Chloroquine inhibits production of TNF-alpha, IL-1beta and IL-6 from lipopolysaccharide-stimulated human monocytes/macrophages by different modes.

OBJECTIVES: TNF-alpha, IL-1 and IL-6 are known to have primary roles in the pathogenesis of rheumatoid arthritis and other inflammatory diseases. The anti-rheumatic drug chloroquine has been shown to inhibit TNF-alpha, IL-1 and IL-6 production from mononuclear phagocytes. We examined the underlying mechanisms involved in the chloroquine-induced inhibition of cytokine production. METHODS: Human peripheral blood mononuclear cells and monocytes/macrophages and monocytic U-937 and THP-1 cells were stimulated with lipopolysaccharide, and TNF-alpha, IL-1beta and IL-6 production was measured by ELISA. Levels of mRNA were measured by northern blotting and reverse transcription-polymerase chain reaction. Synthesis of 26-kDa TNF-alpha precursor was measured by metabolic labelling and immunoprecipitation analysis. Transcription rate was determined by nuclear run-on assay. RESULTS: TNF-alpha release from the cells was inhibited by chloroquine, whereas the steady-state level of TNF-alpha mRNA and synthesis of 26-kDa TNF-alpha precursor were not changed by chloroquine. In contrast, chloroquine-induced inhibition of IL-1beta and IL-6 release was accompanied by a decrease in their steady-state mRNA levels. The transcription rates of the IL-1beta and IL-6 genes were not changed by chloroquine, whereas the stability of IL-1beta and IL-6 mRNA was decreased by chloroquine. Weak-base amines such as methylamine and ammonium chloride had no effect on the production of TNF-alpha, whereas they partially blocked the production of IL-1beta and IL-6. CONCLUSIONS: Our results indicate that chloroquine-mediated inhibition of TNF-alpha, IL-1beta and IL-6 synthesis occurs through different modes in lipopolysaccharide-stimulated human monocytes/macrophages: it blocks the conversion of cell-associated TNF-alpha precursor to mature soluble protein, whereas it reduces the levels of IL-1beta and IL-6 mRNA, at least in part, by decreasing their stability and by a pH-dependent mechanism.

Murine pro-tumor necrosis factor expressed in Saccharomyces cerevisiae HF7c localizes to membrane/particulate.

Tumor necrosis factor (TNF) is a cytokine that is produced by immune cells in response to bacterial and viral stimuli and plays important roles in various inflammatory diseases. TNF is produced as a membrane-bound precursor, which is then cleaved to release soluble mature protein. We expressed murine pro-TNF in Saccharomyces cerevisiae and examined processing and cellular localization of the recombinant protein. Yeast cells were transformed with an expression construct carrying the pro-TNF gene under the control of alcohol dehydrogenase promoter. Immunoblotting analysis of cell homogenate revealed expression of 26 kD pro-TNF in transformed cells. Upon centrifugation, pro-TNF transformed cells fractionated into the membrane/particulate. In a clone that expresses a high level of pro-TNF, mature 17 kD TNF was detected in the culture medium, although the amount was far smaller than that of cell-associated pro-TNF. Flow cytometric analysis of yeast spheroplasts demonstrated the presence of TNF on the cell surface. Our results show that pro-TNF expressed in yeast mainly resides in the cellular membrane with an orientation similar to that of pro-TNF produced in mammalian cells. Our data suggest that the transformed yeast cells can be used for the genetic analysis of pro-TNF processing machinery in immune cells.

Thiol-reactive metal compounds inhibit NF-kappa B activation by blocking I kappa B kinase.

Gold compounds are used in the treatment of rheumatoid arthritis. NF-kappa B is a transcription factor implicated in the expression of many inflammatory genes. NF-kappa B is activated by signal-induced phosphorylation and subsequent degradation of inhibitory I kappa B (inhibitory protein that dissociates from NF-kappa B) proteins, and a multisubunit I kappa B kinase (IKK) has been identified previously. We tested the effect of various gold compounds on the activation of NF-kappa B and IKK in LPS-stimulated RAW 264.7 mouse macrophages. A lipophilic gold compound, auranofin, suppressed the LPS-induced increase of nuclear kappa B-binding activity, degradation of I kappa B proteins, and IKK activation. Auranofin also blocked IKK activation induced by TNF and PMA/ionomycin, suggesting that the target of auranofin action is common among these diverse signal pathways. In vitro IKK activity was suppressed by addition of hydrophilic gold compounds, such as aurothiomalate, aurothioglucose, and AuCl3. Other thiol-reactive metal ions such as zinc and copper also inhibited IKK activity in vitro, and induction of IKK in LPS-stimulated macrophages. In vitro IKK activity required the presence of reducing agent and was blocked by addition of thiol group-reactive agents. Two catalytic subunits of IKK complex, IKK alpha and IKK beta, were both inhibited by these thiol-modifying agents, suggesting the presence of a cysteine sulfhydryl group in these subunits, which is critical for enzyme activity. The antiinflammatory activity of gold compounds in the treatment of rheumatoid arthritis may depend on modification of this thiol group by gold.

Chloroquine inhibits inducible nitric oxide synthase expression in murine peritoneal macrophages.

Induction of inducible nitric oxide synthase in vivo or in vitro in response to stimuli is only temporary. However, chronic localized expression of inducible nitric oxide synthase in certain organs has been associated with the development of autoimmune diseases or chronic inflammatory diseases. Chloroquine is being used as an antiinflammatory drug, and its inhibitory effect on the synthesis of pro-inflammatory cytokines, such as tumour necrosis factor-alpha and interferon-gamma, has been reported. In this study, we examined whether chloroquine could inhibit nitric oxide synthesis in murine peritoneal macrophages stimulated with interferon-gamma and lipopolysaccharide. Although prolonged incubation of cells with high concentrations of chloroquine showed some cytotoxicity, the drug itself was not cytotoxic when macrophages were preincubated with chloroquine for 2 hr, washed and stimulated with interferon-gamma and lipopolysaccharide in the absence of chloroquine for another 48 hr. The nitric oxide production from stimulated macrophages was markedly reduced by chloroquine in a dose-dependent manner and induction of inducible nitric oxide synthase mRNA was also suppressed by chloroquine pretreatment. These results show that chloroquine inhibits the induction of inducible nitric oxide synthase from interferon-gamma and lipopolysaccharide-stimulated macrophages, thereby reducing nitric oxide synthesis.

Nuclear factor kappaB (NF-kappaB) pathway as a therapeutic target in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by persistent joint swelling and progressive destruction of cartilage and bone. Current RA treatments are largely empirical in origin and their precise mechanism of action is uncertain. Increasing evidence shows that chronic inflammatory diseases such as RA are caused by prolonged production of proinflammatory cytokines including tumor necrosis factor (TNF) and interleukin 1 (IL-1). The nuclear factor kappaB (NF-kappaB) plays an essential role in transcriptional activation of TNF and IL-1. NF-kappaB is induced by many stimuli including TNF and IL-1, forming a positive regulatory cycle that may amplify and maintain RA disease process. NF-kappaB and enzymes involved in its activation can be a target for anti-inflammatory treatment. Aspirin and sodium salicylate inhibit activation of NF-KB by blocking IkappaB kinase, a key enzyme in NF-kappaB activation. Glucocorticoids suppress expression of inflammatory genes by binding glucocorticoid receptor with NF-kappaB, and increasing expression of inhibitory protein of NF-kappaB, IkappaBalpha. Sulfasalazine and gold compounds also inhibit NF-kappaB activation. Continuing advances in our understanding of action mechanism of antirheumatic agents will benefit the future development of RA regimens with greater efficacy and less toxicity.

Chloroquine inhibits processing of tumor necrosis factor in lipopolysaccharide-stimulated RAW 264.7 macrophages.

TNF, a potent immunoregulatory cytokine, is associated with inflammatory diseases such as rheumatoid arthritis, multiple sclerosis, and cerebral malaria when produced in excess. Antimalarial agents such as chloroquine and hydroxychloroquine have been used to treat some rheumatic diseases. Chloroquine was reported to inhibit production of TNF, although the underlying mechanism is poorly understood. In RAW 264.7 cells stimulated with LPS, addition of chloroquine at nontoxic concentrations did not inhibit induction of TNF mRNA and NF-kappaB activity. In the same cells, synthesis and steady state level of 26-kDa pro-TNF were also not significantly reduced by addition of chloroquine, while only small amount of 17-kDa mature TNF was detected in the medium. A pulse-chase experiment of pro-TNF produced in chloroquine-treated cells showed significant inhibition of processing of prohormone. Hydroxychloroquine showed similar inhibitory effect, whereas other lysosomal inhibitors such as ammonium chloride and methylamine had no effect on the production of TNF. Our results suggest that chloroquine inhibits production of TNF at the step of processing of membrane-bound pro-TNF to make soluble mature protein in a lysosome-independent manner.

Tosylphenylalanine chloromethyl ketone inhibits TNF-alpha mRNA synthesis in the presence of activated NF-kappa B in RAW 264.7 macrophages.

Serine proteinase inhibitors such as N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and N alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) were shown to inhibit production of tumour necrosis factor-alpha (TNF-alpha) in lipopolysaccharide (LPS)-activated RAW 264.7 macrophages. The proteinase inhibitors were also reported to inhibit activation of the transcription factor nuclear factor-kappa B (NF-kappa B) by blocking the signalling pathway for stimuli-induced phosphorylation of the inhibitory subunit (I kappa B alpha) and thus preventing its degradation. In RAW 264.7 cells TPCK and TLCK significantly suppressed LPS-induced increase in TNF-alpha mRNA, induction of nuclear kappa B-binding activity and degradation of I kappa B alpha. TPCK and TLCK effectively blocked TNF-alpha mRNA synthesis even when they were added after LPS stimulation. In these cells, however, the inhibitory modes of the two inhibitors were found to be different: while addition of TLCK suppressed I kappa B alpha degradation and reduced NF-kappa B activity, a comparable decrease in the nuclear kappa B-binding activity or in I kappa B alpha degradation was not observed in cells treated with TPCK. Our results show that TPCK inhibits LPS-induced TNF-alpha mRNA synthesis in the presence of activated NF-kappa B and suggests that mechanisms other than NF-kappa B activation are involved in the transcriptional regulation of the TNF-alpha gene.

Pro-tumour necrosis factor cleavage enzyme in macrophage membrane/particulate.

Tumour necrosis factor (TNF) is synthesized initially as a membrane-bound precursor which is then cleaved to yield soluble, mature protein. The 26,000 MW TNF precursor isolated from the lysate of activated RAW 264.7 (mouse macrophage) cells by immunoprecipitation was used to identify pro-TNF cleavage enzyme in the same cells. A significant amount of mature protein was formed in samples containing Nonidet P-40 (NP-40)-lysed cells, whereas sonicated cells showed negligible activity. Most of the cleavage activity in macrophages was localized in the membrane/particulate fraction and remained largely insoluble after sonication or treatment with 2 mM EDTA/1 M NaCl, indicating that the enzyme is associated with the membrane/particulate fraction. The crude cleavage activity in membrane/particulate was partially inhibited by a spectrum of serine, cysteine and aspartate proteinase inhibitors, whereas secretion of TNF from activated macrophages was inhibited exclusively by serine and serine/cysteine proteinase inhibitors. This result suggested that, among heterogenous pro-TNF cleavage activities, the enzyme responsible for the processing of TNF is a serine proteinase. Pro-TNF cleavage activity was present in non-stimulated macrophages and decreased significantly 8 hr after lipopolysaccharide (LPS) stimulation, suggesting that it is negatively regulated after an initial burst of TNF synthesis.

Characterization of high molecular weight glycosylated forms of murine tumor necrosis factor.

Tumor necrosis factor (TNF) is synthesized as a prohormone with an unusually long and atypical signal sequence which is absent from the mature secreted cytokine. In addition to mature 17 kDa TNF, LPS-stimulated murine macrophages secrete at least seven TNF-like proteins (isoforms) of differing electrophoretic mobility which appear as a "ladder" on SDS-PAGE. We here present data indicating that these isoforms derive not from sequential clipping of propiece fragments, but rather from differential glycosylation at sites on the mature hormone. Selected isoforms have been isolated and purified by sequential chromatographic and electrophoretic steps. NH2-terminal sequence analysis of two of these isoforms reveal sequences identical to that of mature 17 kDa murine TNF. Characterization of the secretory products of tunicamycin-treated. LPS-stimulated murine macrophages indicate that the "ladder" complex reflects differential glycosylation of mature 17 kDa TNF. Digestion of purified isoforms with a battery of glycosidic enzymes indicate that secreted forms of murine TNF contain both sialic acid and asparagine(N)-linked chains. The biological significance of this heterogeneity is not known.

Processing of newly synthesized cachectin/tumor necrosis factor in endotoxin-stimulated macrophages.

The biosynthesis and processing of cachetin/tumor necrosis-factor (TNF) were examined in the murine macrophage-like cell line RAW 264.7. Lipopolysaccharide-stimulated cells secreted both glycosylated and nonglycosylated 17-kilodalton (kDa) mature cachectin/TNF into the culture medium. Secreted cachectin/TNF was derived from membrane-associated precursors that were precipitated by polyclonal antisera raised against either the mature protein or synthetic peptide fragments of the 79 amino acid cachectin/TNF prohormone sequence. About half of the precursors were N-glycosylated, apparently cotranslationally. The cachectin/TNF precursors were then proteolytically cleaved to release soluble mature cytokine into the medium, while the membrane-bound 14-kDa presequence remained cell associated. During the period of LPS stimulation, the amount of macrophage cell surface cachectin/TNF remained at a low level, suggesting that both nonglycosylated and glycosylated precursors of cachectin/TNF are efficiently cleaved by these cells. These findings suggest the presence of a unique mechanism for the secretion of cachectin/TNF.

Simple spectrophotometric determination of haptoglobin-haemoglobin complex in haemolysed samples.

The concentration of haptoglobin-haemoglobin complex in haemolysed serum was measured by reducing the sample with sodium hydrosulphite. The absorption spectra of serum before and after sodium hydrosulphite treatment were compared, and the absorbance change was used to determine the amount of haemoglobin in the sample. By analysing the difference absorption spectra of various types of haemoglobin-containing samples, it was also possible to determine the haptoglobin level in samples haemolysed severely beyond their haemoglobin-binding capacities. When the present methods are used, along with the spectrophotometric method for free haptoglobin, one can measure separately the amounts of free haptoglobin, haptoglobin-haemoglobin complex, and free haemoglobin in samples with varying degrees of haemolysis.

Simple spectrophotometric determination of haptoglobin level in serum.

Haptoglobin concentration was determined from the change caused in the absorption spectrum of hemoglobin due to binding with haptoglobin. Absorbance changed markedly near the Soret band, and a linear correlation could be observed between the extent of the absorbance change and the amount of free haptoglobin. The levels of serum haptoglobin measured by the present method correlated well with those determined by an electrophoretic or a peroxidase method. High concentrations (more than 10 mg/100 ml) of hematin gave positive interference in the measured haptoglobin level, whereas bilirubin and chylomicron showed low levels of interference. The proposed method could be used as a simple and reliable method for the determination of haptoglobin level in various types of sample.

Inhibition of prostaglandin synthase activity of sheep seminal vesicular gland by human serum haptoglobin.

Serum haptoglobin was added to the reaction mixture of prostaglandin synthase (EC 1.14.99.1) and its inhibitory effect was studied [1-14C]Arachidonic acid was used as substrate and the enzyme activity was estimated by monitoring the radioactivity of the products after thin layer chromatography. With or without addition of hemoglobin to the reaction mixture, both the purified haptoglobin 1-1 and 2-2 showed inhibitory activity. In the presence of 5 microM hematin, however, inhibitory activity haptoglobin was not observed. Inhibition of prostaglandin synthesis in the system depended on the molar ratio of haptoglobin to hemoglobin in the reaction mixture. These results demonstrate that haptoglobin inhibits prostaglandin synthase by restricting available heme group for the enzyme activity through complexing with hemoglobin. However, haptoglobin did not inhibit completely the stimulatory effect of free hemoglobin. Relevant significant of this effect was discussed.