Interfering effect of diphtheria-tetanus-acellular pertussis combined (DTaP) vaccines on the bacterial endotoxin test.

We evaluated applicability of the endotoxin test to DTaP vaccines. Although no DTaP vaccine batches showed immediate interference with the activating activity of spiked endotoxin on the clotting of Limulus amaebocyte lysate (LAL), a gradually progressing interference depending on the time after spiking was seen for some of the batches. The interfering DTaP vaccine batches, however, showed no significant effect onin vitro TNF-alpha induction in RAW264.7 cells and pyrogenicity in rabbits of spiked endotoxin. Aluminium hydroxide gel in the vaccines was suggested to be one of the causes of the interference. Accordingly, a careful evaluation of the interfering effect was assumed crucial for the effective application of the endotoxin test to check residual biological activity of endotoxin in DTaP vaccines.     

A limulus amoebocyte lysate activating activity (LAL activity) that lacks biological activities of endotoxin found in biological products

Pyrogenic substances in influenza HA (IHA) vaccine have been controlled by the pyrogen test or the mouse body weight decreasing toxicity (BWD) test. We examined the possibility of replacing the animal tests with the endotoxin test Commercial IHA vaccines were found to show considerable levels of LAL activity ranging from 0.2 to 160 EU/ml. However, a batch of the vaccine having even 100 EU/ml of LAL activity showed neither pyrogenicity in rabbits nor tumor necrosis factor alpha (TNF-alpha) induction in RAW264.7 cells. The LAL activity of IHA vaccine was abolished by a monoclonal antibody that recognizes LPS-binding epitope of LAL factor C. The activity of IHA vaccine showed different physicochemical properties from those of LAL activity of endotoxin. LAL activity of endotoxin is known to be sensitive to polymyxin B treatment and was found to be resistant to polyoxyethylene 10 cetyl ether (Brij56) treatment. On the contrary, the LAL activity of IRA vaccine was shown to be resistant to polymyxin B but sensitive to Brij56 treatment. The difference in sensitivity of the two LAL activities to polymyxin B and Brij56 might suggest the possibility of their discriminative measurements.     

A quantitative in vitro assay method for detecting biological activity of endotoxin using prostaglandin E2 induction in rabbit peripheral blood

The pyrogen test and the endotoxin test (the LAL test) have been playing crucial roles in detecting endotoxin in parenteral drugs. The current test methods, however, have disadvantages such as requiring a large number of animals or an inadequacy in evaluation of in vivo endotoxin activity. We attempted to establish a new assay method that can overcome the shortcomings of the current methods. We standardized the in vitro assay method by the use of prostaglandin E2 (PGE2) induction from peripheral blood of rabbits for detecting endotoxin activity. A linear dose-response regression was attained from approximately 0.15 to 5 endotoxin units/ml of Japanese national reference standard endotoxin by the in vitro assay. The assay showed a fine correlation with the pyrogen test but not with the LAL test, when endotoxins from various bacterial sources were tested. The in vitro assay was also shown to have the capability of detecting a synergistic effect of endotoxin and parenteral drugs. The in vitro PGE2 induction test using rabbit blood was, therefore, suggested to be the appropriate test method for guaranteeing the same level of safety of parenteral drugs as the pyrogen test does.     

Cytokine-mediated induction of metallothionein in Hepa-1c1c7 cells by oleanolic acid

Oleanolic acid (OA), a pentacyclic triterpene acid, has been reported to possess inducing activity of hepatic metallothionein (MT). However, the mechanism underlying its effects is unknown. This study investigated the effects of OA on the regulation of MT expression in an in vitro model. OA that was added directly to Hepa-1c1c7 cells had no effect on MT induction. However, MT and its mRNA levels increased markedly when the Hepa-1c1c7 cells were cultured with the OA-treated conditioned media from the RAW 264.7 cells. Co-treating the RAW 264.7 cells with OA and pentoxifylline, a TNF-alpha synthesis inhibitor, resulted in a decrease in the effects of OA on the MT induction. In the OA-exposed RAW 264.7 cell cultures, production and mRNA levels of TNF-alpha and IL-6 were increased. However, the MT induction activity was inhibited when antibodies to TNF-alpha and/or IL-6 were added to the OA-treated conditioned media from the RAW 264.7 cells. These results suggest that the up-regulation of MT expression by OA was mediated by the TNF-alpha and IL-6 released from UA-activated macrophages.     

Lipopolysaccharide induces hyporesponsiveness to its own action in RAW 264.7 cells

Bacterial endotoxin (lipopolysaccharide, LPS) has the property of inducing hyporesponsiveness or tolerance to its own effects. This phenomenon has been demonstrated in man and experimental animals. The cellular changes that contribute to LPS tolerance are not understood. One mechanism of tolerance could involve a diminished response to LPS by key effector cells such as macrophages. Here we describe experiments designed to determine the mechanism whereby LPS produces a hyporesponsive state to its own effects. Because of the importance of the monokine known as tumor necrosis factor-alpha (TNF-alpha) in mediating many of the diverse effects of LPS, we have studied induction of TNF-alpha at the mRNA and activity level in the murine macrophage-like cell line RAW 264.7. Hyporesponsiveness can be induced by exposure of RAW 264.7 cells to low doses of LPS for more than 6 h prior to challenge with a second, normally stimulatory dose of LPS. This hyporesponsiveness is characterized by a diminished ability of LPS to increase steady state levels of TNF-alpha mRNA, is not due to an increased rate of TNF-alpha mRNA degradation, and is specific for LPS since LPS-pretreated and control cells produce similar amounts of TNF-alpha in response to challenge with heat-killed Staphylococcal aureus. The presence of indomethacin during the primary and/or challenge LPS treatment has no effect on the induction of acquired hyporesponsiveness. Thus, cyclooxygenase products are probably not involved in the development of LPS-induced hyporesponsiveness. These studies provide the basis for a better understanding of the cellular mechanisms that contribute to LPS tolerance.     

Identification of a novel inhibitor of LPS-induced TNF-alpha production with antiproliferative activity in monocyte/macrophages

An isoquinoline derivative, 5-methyl-7,8-dimethoxy-1-phenylpyrazolo[5,4-c]isoquinoline (compound 1), was identified as a novel inhibitor of LPS-induced TNF-alpha production by cell-based screening. Compound 1 suppressed LPS-induced TNF-alpha production in RAW264.7 cells and murine peritoneal macrophages in a dose-dependent manner similar to SB203580, known as a specific inhibitor of p38 MAPK. It also inhibited an LPS-induced increase in serum TNF-alpha in a mouse endotoxic shock model with an ED(50) of approximately 10 mg/kg. Compound 1 had little effect on the incorporation of [3H]-leucine into the cells, while it suppressed LPS-induced TNF-alpha mRNA levels in RAW264.7 cells. The results indicate that suppression of TNF-alpha production was not a result of nonspecific inhibition of de novo translation but was based on the decreased TNF-alpha mRNA levels. The in vitro kinase assay revealed that compound 1 did not strongly inhibit p38 MAPK activity, its potency being much lower than that of SB203580, suggesting that the TNF-alpha-suppressive action of compound 1 cannot be attributed to the inhibition of p38 MAPK. Furthermore, in contrast to SB203580, it significantly inhibited the growth of RAW264.7 and THP-1 cells in a cytostatic manner. Compound 1 is likely to have antiinflammatory and antiproliferative effects by acting on some molecule other than p38 MAPK that contributes to both LPS-induced TNF-alpha production and the cell growth of monocyte/macrophages.     

Cathelicidin family of antibacterial peptides CAP18 and CAP11 inhibit the expression of TNF-alpha by blocking the binding of LPS to CD14(+) cells.

Mammalian myeloid and epithelial cells express several kinds of antibacterial peptides (alpha-/beta-defensins and cathelicidins) that contribute to the innate host defense by killing invading micro-organisms. In this study we evaluated the LPS-neutralizing activities of cathelicidin peptides human CAP18 (cationic antibacterial proteins of 18 kDa) and guinea pig CAP11 using the CD14(+) murine macrophage cell line RAW264.7 and the murine endotoxin shock model. Flow cytometric analysis revealed that CAP18 and CAP11 inhibited the binding of FITC-conjugated LPS to RAW264.7 cells. Likewise, Northern and Western blot analyses indicated that CAP18 and CAP11 suppressed LPS-induced TNF-alpha mRNA and protein expression by RAW264.7 cells. Interestingly, CAP18 and CAP11 possessed LPS-binding activities, and they strongly suppressed the interaction of LPS with LPS binding protein that mediates the transport of LPS to CD14 to facilitate the activation of CD14(+) cells by LPS. Moreover, when CAP18 and CAP11 were preincubated with RAW264.7 cells, they bound to the cell surface CD14 and inhibited the binding of FITC-LPS to the cells. Furthermore, in the murine endotoxin shock model, CAP18 or CAP11 administration inhibited the binding of LPS to CD14(+) cells (peritoneal macrophages) and suppressed LPS-induced TNF-alpha expression by these cells. Together these observations indicate that cathelicidin peptides CAP18 and CAP11 probably exert protective actions against endotoxin shock by blocking the binding of LPS to CD14(+) cells, thereby suppressing the production of cytokines by these cells via their potent binding activities for LPS and CD14.     

Applicability of bacterial endotoxins test to various blood products by the use of endotoxin-specific lysates

Endotoxin contamination is a serious threat to the safety of parenteral drugs, and the rabbit pyrogen test has played a crucial role in controlling this contamination. Although the highly sensitive endotoxin test has replaced the pyrogen test for various pharmaceuticals, the pyrogen test is still implemented as the control test for most blood products in Japan. We examined the applicability of the endotoxin test to blood products for reliable detection and quantification of endotoxin. Nineteen types of blood products were tested for interfering factors based on spike/recovery of endotoxin by using 2 types of endotoxin-specific lysate reagents for photometric techniques. Interfering effects on the endotoxin test by the products could be eliminated by diluting from 1/2 to 1/16, with the exception of antithrombin III. However, conventional lysate reagents that also react with non-pyrogenic substances, such as (1–3)-β-d-glucan, produced results that were not relevant to endotoxin content or pyrogenicity. Our results showed that the endotoxin test would be applicable to most blood products if used with appropriate endotoxin-specific lysate reagents.     

二氢青蒿素对超高分子量聚乙烯颗粒诱导的巨噬细胞源性炎性因子释放的影响

目的:研究二氢青蒿素(Dihydroartemisinin,DHA)对超高分子量聚乙烯(Ultra highmolecularweightpolyethylene,UHMWPE)颗粒诱导的小鼠巨噬细胞系RAW264.7细胞源性炎性因子释放的影响。方法:建立UHMWPE颗粒诱导的小鼠巨噬细胞系RAW264.7细胞源性炎性因子释放模型;施加不同浓度的二氢青蒿素观察药物对细胞的影响,酶联免疫分析法(Enzyme-linked immuno sorbent assay,ELISA)检测细胞培养液上清中TNF-α,IL-1β,IL-6和IL-10含量,MTT法检测细胞毒性反应。结果:酶联免疫分析方法结果表明,二氢青蒿素可以显著抑制由UHMWPE颗粒诱导的小鼠RAW264.7细胞促炎细胞因子TNF-α,IL-1和IL-6的表达,并显著促进抗炎因子IL-10的释放,其效应具有剂量依赖性。结论:二氢青蒿素具有显著的抗炎作用,可以抑制UHMWPE颗粒诱导的巨噬细胞炎症反应,其在预防人工关节置换术后假体无菌性松动的药物治疗方面具有潜在的作用。     

Upregulation of vascular endothelial growth factor by heat-killed Listeria monocytogenes in macrophages

We investigated the effect of heat-killed Listeria monocytogenes (HKLM) on the expression of vascular endothelial growth factor (VEGF) in RAW264.7 macrophage-like cells. The expression of VEGF was induced in RAW264.7 cells treated with HKLM. Pretreatment of cells with cycloheximide, a protein synthesis inhibitor, inhibited the induction of VEGF mRNA by HKLM. Induction of VEGF by HKLM was partially inhibited by treatment of cells with SB203580, a p38 mitogen-activated protein kinase (MAPK) inhibitor, or a neutralizing antibody against tumor necrosis factor-alpha (TNF-alpha). In addition, HKLM induced phosphorylation of p38 MAPK. These results suggest that p38 MAPK and TNF-alpha are involved in the VEGF expression induced by HKLM in RAW264.7 cells. We confirmed that increased VEGF expression is immunohistochemically detected in splenic macrophages of mice infected with L. monocytogenes (L. monocytogenes). VEGF is thought to be involved in inflammatory reactions induced by L. monocytogenes infection.     

Cross-talk between the pathways leading to the induction of apoptosis and the secretion of tumor necrosis factor-alpha in ricin-treated RAW 264.7 cells

Ricin induced apoptotic nuclear morphological changes in mouse macrophage cell line RAW264.7 cells at concentrations sufficient to cause severe protein synthesis inhibition. Ricin also induced the release of tumor necrosis factor-alpha (TNF-alpha) from this cell line in a dose-dependent manner but the profile was bell-shaped. However, the isolated galactose-specific ricin B-chain had no such effects. These results suggest that the receptor-binding of ricin through the B-chain is not enough, and subsequent attack on the intracellular target, i.e., the 28S ribosomal RNA (rRNA), by the A-chain of internalized ricin is required for the effects of ricin. Z-D-CH2-DCB, a caspase family inhibitor, showed potent inhibition of the release of TNF-alpha from RAW264.7 cells as well as blockage of the induction of apoptosis by ricin. Furthermore, SB202190, a specific P38 mitogen-activated protein (MAP) kinase inhibitor that strongly inhibits the release of TNF-alpha, also showed significant inhibition of ricin-induced apoptosis. These results suggest that there may be cross-talk between the pathways leading to the release of TNF-alpha and apoptosis. Time course analysis revealed that the activation of p38 MAP kinase started prior to the induction of TNF-alpha release and apoptosis. Since the activation of p38 MAP kinase in ricin-treated RAW264.7 cells was not prevented by Z-D-CH2-DCB, the activation of p38 MAP kinase may occur upstream of the caspase cascade. Among the other protein synthesis inhibitors examined, modeccin and anisomycin, which can trigger a ribotoxic stress response similar to ricin, induced the release of TNF-alpha, but emetine and cycloheximide did not. These results suggest that the specific attack on the 28S ribosomal RNA and the resulting ribotoxic stress response may trigger the multiple signal transduction pathways through the activation of p38 MAP kinase, which in turn leads to TNF-alpha release and apoptosis.     

Hydrogen peroxide induces the production of tumor necrosis factor-alpha in RAW 264.7 macrophage cells via activation of p38 and stress-activated protein kinase

The effect of hydrogen peroxide (H(2)O(2)) on production of tumor necrosis factor (TNF)-alpha was examined in RAW 264.7 murine macrophage cells. H(2)O( 2) led to production of TNF-alpha up to 24 h after the treatment, but not nitric oxide in RAW 264.7 cells. H(2)O(2) induced TNF-alpha production in mouse peritoneal macrophages as well as RAW 264.7 cells. The H(2)O(2)induced TNF-alpha production was prevented by inhibitors of p38 and stress-activated protein kinase (SAPK/JNK), and H(2)O( 2) induced the phosphorylation of p38 and SAPK. Further, H(2)O( 2) significantly augmented the AP-1 activity, but not nuclear factor (NF)-kappaB activity in RAW 264.7 cells. A high level of intracellular reactive oxygen radicals (ROS) was detected in H(2)O(2)-exposed RAW 264.7 cells. Ebselen, a cell permeable antioxidant, prevented the H( 2)O(2)-induced TNFalpha production. H(2)O(2) significantly enhanced lipopolysaccharide (LPS)-induced TNF-alpha production. Therefore, H( 2) O(2) was suggested to induce TNF-alpha production in macrophages via activating p38 and SAPK/JNK as oxidative stress-related signal pathways.     

Comparison of the rabbit pyrogen test and Limulus amoebocyte lysate (LAL) assay for endotoxin in hepatitis B vaccines and the effect of aluminum hydroxide.

The rabbit pyrogen test and Limulus amoebocyte lysate (LAL) assay have been used to detect endotoxins in vaccines, but interactions between the endotoxins and proteins or aluminum hydroxide can interfere with the results. Currently, the rabbit pyrogen test is used to detect endotoxin in hepatitis B (HB) vaccines even though the HB surface protein, the active ingredient, is over-expressed in and purified from eukaryotic cells which lack endotoxin. Therefore, we examined the possibility of replacing the animal tests with the more efficient LAL test. To this end, we determined whether the aluminum hydroxide in the HB vaccines affects the rabbit pyrogen test and the LAL assay. HB vaccines and HB protein solutions spiked with lipopolysaccharide (LPS) produced almost the same dose-dependent temperature rise in rabbits, indicating that the aluminum hydroxide in the HB vaccine does not interfere with the pyrogenic response in rabbit. In contrast, a spike recovery study showed that aluminum hydroxide interfered with the LAL clot and kinetic assays; however, the LAL clot assay was effective at detecting endotoxin without loss of LAL activity after serial dilution of the samples. Furthermore, there was good correlation in the LAL clot assay between the amount of LPS added and the amount recovered. However, both turbidimetric and chromogenic kinetic assays displayed no correlation between the LPS amount added and recovered. Our results suggest that the LAL clot assay is sensitive and reliable when samples are properly prepared, and can be used to replace the rabbit pyrogen test for the detection of endotoxin in HB vaccines.     

CEL-I, an invertebrate N-acetylgalactosamine-specific C-type lectin, induces TNF-alpha and G-CSF production by mouse macrophage cell line RAW264.7 cells

Our previous studies demonstrated that CEL-I, an N-acetylgalactosamine (GalNAc)-specific C-type lectin purified from the marine invertebrate Cucumaria echinata (Holothuroidea) showed potent cytotoxicity to several cell lines such as HeLa, MDCK and XC cells. In this study, we found that CEL-I induced increased secretion of tumour necrosis factor-alpha (TNF-alpha) and granulocyte colony stimulation factor (G-CSF) by mouse macrophage cell line RAW264.7 cells in a dose-dependent manner, whereas this cell line was highly resistant to CEL-I cytotoxicity. The cytokine-inducing activity of CEL-I was stronger than that of phytohaemagglutinin (PHA-L). A binding study using FITC-labelled CEL-I (F-CEL-I) indicated that the amount of bound F-CEL-I on RAW264.7 cells was greater than that of F-PHA-L, suggesting that the greater activity of CEL-I to induce cytokine secretion by RAW264.7 cells is partly due to the higher binding ability. Since the cell binding and cytokine-inducing activity of CEL-I were partly but significantly inhibited by the specific sugar (GalNAc), it is considered that the binding of CEL-I to cell-surface-specific saccharide moieties, which may be recognized by CEL-I with higher affinity than GalNAc, is essential for the induction of cytokine secretion. The secretion of TNF-alpha and G-CSF from CEL-I-treated RAW264.7 cells were almost completely prevented by brefeldin A (BFA), whereas increase in mRNA levels of these cytokines were not affected by BFA. Bio-Plex beads assay suggested that temporal increase in phosphorylation of extracellular-regulated kinase (ERK), c-jun NH(2)-terminal kinase (JNK) and p38 MAP kinase occurred at relatively early time following CEL-I treatment. Furthermore, the secretion of TNF-alpha and G-CSF were inhibited by specific inhibitors for these MAP kinases. These results suggest that the intracellular signal transduction through the activation of MAP kinase system is involved in CEL-I-induced cytokine secretion.