Effect of lactoferrin on the production of tumor necrosis factor‐α and nitric oxide

One of the biological functions of lactoferrin is the modulation of the host defense systems, including cytokine production and immune responses. We have tested the effect of lactoferrin on the productions of tumor necrosis factor‐α and nitric oxide in some cells. Lactoferrin itself did not induce either tumor necrosis factor‐α production or nitric oxide production, but lipopolysaccharide‐stimulated tumor necrosis factor‐α production of macrophages and monocytes were inhibited by lactoferrin treatment combined with stimulant. The induction of nitric oxide synthesis in stimulated macrophages and vascular smooth muscle cells was not affected by the lactoferrin. J. Cell. Biochem. 76:30–36, 1999. © 1999 Wiley‐Liss, Inc.     

Excretory/secretory products from plerocercoids of Spirometra erinaceieuropaei suppress gene expressions and production of tumor necrosis factor-alpha in murine macrophages stimulated with lipopolysaccharide or lipoteichoic acid

We previously reported that the ES products from the plerocercoids of Spirometra erinaceieuropaei reduce nitric oxide synthase and chemokine gene expression in macrophages. In this study, we show that ES products suppressed tumor necrosis factor-alpha mRNA expression and tumor necrosis factor-alpha production in murine peritoneal macrophages stimulated with lipopolysaccharide or lipoteichoic acid in vitro. When macrophages from ES product-injected mice were stimulated with lipopolysaccharide in vitro, these cells produced smaller amounts of tumor necrosis factor-alpha compared with those taken from control mice. The suppressive effects of ES products were not restored by the treatment of indomethacin or anti-IL-10 antibody, and the ES products did not induce mRNA expression of secretory leukocyte protease inhibitor. Macrophages from C3H/HeJ mice, which have a single point mutation in the Toll-like receptor 4 gene, expressed tumor necrosis factor-alpha and IL-1alpha mRNA in the presence of lipopolysaccharide, but these expressions were less than those of macrophages from C3H/HeN. ES products significantly suppressed tumor necrosis factor-alpha gene expression and tumor necrosis factor-alpha production in macrophages from C3H/HeN and C3H/HeJ mice stimulated with lipopolysaccharide. However, ES products had no effect on IL-1 mRNA expression. Our data suggest that the plerocercoids secrete the tumor necrosis factor-alpha inhibitory products to evade the host's immune system, and that tumor necrosis factor-alpha mRNA expression might be inhibited downstream from Toll-like receptor 4 in the lipopolysaccharide signaling pathway.     

Antimicrobial peptide P18 inhibits inflammatory responses by LPS- but not by IFN-γ-stimulated macrophages

Antimicrobial peptide P18 markedly inhibited the expression of inducible nitric oxide synthase (iNOS), tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1beta) in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells, whereas magainin 2 did not inhibit these activities. P18 dose-dependently reduced nitric oxide (NO) production by LPS-stimulated RAW 264.7 macrophage cells, with complete inhibition at 20 microg P18 ml(-1). In contrast, P18 had no effect on NO production and the expression of iNOS mRNA and iNOS protein by interferon-gamma (IFN-gamma)-stimulated RAW264.7 cells, suggesting P18 selectively inhibits LPS-stimulated inflammatory responses in macrophages. An LAL assay showed that P18 has strong LPS-neutralizing activity, indicating that P18 inhibits the inflammatory responses in LPS-stimulated macrophages by direct binding to LPS. Collectively, our results indicate that P18 has promising therapeutic potential as a novel anti-inflammatory as well as antimicrobial agent.     

Peloruside A, an antimitotic agent, specifically decreases tumor necrosis factor-alpha production by lipopolysaccharide-stimulated murine macrophages

Peloruside A (peloruside) is a naturally occurring compound isolated from a New Zealand marine sponge that, like the anticancer drug paclitaxel, stabilizes microtubules and inhibits mitosis. Paclitaxel is known to induce a proinflammatory response in murine macrophages; whereas, peloruside has never been tested for its immunomodulatory effects in these cells. Although the antimitotic effects of the two drugs appear to be similar, we found that peloruside, unlike paclitaxel, does not induce murine macrophages to produce the proinflammatory mediators interleukin-12p40 (IL-12p40), tumor necrosis factor-alpha (TNF-alpha), and nitric oxide. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay confirmed that the absence of cytokine production was not caused by cytotoxicity in these nondividing cells. Additionally, there was no effect on unstimulated splenocytes; whereas, both compounds inhibited proliferation after concanalavin A (Con A) stimulation. Finally, there was a significant decrease in TNF-alpha and nitric oxide but not IL-12p40 when macrophages were cultured with lipopolysaccharide (LPS) and either paclitaxel or peloruside. These results suggest that peloruside may prove to be an effective anti-inflammatory treatment, since it does not induce the production of proinflammatory mediators yet can downregulate TNF-alpha and nitric oxide production by LPS-stimulated macrophages, as well as inhibit lymphocyte proliferation.     

Leonurus sibiricus induces nitric oxide and tumor necrosis factor-alpha in mouse peritoneal macrophages

Using mouse peritoneal macrophages, we have examined the mechanism by which Leonurus sibiricus (LS) regulates nitric oxide (NO) production. When LS was used in combination with recombinant interferon-gamma (rIFN-gamma), there was a marked cooperative induction of NO production; however, LS by itself had no effect on NO production. The increased production of NO from rIFN-gamma plus LS-stimulated cells was almost completely inhibited by pretreatment with pyrrolidine dithiocarbamate (PDTC), an inhibitor of nuclear factor kappaB. Furthermore, treatment of peritoneal macrophages with rIFN-gamma plus LS caused a significant increase in tumor necrosis factor-alpha (TNF-alpha) production. PDTC also decreased the effect of LS on TNF-alpha production significantly. Because NO and TNF-alpha play an important role in immune function and host defense, LS treatment could modulate several aspects of host defense mechanisms as a result of stimulation of the inducible nitric oxide synthase.     

Two neolignans from Perilla frutescens and their inhibition of nitric oxide synthase and tumor necrosis factor-alpha expression in murine macrophage cell line RAW 264.7

Two neolignans were isolated from leaves of Perilla frutescens (Labiatae) as inhibitors of nitric oxide syntheses (IC50 5.9 microM and 53.5 microM, respectively) and tumor necrosis factor-alpha in lipopolysaccharide-activated RAW 264.7 cells. Their structures were identified as 1beta,2alpha,3beta,4alpha-1,2-dimethyl-3,4-bis-(2,4,5-trimethoxyphenyl)-cyclobutane (magnosalin), and 1alpha,2beta,3beta,4alpha-1,2-dimethyl-3,4-bis-(2,4,5-trimethoxyphenyl)-cyclobutane (andamanicin), and their activities were confirmed as resulted from the suppressed expression of inducible nitric oxide synthase enzyme and from the secretion of tumor necrosis factor-alpha from activated macrophages.     

Crucial cytokine interactions in nitric oxide production induced by Mycoplasma arthritidis superantigen

Mycoplasma arthritidis causes autoimmune arthritis in rodents. It produces a superantigen (MAM) that simultaneously activates antigen presenting cells and T cells inducing nitric oxide and cytokine release. Nitric oxide is a key inducer and regulator of the immune system activation. Here, we investigated nitric oxide and cytokine production and interactions of these molecules in MAM-stimulated co-cultures of macrophages (J774A.1 cell line) with spleen lymphocytes. We found that: a) MAM-induced nitric oxide, interferon-gamma, membrane-associated tumor necrosis factor and interleukin-2 production in co-cultures of macrophages with lymphocytes from BALB/c and C3H/HePas but not from C57Bl/6 mice; b) production of nitric oxide was dependent on interferon-gamma whereas that of interferon-gamma was dependent on interleukin-2 and membrane-associated tumor necrosis factor; c) these cytokines up regulated MAM-induced nitric oxide production. Unraveling the mechanisms of cell activation induced by MAM might be helpful to design strategies to prevent immune system activation by superantigens and therefore in seeking amelioration of associated immunopathologies.     

Effects of centimeter waves on the immune system of mice in endotoxic shock

The effects of centimeter waves (8.15-18 GHz, 1 microW/cm2, 1 h daily for 10 days; MW) on the production of the tumor necrosis factor alpha, interleukin-lalpha, interleukin-1beta, interleukin-2, and the expression of interleukin-6, interleukin-10, interferon-gamma, nitric oxide and HSP27, HSP72 and HSP90alpha in mice irradiated before or after LPS injection were studied. An acute endotoxic model was produced by a single LPS injection. The effects of microwaves on nitric oxide, interleukin-6, tumor necrosis factor-alpha, and interferon-gamma were dependent on the functional status of exposed animals. Thus, an exposure of healthy mice to microwaves for 10 days was followed by a decrease in nitric oxide and interferon-gamma production, and an increase in the production of the tumor necrosis factor-alpha and interleukin-6. On the contrary, an exposure to MW before intoxication resulted in an increase in the synthesis of nitric oxide and interferon-gamma as well as a decrease in the concentration of the tumor necrosis factor-alpha and interleukin-6 in blood of mice in endotoxic shock. When microwave exposure was used after LPS injection, it did not provide any protective effect, and preliminary irradiation enhanced the resistance of the organism to endotoxic shock.     

Priming and activation of mouse macrophages by trehalose 6,6'-dicorynomycolate vesicles from Corynebacterium glutamicum

Vesicles consisting of pure trehalose dicorynomycolate (TDCM), the corynebacterial analog of the most studied mycobacterial glycolipid 'cord factor', were isolated from Corynebacterium glutamicum cells by mild detergent treatment; these induced in vivo a macrophage priming similar to that obtained with mycobacterial-derived trehalose dimycolate. In vitro, both TDCM and bacterial lipopolysaccharide (LPS) induced in macrophages the production of nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha), endotoxin tolerance, and were primed for an enhanced secondary NO response to LPS. Interferon-gamma pretreatment did not influence the LPS-induced TNF-alpha response, but considerably increased the TDCM-induced response.     

4-dimethylamino-3',4'-dimethoxychalcone downregulates iNOS expression and exerts anti-inflammatory effects

Reactive oxygen and nitrogen species contribute to the pathophysiology of inflammatory conditions. We have studied the effects of a novel superoxide scavenger, 4-dimethylamino-3', 4'-dimethoxychalcone (CH11) in macrophages and in vivo. CH11 has been shown to inhibit the chemiluminescence induced by zymosan in mouse peritoneal macrophages and the cytotoxic effects of superoxide. In the same cells, the modulation by superoxide of nitric oxide (NO) production in response to zymosan was investigated. CH11 was more effective than the membrane-permeable scavenger Tiron for inhibition of inducible nitric oxide synthase (iNOS) protein expression and nitrite production. We have shown that CH11 inhibited chemiluminescence in vivo, as well as cell migration, and eicosanoid and tumor necrosis factor-alpha (TNF-alpha) levels in the mouse air pouch injected with zymosan. This chalcone derivative also exerted anti-inflammatory effects in the carrageenan paw oedema.     

Production of nitric oxide and tumor necrosis factor-alpha by Smilacis rhizoma in mouse peritoneal macrophages

Using mouse peritoneal macrophages, we have examined the mechanism by which, Smilacis rhizoma (SR) regulates nitric oxide (NO) production. When SR was used in combination with recombinant interferon-gamma (rIFN-gamma), there was a marked cooperative induction of NO production. However, SR had no effect on NO production by itself. The increased production of NO from rIFN-gamma plus SR-stimulated cells was almost completely inhibited by pre-treatment with pyrrolidine dithiocarbamate (PDTC), an inhibitor of nuclear factor kappa B (NF-kappaB). Furthermore, treatment of peritoneal macrophages with rIFN-gamma plus SR caused a significant increase in tumor necrosis factor-alpha (TNF-alpha) production. PDTC also decreased the effect of SR on TNF-alpha production significantly. These findings demonstrate that SR increases the production of NO and TNF-alpha by rIFN-gamma-primed macrophages and suggest that NF-kappaB plays a critical role in mediating these effects of SR.     

NK cells activated in vivo by bacterial DNA control the intracellular growth of Francisella tularensis LVS

We demonstrated previously that mice treated with bacterial or oligonucleotide DNA containing unmethylated CpG motifs are transiently protected against lethal parenteral challenge with the intracellular bacterium Francisella tularensis Live Vaccine Strain (LVS). Here we explore the cellular basis of this protection. Wild-type mice that were treated with CpG oligonucleotide DNA and challenged with a lethal dose of LVS survived, while mice lacking TLR9 did not. In vitro, treatment of LVS-infected macrophages and/or naive splenocytes with oligo DNA had no impact on intracellular bacterial replication. In contrast, in vitro co-culture of LVS-infected macrophages with splenocytes obtained from mice treated with oligo DNA in vivo resulted in control of intracellular LVS growth. Control was reversed by antibodies to interferon-gamma or to tumor necrosis factor-alpha and by inhibition of nitric oxide, and to a lesser degree by antibodies to Interleukin-12. Further, splenocytes from DNA-primed normal, T cell KO, B cell KO, lymphocyte-deficient scid, or perforin KO mice all controlled intra-macrophage LVS growth. Enriched DNA-primed natural killer cells, but not B cells, clearly controlled intracellular LVS growth. Thus, NK cells contribute to DNA-mediated protection by production of cytokines including IFN-gamma and TNF-alpha, resulting in nitric oxide production and control of intracellular Francisella replication.     

Thrombin-induced activation of cultured rodent microglia

Microglia are the resident immune cells of the CNS. Upon brain damage, these cells are rapidly activated and function as tissue macrophages. The first steps in this activation still remain unclear, but it is widely believed that substances released from damaged brain tissue trigger this process. In this article, we describe the effects of the blood coagulation factor thrombin on cultured rodent microglial cells. Thrombin induced a transient Ca(2+) increase in microglial cells, which persisted in Ca(2+)-free media. It was blocked by thapsigargin, indicating that thrombin caused a Ca(2+) release from internal stores. Preincubation with pertussis toxin did not alter the thrombin-induced [Ca(2+)](i) signal, whereas it was blocked by hirudin, a blocker of thrombin's proteolytic activity. Incubation with thrombin led to the production of nitric oxide and the release of the cytokines tumor necrosis factor-alpha, interleukin-6, interleukin-12, the chemokine KC, and the soluble tumor necrosis factor-alpha receptor II and had a significant proliferative effect. Our findings indicate that thrombin, a molecule that enters the brain at sites of injury, rapidly triggered microglial activation.     

Leishmania major proteophosphoglycan is expressed by amastigotes and has an immunomodulatory effect on macrophage function

Proteophosphoglycan (PPG) is a newly described mucin-like glycoprotein found on the surface of Leishmania major promastigotes and secreted in the culture supernatant. We show here that antigenically similar PPGs are present in several Leishmania species. PPG could also be detected on the surface of amastigotes and in small, parasite-free vesicles in infected macrophages. Because of the similarity of its carbohydrate chains to lipophosphoglycan, a parasite receptor for host macrophages, PPG was tested for binding to macrophages. PPG bound to macrophages and was internalized in a time-dependent manner. PPG inhibited the production of tumor necrosis factor-alpha and synergized with interferon-gamma to stimulate the production of nitric oxide by macrophages. PPG may contribute to the binding of Leishmania to host cells and may play a role in modulating the biology of the infected macrophage at the early stage of infection.     

Endotoxin-refractory liver macrophages secrete tumor necrosis factor-alpha upon viral infection

Rat liver macrophages (Kupffer cells) secrete tumor necrosis factor-alpha (cachectin) after exposure to Newcastle disease virus or bacterial endotoxin. Macrophages treated with endotoxin become refractory and fail to release tumor necrosis factor-alpha to a secondary challenge with endotoxin. The acquisition of the refractory state is dose-dependent, requires the continuous presence of endotoxin for a minimum of 8 h, is transient, and reversible. Endotoxin, however, renders Kupffer cells unresponsive only to itself. When endotoxin-refractory macrophages are activated by Newcastle disease virus, they still secrete tumor necrosis factor-alpha in amounts expected with this stimulus. Immunoprecipitation studies show that the precursor of tumor necrosis factor-alpha is found only in lysates of endotoxin-sensitive, but not in refractory macrophages, thus arguing against a post-translational regulatory process. Whereas prostaglandin E2 inhibits the production of tumor necrosis factor-alpha in response to endotoxin and viruses, it does not appear to mediate the refractory state.     

Modulation of macrophage activation by prostaglandins

The effect of prostaglandtn E(2), iloprost and cAMP on both nitric oxide and tumour necrosis factor-alpha release in J774 macrophages has been studied. Both prostaglandin E(2) and iloprost inhibited, in a concentration-dependent fashion, the lipopolysaccharide-induced generation of nitric oxide and tumour necrosis factor-alpha. The inhibitory effect of these prostanoids seems to be mediated by an increase of the second messenger cAMP since it was mimicked by dibutyryl cAMP and potentiated by the selective type IV phosphodiesterase inhibitor RO-20-1724. Our results suggest that the inhibition of nitric oxide release by prostaglandin E(2) and iloprost in lipopolysaccharide-activated J774 macrophages may be secondary to the inhibition of tumour necrosis factor-alpha generation, which in turn is likely to be mediated by cAMP.     

Surfactant protein A enhances mycobacterial killing by rat macrophages through a nitric oxide-dependent pathway

Surfactant-associated protein A (SP-A) is involved in surfactant homeostasis and host defense in the lung. We have previously demonstrated that SP-A specifically binds to and enhances the ingestion of bacillus Calmette-Guerin (BCG) organisms by macrophages. In the current study, we investigated the effect of SP-A on the generation of inflammatory mediators induced by BCG and the subsequent fate of ingested BCG organisms. Rat macrophages were incubated with BCG in the presence and absence of SP-A. Noningested BCG organisms were removed, and the release of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide were measured at varying times. TNF-alpha and nitric oxide production induced by BCG were enhanced by SP-A. In addition, SP-A enhanced the BCG-induced increase in the level of inducible nitric oxide synthase protein. Addition of antibodies directed against SPR210, a specific macrophage SP-A receptor, inhibited the SP-A-enhanced mediator production. BCG in the absence of SP-A showed increased growth over a 5-day period, whereas inclusion of SP-A dramatically inhibited BCG growth. Inhibition of nitric oxide production blocked BCG killing in the presence and absence of SP-A. These results demonstrate that ingestion of SP-A-BCG complexes by rat macrophages leads to production of inflammatory mediators and increased mycobacterial killing.     

Functional alterations in macrophages after hypoxia selection

Regions of low oxygen tension are common features of inflamed and infected tissues and provide physiologic selective pressure for the expansion of cells with enhanced hypoxia tolerance. The aim of this study was to investigate whether macrophages resistant to death induced by hypoxia were accompanied by functional alterations. A mouse macrophage cell line (J774 cells) was used to obtain subpopulations of death-resistant macrophages induced by long-term exposure to severe hypoxia (<1% O(2)). The results indicated that exposing J774 macrophages to periods of severe hypoxia results in the selection of cells with phenotypes associated with the modulation of heat-shock protein 70 kDa (HSP70) expression, tumor necrosis factor-alpha (TNF-alpha), and nitric oxide (NO) production and reduced susceptibility to parasite Leishmania infection. Thus, we suggest that hypoxia-selected macrophages may influence the outcome of inflammation and infection.     

Caffeic acid phenethyl ester protects mice from lethal endotoxin shock and inhibits lipopolysaccharide-induced cyclooxygenase-2 and inducible nitric oxide synthase expression in RAW 264.7 macrophages via the p38/ERK and NF-kappaB pathways

Caffeic acid phenethyl ester has been shown to have anti-inflammatory and anti-cancer effects. We examined the effects of caffeic acid phenethyl ester on lipopolysaccharide-induced production of nitric oxide and prostaglandin E(2), and expression of inducible nitric oxide synthase and cyclooxygenase-2 in RAW 264.7 macrophages. We also investigated the effects of caffeic acid phenethyl ester on lipopolysaccharide-induced septic shock in mice. Our results indicate that caffeic acid phenethyl ester inhibits lipopolysaccharide-induced nitric oxide and prostaglandin E(2) production in a concentration-dependent manner and inhibits inducible nitric oxide synthase and cyclooxygenase-2 in RAW 264.7 cells, without significant cytotoxicity. To further examine the mechanism responsible for the inhibition of inducible nitric oxide synthase and cyclooxygenase-2 expression by caffeic acid phenethyl ester, we examined the effect of caffeic acid phenethyl ester on lipopolysaccharide-induced nuclear factor-kappaB activation and the phosphorylation of mitogen-activated protein kinases. Caffeic acid phenethyl ester treatment significantly reduced nuclear factor-kappaB translocation and DNA-binding in lipopolysaccharide-stimulated RAW 264.7 cells. This effect was mediated through the inhibition of the degradation of inhibitor kappaB and by inhibition of both p38 mitogen-activated protein kinase and extracellular signal-regulated kinase phosphorylation, at least in part by inhibiting the generation of reactive oxygen species. Furthermore, caffeic acid phenethyl ester rescued C57BL/6 mice from lethal lipopolysaccharide-induced septic shock, while decreasing serum levels of tumor necrosis factor-alpha and interleukin-1beta. Collectively, these results suggest that caffeic acid phenethyl ester suppresses the induction of cytokines by lipopolysaccharide, as well as inducible nitric oxide synthase and cyclooxygenase-2 expression, by blocking nuclear factor-kappaB and p38/ERK activation. These findings provide mechanistic insights into the anti-inflammatory and chemopreventive actions of caffeic acid phenethyl ester in macrophages.