNF-alpha), which in an autocrine fashion activates these cells. The aim of the present study was to determine whether endogenously formed TNF-alpha also is required for rIFN-gamma-induced macrophage activation and enhanced antimicrobial activity in vivo. After an intraperitoneal injection of rIFN-gamma into CBA/J mice, their peritoneal macrophages released enhanced amounts of NO2- and inhibited the intracellular proliferation of Toxoplasma gondii. Injection of neutralizing antibodies against TNF-alpha simultaneously with the rIFN-gamma completely inhibited both the release of NO2- by macrophages and their toxoplasmastatic activity. Similar results were observed after intraperitoneal injection of a competitive inhibitor of L-arginine, NG-monomethyl-L-arginine, together with rIFN-gamma, demonstrating that in vivo L-arginine-derived reactive nitrogen intermediates are essential for the induction of toxoplasmastatic activity. Intravenous injection of rIFN-gamma inhibited the growth of Listeria monocytogenes in the livers and spleens of mice; this effect was abrogated by antibodies against TNF-alpha. Intravenous injection of a large dose of rTNF-alpha resulted in a decrease in the number of bacteria in the liver and spleen, but an injection of rIFN-gamma and rTNF-alpha did not result in enhanced inhibition of the proliferation of L. monocytogenes. Together, the results of the present study are the first to demonstrate that endogenous TNF-alpha is required in vivo for the expression of macrophage activation with respect to the release of reactive nitrogen intermediates and toxoplasmastatic activity and for enhanced listericidal activity in the livers and spleens of mice stimulated with rIFN-gamma.     

Nitric oxide production by high molecular weight water-soluble chitosan via nuclear factor-kappaB activation

High molecular weight water-soluble chitosan (WSC), having an average molecular weight of 300000 Da and a degree of deacethylation over 90%, can be produced using a simple multi-step membrane separation process. In this study, the effect of WSC on the production of nitric oxide (NO) in RAW 264.7 macrophages was evaluated. Water-insoluble chitosan alone has been previously shown to exhibit in vitro stimulatory effect on macrophages NO production. However, WSC had no effect on NO production by itself. When WSC was used in combination with recombinant interferon-gamma (rIFN-gamma), there was a marked cooperative induction of NO synthesis in a dose-dependent manner. The optimal effect of WSC on NO synthesis was shown 24 h after treatment with rIFN-gamma. The increased production of NO from rIFN-gamma plus WSC-stimulated RAW 264.7 macrophages was decreased by the treatment with N(G)-monomethyl-L-arginine (N(G)MMA). The increase in NO synthesis was reflected, as an increased amounts of inducible NO synthase protein. In addition, synergy between rIFN-gamma and WSC was mainly dependent on WSC-induced tumor necrosis factor-alpha (TNF-alpha) and nuclear factor-kappaB (NF-kappaB) activation. The present results indicate that the capacity of WSC to increase NO production from rIFN-gamma-primed RAW 264.7 macrophages is the result of WSC-induced TNF-alpha secretion via the signal transduction pathway of NF-kappaB activation.     

LY294002 inhibits LPS-induced NO production through a inhibition of NF-kappaB activation: independent mechanism of phosphatidylinositol 3-kinase

Phosphatidylinositol 3-kinase (PI3K) is critical player in cell proliferation and survival. The effects of LY294002 and wortmannin, inhibitors of PI3K, on nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in lipoploysaccharide (LPS)-induced Raw 264.7 cells were investigated. Significant inhibition of LPS-induced protein kinase B (PKB, Akt) phosphorylation occurred at 25 microM LY294002 or 0.5 microM wortmannin. At the same concentrations, LY294002, but not wortmannin, significantly inhibited NO production and iNOS expression. LY303511, an inactive analogue of LY294002, also inhibited NO production and iNOS expression. In addition, LY294002 and LY303511 significantly inhibited the DNA binding activity of NF-kappaB and NF-kappaB dependent reporter gene expression. These results suggest that LY294002 inhibits iNOS expression at least in part via inhibition of NF-kappaB activation, independent of PI3K.     

Modulation and function of intercellular adhesion molecule-1 (CD54) on human retinal pigment epithelial cells.

As part of the blood-retina barrier, the neuroectodermally-derived retinal pigment epithelial (RPE) monolayer is strategically positioned to interact with circulating leukocytes and regulate their access to the retina. We, therefore, studied whether human RPE cells express intercellular adhesion molecule-1 (ICAM-1), a specialized cell surface glycoprotein that binds the leukocyte function antigen-1 receptor present on all leukocytes. Using specific monoclonal antibody to ICAM-1, immunohistochemical staining of freshly-isolated primary and fourth passaged human RPE cells resulted in delicate reaction product that increased dramatically upon exposure to human recombinant (r) interferon-gamma (rIFN-gamma), interleukin-1-beta (rIL-1 beta), or tumor necrosis factor-alpha (rTNF-alpha). Fluorescence-activated cell sorting analysis demonstrated 2-fold increases in constitutive RPE ICAM-1 expression within 6 hours of exposure to physiologic concentrations of rIFN-gamma, rIL-1 beta, or rTNF-alpha. In standardized leukocyte adherence assays, cultured RPE cells showed avid binding of neutrophils that increased significantly after stimulation with rIFN-gamma, rIL-1 beta, or rTNF-alpha (p less than 0.001). In parallel assays, monoclonal antibody to either ICAM-1 on RPE cells, or subunits of leukocyte function antigen-1 receptors on leukocytes significantly blocked leukocyte binding to unstimulated (p less than 0.001) or rIFN-gamma-stimulated RPE cells (p less than 0.001). To demonstrate RPE ICAM-1 expression in intact human tissue, fresh uveoretinal explants were exposed to rIFN-gamma, rIL-1 beta, or rTNF-alpha and stained using mAb to ICAM-1. Tissue sections of cytokine-stimulated explants revealed dramatic increases in RPE ICAM-1 immunoreactivity over the low levels observed in unstimulated uveoretinal tissue. Our results indicate that: (a) ICAM-1 is expressed at low levels on unstimulated RPE cells, (b) RPE ICAM-1 may be augmented by inflammatory cytokines, and (c) RPE ICAM-1 is a functional receptor mediating leukocyte binding. ICAM-1 on RPE cells at the blood-retina barrier may regulate leukocytic infiltration in ocular diseases in which leukocytes are important pathogenetically and may be important to the generation of ocular immune responses.     

Activation of murine macrophages by tumor necrosis factor, interleukin-1, interferon-gamma and cisplatin

Murine peritoneal macrophages were rendered tumoricidal to Dalton's lymphoma (DL) cells on incubation with recombinant tumor necrosis factor alpha (rTNF-alpha), recombinant interleukin-1 (rIL-1) and cisplatin in vitro. Simultaneous treatment of macrophages with suboptimal doses of rTNF-alpha and rIL-1 had additive effect on the activation of macrophages. Priming of macrophages with recombinant interferon gamma (rIFN-gamma) significantly enhanced the rTNF-alpha and rIL-1-induced macrophage cytotoxicity. Cisplatin was found to up-regulate rIL-1-induced macrophage activation but inhibited the activation of macrophages with rTNF-alpha. These studies indicate the potential of appropriate combination of these Biological Response Modifiers (BRMs) against neoplasia.     

The nitric oxide-producing properties of Ulmi radicis cortex extract.

We investigated the effect of Ulmi radicis cortex extract (UrCE) on the production of nitric oxide (NO). Stimulation of mouse peritoneal macrophages with UrCE after the treatment of recombinant interferon-gamma (rIFN-gamma) resulted in the increased NO synthesis. UrCE had no effect on NO synthesis by itself. When UrCE was used in combination with rIFN-gamma, there was a marked cooperative induction of NO synthesis in a dose-dependent manner. The optimal effect of UrCE on NO synthesis was shown 6 h after treatment with rIFN-gamma. NO production was inhibited by NG-monomethyl-L-arginine. The increased production of NO from rIFN-gamma plus UrCE-stimulated cells was decreased by the treatment of protein kinase C inhibitor such as staurosporin. In addition, synergy between rIFN-gamma and UrCE was mainly dependent on UrCE-induced tumor necrosis factor-alpha (TNF-alpha) secretion. All the preparations of UrCE were endotoxin free. These results suggest that the capacity or UrCE to increase NO production from rIFN-gamma-primed mouse peritoneal macrophages is the result of UrCE-induced TNF-alpha secretion.     

Intercellular adhesion molecule-1 on eosinophils is involved in eosinophil protein X release induced by cytokines.

Recent evidence suggests that adhesion molecules play important roles in eosinophil functions such as degranulation and superoxide anion production. CD11b/CD18 (Mac-1) and CD49d/CD29 (VLA-4) are involved in eosinophil-endothelial adhesion through their counterligands, intercellular adhesion molecule-1 (ICAM-1; CD54) and vascular cell adhesion molecule-1 (VCAM-1), respectively. CD54 is also induced on eosinophils by cytokine stimulation. We hypothesized that CD54 on human eosinophils may participate in eosinophil degranulation. CD54 was induced on eosinophils by a combination of human recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF) and human recombinant tumour necrosis factor-alpha (rTNF-alpha) within 2 hr of incubation, as determined by flow cytometric analysis. Recombinant GM-CSF alone induced a slight but significant CD54 expression on eosinophils. Release of eosinophil protein X, an indicator of eosinophil degranulation, was induced by rGM-CSF and this effect was synergistically enhanced by adding rTNF-alpha. To determine the role of newly expressed CD54 in eosinophil degranulation, a blocking assay was performed using monoclonal antibody (mAb) against CD54 and CD18. Anti-CD18 mAb and anti-CD54 mAb markedly inhibited eosinophil degranulation induced by rGM-CSF or a combination of rGM-CSF and rTNF-alpha. On the other hand, anti-CD54 mAb had little effect on rGM-CSF- or rGM-CSF/rTNF-alpha-induced adhesion of eosinophils, whereas anti-CD18 mAb significantly inhibited eosinophil adhesion. These results indicate that CD54 on eosinophils plays an important role in the eosinophil degranulation and that eosinophils are capable of interacting with other beta 2 integrin-positive cells.     

Effects of biological response modifiers on lung natural killer activity.

Natural killer (NK) activity plays an important role in host defense against tumors, especially once augmented by immunomodulators. It is likely that the modulation of NK cells is a reflection of the environment in which they reside. The current study was undertaken to characterize the response profile of lung interstitial lymphocyte natural killer (LLNK) activity to various biological response modifiers (BRM) in vitro after short term incubation (18h). The presented data show that treatment of lung lymphocytes with human recombinant interleukin 2 (rIL-2), purified rat interferon alpha/beta (IFN-alpha/beta), or murine recombinant tumor necrosis factor alpha (rTNF-alpha) resulted in a dose-dependent increase in LLNK activity. The maximum stimulation was similar for rIL-2 and IFN-alpha/beta, although a much higher concentration of IFN-alpha/beta was required to reach this level of stimulation. The maximum response to rTNF-alpha treatment was about half that seen with rIL-2 or IFN-alpha/beta and it, too, required a high concentration. By contrast, rat recombinant interferon gamma (rIFN-gamma) or murine recombinant interleukin 1 (rIL-1) failed to alter LLNK activity significantly when used alone. Furthermore, doses of IFN-alpha/beta and rTNF-alpha that had little enhancing effect were able to synergize with a suboptimal dose of rIL-2, whereas rIL-1 and rIFN-gamma failed to do so. These data demonstrate the response of lung NK activity to BRM treatment, which is important for the responsible and effective use of BRM. However the spectrum of lung NK cell response to BRM is smaller than that previously reported for NK cells from other anatomic compartments.