Effects of Kumaizasa (Sasa senanensis) Leaf Extract on Innate Immune Regulation in HEK293 Cells and Macrophages.

BACKGROUND/AIM: We investigated the effect of Kumaizasa leaf extract (KLE) on innate immunity using the HEK293 and RAW 264.7 cell lines. MATERIALS AND METHODS: KLE, lipopolysaccharides (LPS), or KLE with LPS were added to RAW 264.7 cells. The TNF-α and IL-1ß mRNA expression was then quantified. The expression of MAPKs, NFĸB, TNF-α and IL-1ß proteins was also quantified. In addition, KLE was added to HEK293 cells and the IL-8 concentration was measured. RESULTS: In RAW 264.7 cells, KLE increased the levels of TNF-α and IL-1ß mRNA. By contrast, when KLE and LPS were added to RAW 264.7 cells, the increase in TNF-α and IL-1ß mRNA was ameliorated. Similarly, the expression of JNK and ERK proteins was reduced. The addition of KLE to HEK293 cells induced IL-8 production. CONCLUSION: Based on these results, a KLE-mediated mechanism may regulate immunity by suppressing the expression of JNK and ERK, which are involved in inflammatory signal transduction.

Sorafenib attenuated the function of natural killer cells infiltrated in HCC through inhibiting ERK1/2.

Sorafenib has been systemically utilized to therapy the advanced hepatocellular carcinoma (HCC). Natural killer cells (NKs) are important cytotoxic innate lymphocytes, which can exert effector functions especially in liver and tumor. However, how Sorafenib affects the function of NKs remains to be elucidated. Here, we utilized the subcutaneous and in situ tumor bearing mice with mouse hepatoma cell line hepa 1-6. At the endpoint, the number and function of NKs in blood, liver, TDLN, and tumor were explored using FACS, ELISA, WB, etc. To confirm the direct effects of Sorafenib on NKs, the NKs were sorted using FACS, which were then stimulated with Sorafenib to detect the functions and the relevant mechanisms using qPCR, western blot, and FACS in vitro. Finally, we found that Sorafenib led to a significant block of tumor progression, but reduced the number of NK cells through suppressing the proliferation of NK cells. This phenotype made us study the terminal function of NK cells, revealing that Sorafenib could decrease the production of effector molecules and cytokines, such as perforin, granzyme B, TNF-α, IFN-γ, etc. Besides, p-ERK1/2 in NK cells was inhibited after treatment with Sorafenib, and a similar tendency of NK cells could be achieved using ERK1/2 inhibitor. Collectively, our data suggested that Sorafenib functioned as a critical inhibitor that controlled the number and function of NK cells through inhibiting ERK1/2.

Javamide-II Found in Coffee Is Better than Caffeine at Suppressing TNF-α Production in PMA/PHA-Treated Lymphocytic Jurkat Cells.

Recent studies have suggested positive benefits of coffee consumption on inflammation-related diseases, such as liver diseases and diabetes, where activated lymphocytes and TNF-α are critically implicated. Interestingly, some reports suggested that javamide-II found in coffee may have anti-inflammatory activity greater than that of caffeine, but there is limited information about its effect on TNF-α production by activated lymphocytes. Therefore, the inhibitory effect of javamide-II on TNF-α was investigated in PMA/PHA-treated lymphocytic Jurkat cells. At 5 µM, javamide-II, not caffeine, inhibited TNF-α production in the cells (45 ± 4%, P < 0.001). To elucidate the underlying mechanism, the phosphorylation of MAP kinases (ERK, p38, and JNK) was investigated in the Jurkat cells. Javamide-II had little effect on JNK or p38 phosphorylation, but javamide-II (<20 µM) decreased ERK phosphorylation, consequently reducing TNF-α mRNA expression in the cells ( P < 0.001). The involvement of ERK phosphorylation was also confirmed by an ERK1/2 inhibitor (SCH772984). Furthermore, javamide-II was also found to inhibit IL-2 production, which is up-regulated by ERK phosphorylation in cells ( P < 0.001). These data suggested that javamide-II may be a potent compound to suppress TNF-α production more efficiently than caffeine by inhibiting ERK phosphorylation in Jurkat cells.

In Vitro Anti-Inflammatory Activity of Ilex cornuta Extract Mediated by Inhibition of Extracellular Signal-Regulated Kinase Phosphorylation.

Ilex cornuta, commonly known as Chinese holly, is an evergreen shrub from the family Aquifoliaceae, and it is widely distributed in Korea and China. In folk medicine, the leaves of I. cornuta are used for treatment of several disorders, including weakness of the waist and knees, arthrodynia, headache, acute conjunctivitis, toothache, urticaria, rheumatic arthralgia, and cardiovascular diseases. In this study, we investigated the anti-inflammatory effects of an I. cornuta leaf ethanol extract (ILE) and its underlying mechanisms of action. The anti-inflammatory activities of ILE were evaluated in murine RAW 264.7 macrophages, using lipopolysaccharide (LPS) stimulation. ILE treatment-related changes in the production of nitric oxide (NO), prostaglandin E2 (PGE2), and proinflammatory cytokines were also measured. Finally, the expression of signaling molecules involved in inflammatory reactions was also assessed. Pretreatment of macrophages with ILE attenuated the expression of inducible NO synthase and cyclooxygenase-2, resulting in a decrease in NO and PGE2 production. The secretion of proinflammatory cytokines such as interleukin (IL)-6 and IL-1ß was also reduced. Furthermore, ILE reduced extracellular signal-regulated kinases (ERK) phosphorylation, without affecting the inhibitor of kappa Bα and other mitogen-activated protein kinases. Liquid chromatography-tandem mass spectrometry analysis (LC-MS/MS) demonstrated that 1 g of ILE contains 27 mg of kaempferol, 0.3 mg of vanillic acid, and 21 mg of combined amount of isoquercetin and hyperin, among which isoquercetin and kaempferol significantly suppressed IL-6, IL-1ß, and PGE2 production. Our results demonstrated that ILE possesses anti-inflammatory effects mediated through inhibition of ERK phosphorylation.

Tonggyu-tang, a traditional Korean medicine, suppresses pro-inflammatory cytokine production through inhibition of MAPK and NF-κB activation in human mast cells and keratinocytes.

BACKGROUND: Allergic diseases including allergic rhinitis, asthma, and atopic dermatitis are increasing worldwide. Common medications used to treat these inflammatory disorders are anti-histamines and corticosteroids, but they have their own limitations such as short duration and severe side effects. Thus, interest in complementary and alternative medicine is continually growing. Here, we investigate the anti-inflammatory mechanisms of Tonggyu-tang (TGT), a traditional Korean medicine that has been used to treat patients with allergic nasal disorders. METHODS: We measured mRNA expressions and production of pro-inflammatory cytokines such as interleukin (IL)-4, IL-6, IL-8 and tumor necrosis factor alpha (TNF-α) by RT-PCR and ELISA assays in HMC-1 (human mast cell line-1) and HaCaT cells, immortalized human keratinocytes. Moreover, we evaluated the effect of TGT on two major inflammation-related pathways, mitogen activated protein kinase (MAPK) and NF-κB signaling pathway in these two cells. RESULTS: Our results revealed that that TGT significantly reduced the expression and production of inflammatory cytokines such as IL-4, IL-6, IL-8, and TNF-α in the agonist-treated HMC-1 and HaCaT cells. We also found that TGT suppressed MAPK signaling pathway including extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (p38), and c-Jun N-terminal kinase (JNK) as well as NF-κB pathway, which are known to regulate inflammatory cytokine expression. CONCLUSION: Taken together, our results demonstrate that TGT inhibits expression of pro-inflammatory cytokines by suppressing MAPK and NF-kB pathway in both mast cells and keratinocytes, suggesting the potential use of TGT in treating allergic inflammatory diseases.

Pinellia ternata Attenuates Mucus Secretion and Airway Inflammation after Inhaled Corticosteroid Withdrawal in COPD Rats.

Inhaled corticosteroids (ICS) are widely used to manage chronic obstructive pulmonary disease (COPD). However, withdrawal of ICS generally causes various adverse effects, warranting careful management of the ICS withdrawal. Pinellia ternata, a traditional Chinese herbal medicine, has been used to treat respiratory diseases in China for centuries. Here, we investigated its role in antagonizing ICS withdrawal-induced side effects, and explored the underlying mechanisms. The rat COPD model was established using a combination of passive cigarette smoking and intratracheal instillation of lipopolysaccharide (LPS). COPD rats were treated with saline or budesonide inhalation, or with budesonide inhalation followed by saline inhalation or Pinellia ternata gavage. The number of goblet cells and the level of mucin 5AC (MUC5AC) were enhanced by budesonide withdrawal. Pinellia ternata treatment significantly blocked these effects. Further, Pinellia ternata treatment reversed budesonide withdrawal-induced increase of interleukin 1[Formula: see text] (IL-1[Formula: see text] and tumor necrosis factor [Formula: see text] (TNF-[Formula: see text]) levels in bronchoalveolar lavage fluid (BALF). Extracellular signal-regulated kinase (ERK), but neither p38 nor c-Jun N-terminal kinase (JNK), was activated by budesonide withdrawal, and the activation was blocked by Pinellia ternata treatment. The MUC5AC expression was positively correlated with goblet cell number, IL-1[Formula: see text] and TNF-[Formula: see text] levels, and ERK activity. Pinellia ternata treatment protected the airway from ICS withdrawal-induced mucus hypersecretion and airway inflammation by inhibiting ERK activation. Pinellia ternata treatment may represent a novel therapeutic strategy to prevent ICS withdrawal-induced side effects in COPD patients.

Isoquercitrin suppresses the expression of histamine and pro-inflammatory cytokines by inhibiting the activation of MAP Kinases and NF-κB in human KU812 cells.

Mast cells and basophils are multifunctional effector cells that contain abundant secretory granules in their cytoplasm. Both cell types are involved in a variety of inflammatory and immune events, producing an array of inflammatory mediators, such as cytokines. The aim of the study was to examine whether isoquercitrin modulates allergic and inflammatory reactions in the human basophilic KU812 cells and to elucidate its influence on the phosphorylation of mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-κB activation. The KU812 cells were stimulated with phorbol-12-myristate 13-acetate plus the calcium ionophore A23187 (PMACI). The inhibitory effects of isoquercitrin on the productions of histamine and pro-inflammatory cytokines in the stimulated KU812 cells were measured using cytokine-specific enzyme-linked immunosorbent (ELISA) assays. Western blotting analysis was used to assess the effects of isoquercitrin on the MAPKs and NF-κB protein levels. Our results indicated that the isoquercitrin treatment of PMACI-stimulated KU812 cells significantly reduced the production of histamine and the pro-inflammatory cytokines, such as interleukin (IL)-6, IL-8, IL-1ß, and tumor necrosis factor (TNF)-α. The treated cells exhibited decreased phosphorylation of extracellular signal-regulated kinase (ERK), revealing the role of ERK MAPK in isoquercitrin-mediated allergy inhibition. Furthermore, isoquercitrin suppressed the PMACI-mediated activation of NF-κB in the human basophil cells. In conclusion, the results from the present study provide insights into the potential therapeutic use of isoquercitrin for the treatment of inflammatory and allergic reactions.

Inhibitory effects of geranium essential oil and its major component, citronellol, on degranulation and cytokine production by mast cells.

We investigated the effects of geranium essential oil (GEO) on anaphylaxis. GEO can exert antioxidant and anti-inflammatory effects, but its roles in allergic reactions are incompletely understood. Here, we used mouse cells to show that GEO inhibited the degranulation of cultured mast cells (CMCs). Citronellol is the major component of GEO and inhibited CMC degranulation. The l-enantiomer of citronellol more effectively suppressed CMC degranulation than did d-citronellol. We also examined whether citronellol could inhibit the immunoglobulin (Ig) E-induced production of tumor necrosis factor (TNF)-α. Treatment with various concentrations of citronellol before CMC activation with IgE significantly inhibited the induction of TNF-α in a dose-dependent manner. Mechanistically, citronellol suppressed the phosphorylation of mitogen-activated protein kinase (ERK), which is critical for ERK activation and the production of inflammatory cytokines in mast cells. These findings suggest that citronellol may represent a candidate compound for the effective treatment of allergic diseases.

ETP-46321, a dual p110α/δ class IA phosphoinositide 3-kinase inhibitor modulates T lymphocyte activation and collagen-induced arthritis.

Class IA phosphoinositide 3-kinases (PI3Ks) are essential to function of normal and tumor cells, and to modulate immune responses. T lymphocytes express high levels of p110α and p110δ class IA PI3K. Whereas the functioning of PI3K p110δ in immune and autoimmune reactions is well established, the role of p110α is less well understood. Here, a novel dual p110α/δ inhibitor (ETP-46321) and highly specific p110α (A66) or p110δ (IC87114) inhibitors have been compared concerning T cell activation in vitro, as well as the effect on responses to protein antigen and collagen-induced arthritis in vivo. In vitro activation of naive CD4(+) T lymphocytes by anti-CD3 and anti-CD28 was inhibited more effectively by the p110δ inhibitor than by the p110α inhibitor as measured by cytokine secretion (IL-2, IL-10, and IFN-γ), T-bet expression and NFAT activation. In activated CD4(+) T cells re-stimulated through CD3 and ICOS, IC87114 inhibited Akt and Erk activation, and the secretion of IL-2, IL-4, IL-17A, and IFN-γ better than A66. The p110α/δ inhibitor ETP-46321, or p110α plus p110δ inhibitors also inhibited IL-21 secretion by differentiated CD4(+) T follicular (Tfh) or IL-17-producing (Th17) helper cells. In vivo, therapeutic administration of ETP-46321 significantly inhibited responses to protein antigen as well as collagen-induced arthritis, as measured by antigen-specific antibody responses, secretion of IL-10, IL-17A or IFN-γ, or clinical symptoms. Hence, p110α as well as p110δ Class IA PI3Ks are important to immune regulation; inhibition of both subunits may be an effective therapeutic approach in inflammatory autoimmune diseases like rheumatoid arthritis.

The Impact of Vitamin D Levels on Inflammatory Status: A Systematic Review of Immune Cell Studies.

Chronic low-grade inflammation accompanies obesity and its related chronic conditions. Both peripheral blood mononuclear cells (PBMCs) and cell lines have been used to study whether vitamin D has immune modulating effects; however, to date a detailed systematic review describing the published evidence has not been completed. We therefore conducted a systematic review on the effect of vitamin D on the protein expression and secretion of inflammatory markers by human-derived immune cells. The review was registered at the International Prospective Register for Systematic Reviews (PROSPERO, Registration number CRD42015023222). A literature search was conducted using Pubmed, Science Direct, Scopus, Web of Science and Medline. The search strategy used the following search terms: Vitamin D or cholecalciferol or 1,25-dihydroxyvitamin or 25-hydroxy-Vitamin D and Inflam* or cytokine* and supplement* or cell*. These terms were searched in the abstract, title and keywords. Inclusion criteria for study selection consisted of human-derived immune cell lines or cellular studies where PBMCs were obtained from humans, reported in the English language, and within the time period of 2000 to 2015. The selection protocol was mapped according to PRISMA guidelines. Twenty three studies (7 cell line and 16 PBMCs studies) met our criteria. All studies selected except one used the active metabolite 1,25(OH)2, with one study using cholecalciferol and two studies also using 25(OH)D. Four out of seven cell line studies showed an anti-inflammatory effect where suppression of key markers such as macrophage chemotactic protein 1, interleukin 6 and interleukin 8 were observed. Fourteen of sixteen PBMC studies also showed a similar anti-inflammatory effect based on common inflammatory endpoints. Mechanisms for such effects included decreased protein expression of toll-like receptor-2 and toll-like receptor-4; lower levels of phosphorylated p38 and p42/42; reduced expression of phosphorylated signal transducer and activator of transcription 5 and decreased reactive oxygen species. This review demonstrates that an anti-inflammatory effect of vitamin D is a consistent observation in studies of cell lines and human derived PBMCs.

The Pelargonium sidoides Extract EPs 7630 Drives the Innate Immune Defense by Activating Selected MAP Kinase Pathways in Human Monocytes.

Pelargonium sidoides is a medical herb and respective extracts are used very frequently for the treatment of respiratory tract infections. However, the effects of Pelargonium sidoides and a special extract prepared from its roots (EPs 7630) on human immune cells are not fully understood. Here we demonstrate that EPs 7630 induced a rapid and dose-dependent production of TNF-α, IL-6, and IL-10 by human blood immune cells. This EPs 7630-induced cytokine profile was more pro-inflammatory in comparison with the profile induced by viral or bacterial infection-mimicking agents. The search for EPs 7630 target cells revealed that T-cells did not respond to EPs 7630 stimulation by production of TNF-α, IL-6, or IL-10. Furthermore, pretreatment of T-cells with EPs 7630 did not modulate their TNF-α, IL-6, and IL-10 secretion during subsequent activation. In contrast to lymphocytes, monocytes showed clear intracellular TNF-α staining after EPs 7630 treatment. Accordingly, EPs 7630 predominantly provoked activation of MAP kinases and inhibition of p38 strongly reduced the monocyte TNF-α production. The pretreatment of blood immune cells with EPs 7630 lowered their secretion of TNF-α and IL-10 and caused an IL-6 dominant response during second stimulation with viral or bacterial infection-mimicking agents. In summary, we demonstrate that EPs 7630 activates human monocytes, induces MAP kinase-dependent pro-inflammatory cytokines in these cells, and specifically modulates their production capacity of mediators known to lead to an increase of acute phase protein production in the liver, neutrophil generation in the bone marrow, and the generation of adaptive Th17 and Th22 cells.

Chloroform extract of alfalfa (Medicago sativa) inhibits lipopolysaccharide-induced inflammation by downregulating ERK/NF-κB signaling and cytokine production.

Alfalfa (Medicago sativa L.) is commonly used as a traditional medicine and functional food. This study investigated the anti-inflammatory potential of alfalfa and the mechanisms involved. The chloroform extract of alfalfa aerial parts inhibited lipopolysaccharide (LPS)-stimulated immune responses more than ether, butanol, or water soluble extracts. Treatment with 1 µg/mL LPS increased nitrite concentrations to 44.3 µM in RAW267.4 macrophages, but it was reduced to 10.6 µM by adding 100 µg/mL chloroform extract. LPS treatment also increased the concentrations of tumor necrosis factor-α, interleukin (IL)-6, and IL-1ß to 41.3, 11.6, and 0.78 ng/mL in culture supernatants of the cells, but these cytokine levels decreased to 12.5, 3.1, and 0.19 ng/mL, respectively, by pretreating with 100 µg/mL of the extract. ICR mice injected with LPS (30 mg/kg body weight) alone showed a 0% survival rate after 48 h of the injection, but 48-h survival of the mice increased to 60% after oral administration of the extract. Subfractions of the chloroform extract markedly suppressed LPS-mediated activation of the extracellular signal-regulated kinase and nuclear factor kappa-B. Cinnamic acid derivatives and fatty acids were found to be active constituents of the extract. This research demonstrated that alfalfa aerial parts exert anti-inflammatory activity and may be useful as a functional food for the prevention of inflammatory disorders.

Anti-inflammatory activities of Reynoutria elliptica through suppression of mitogen-activated protein kinases and nuclear factor-κB activation pathways.

Reynoutria elliptica has been used in traditional Korean medicine to promote blood circulation, relieve pain, increase dieresis, and alleviate respiratory problems, through as yet undefined mechanisms. We set out to determine whether the anti-inflammatory effects of this plant are linked with its ability to suppress mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB) activation in lipopolysaccharide (LPS)-activated RAW 264.7 cells. We found for the first time that the hexane fraction of Reynoutria elliptica (HRE) significantly inhibited LPS-stimulated NO and PGE2 synthesis. This is due to the diminishing of the mRNA and protein expression of iNOS and COX-2, respectively. HRE also suppressed LPS-stimulated TNF-α secretion in a dose-dependent manner, which might be due to the suppression of LPS-induced MAPKs and NF-κB activation. Moreover, our HPLC data demonstrated that the major components of the HRE were bioactive compounds such as emodin-6-Glc, emodin, and physcion. Overall, our results indicate that Reynoutria elliptica could be provided as a potential candidate for anti-inflammation treatment.

Attenuation of LPS-induced inflammation by ICT, a derivate of icariin, via inhibition of the CD14/TLR4 signaling pathway in human monocytes.

OBJECTIVE: To evaluate the anti-inflammatory potential of ICT in LPS stimulated human innate immune cells. BACKGROUND: 3, 5, 7-Trihydroxy-4'-methoxy-8-(3-hydroxy-3- methylbutyl)-flavone (ICT) is a novel derivative of icariin, the major active ingredient of Herba Epimedii, an herb used in traditional Chinese medicine. We previously demonstrated its anti-inflammatory potential in a murine macrophage cell line as well as in mouse models. METHODS: We measured TNF-α production by ELISA, TLR4/CD14 expression by flow cytometry, and NF-κB and MAPK activation by western blot all in LPS-stimulated PBMC, human monocytes, or THP-1 cells after treatment with ICT. RESULTS: ICT inhibited LPS-induced TNF-α production in THP-1 cells, PBMCs and human monocytes in a dose-dependent manner. ICT treatment resulted in down-regulation of the expression of CD14/TLR4 and attenuated NF-κB and MAPK activation induced by LPS. CONCLUSION: We illustrate the anti-inflammatory property of ICT in human immune cells, especially in monocytes. These effects were mediated, at least partially, via inhibition of the CD14/TLR4 signaling pathway.

Sauchinone suppresses pro-inflammatory mediators by inducing heme oxygenase-1 in RAW264.7 macrophages.

Sauchinone, a biologically active lignan isolated from the roots of Saururus chinensis (LOUR.) BAILL. (Saururaceae), is reported to exert a variety of biological activities, such as hepatoprotective, anti-inflammatory actions and inhibitory effects on bone resorption. In this study, we investigated the effect of sauchinone in suppressing cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) expression, leading to a reduction in COX-2-derived prostaglandin E(2) (PGE(2)) and iNOS-derived nitric oxide (NO) production in lipopolysaccharide (LPS) stimulated RAW264.7 macrophages. Present study also demonstrates the effects of sauchinone in inducing heme oxygenase-1 (HO-1) expression and an increase in heme oxygenase (HO) activity in RAW264.7 macrophages. The effects of sauchinone on LPS-induced PGE(2), NO, tumor necrosis factor-α (TNF-α) and interlukine-1ß (IL-1ß) production were partially reversed by the HO-1 inhibitor Tin protoporphyrin was also seen in this study. In addition, we found that treatment with extracellular signal-regulated kinase (ERK) inhibitor (PD98059) reduced sauchinone-induced HO-1 expression. Sauchinone also increased ERK phosphorylation. These results suggest that sauchinone inhibits pro-inflammatory mediators through expression of anti-inflammatory HO-1 via ERK pathway.

Immune stimulatory effects of Loranthi ramulus on macrophages through the increase of NO and TNF-alpha.

The activation of macrophages by microorganisms plays an important role in host defense and immunopathology. Loranthi ramulus (LR) is commonly used as a traditional drug and health food in Korea. Here, we investigated the regulatory effects of LR on macrophage-mediated immune responses. Treatment of macrophages with LR resulted in the enhanced cell-surface expression of CD80, CD86 and major histocompatibility complex (MHC) class II, as well as the enhanced production of nitric oxide (NO) and tumor necrosis factor (TNF)-alpha, and also iNOS and TNF-alpha mRNA expression. These alterations of LR-treated cells were associated with the activation of NF-kappaB and mitogen-activated protein kinases (MAPKs). LR increased the phosphorylation of MAPKs (JNK, ERK1/2, p38 MAPK) and the activation of NF-kappaB in Raw 264.7 cells. These results suggest that LR has increased NO and TNF-alpha production through phosphorylation of all three MAPKs following IkappaBalpha degradation and NF-kappaB activation. In conclusion, our results demonstrate that LR can effectively promote the activation of macrophages, suggesting that LR may possess the potential to regulate immune responses.

Potent inhibition of superoxide anion production in activated human neutrophils by isopedicin, a bioactive component of the Chinese medicinal herb Fissistigma oldhamii.

Fissistigma oldhamii is widely used in traditional Chinese medicine to treat rheumatoid arthritis. Activation of neutrophils is a key feature of inflammatory diseases. Herein, the anti-inflammatory functions of isopedicin, a flavanone derived from F. oldhamii, and its underlying mechanisms were investigated in human neutrophils. Isopedicin potently and concentration-dependently inhibited superoxide anion (O(2)(*)(-)) production in formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP)-activated human neutrophils with an IC(50) value of 0.34+/-0.03 microM. Furthermore, isopedicin displayed no superoxide-scavenging ability, and it failed to alter subcellular NADPH oxidase activity. The inhibitory effect of isopedicin on O(2)(*)(-) production was reversed by protein kinase A (PKA) inhibitors. Moreover, isopedicin increased cAMP formation and PKA activity in FMLP-activated human neutrophils, which occurred through the inhibition of phosphodiesterase (PDE) activity but not an increase in adenylate cyclase function. In addition, isopedicin reduced FMLP-induced phosphorylation of extracellular regulated kinase and c-Jun N-terminal kinase, which was reversed by the PKA inhibitor. In contrast, isopedicin failed to alter FMLP-induced phosphorylation of p38 mitogen-activated protein kinase and calcium mobilization. In summary, these results demonstrate that inhibition of O(2)(*)(-) production in human neutrophils by isopedicin is associated with an elevation of cellular cAMP and activation of PKA through its inhibition of cAMP-specific PDE.