María A. Hidalgo

Andrographolide interferes with binding of nuclear factor-κB to DNA in HL-60-derived neutrophilic cells

1 Andrographolide, the major active component from Andrographis paniculata, has shown to possess anti‐inflammatory activity. Andrographolide inhibits the expression of several proinflammatory proteins that exhibit a nuclear factor kappa B (NF‐κB) binding site in their gene. 2 In the present study, we analyzed the effect of andrographolide on the activation of NF‐κB induced by platelet‐activating factor (PAF) and N‐formyl‐methionyl‐leucyl‐phenylalanine (fMLP) in HL‐60 cells differentiated to neutrophils. 3 PAF (100 nM) and fMLP (100 nM) induced activation of NF‐κB as determined by degradation of inhibitory factor B α (IκBα) using Western blotting in cytosolic extracts and by binding to DNA using electrophoretic mobility shift assay (EMSA) in nuclear extracts. 4 Andrographolide (5 and 50 μM) inhibited the NF‐κB‐luciferase activity induced by PAF. However, andrographolide did not reduce phosphorylation of p38 MAPK or ERK1/2 and did not change IκBα degradation induced by PAF and fMLP. 5 Andrographolide reduced the DNA binding of NF‐κB in whole cells and in nuclear extracts induced by PAF and fMLP. 6 Andrographolide reduced cyclooxygenase‐2 (COX‐2) expression induced by PAF and fMLP in HL‐60/neutrophils. 7 It is concluded that andrographolide exerts its anti‐inflammatory effects by inhibiting NF‐κB binding to DNA, and thus reducing the expression of proinflammatory proteins, such as COX‐2.

Andrographolide reduces IL-2 production in T-cells by interfering with NFAT and MAPK activation

The nuclear factor of activated T cells (NFAT) is a transcription factor essential for cytokine production during T-cell activation and is the target of several immunosuppressive drugs. Andrographolide is a diterpenic labdane that possesses anti-inflammatory and immunomodulatory effects. Several studies propose that andrographolide can reduce the immune response through inhibition of the nuclear factor kappa B (NF-kappaB) and mitogen-activated protein kinases (MAPK) such as extracellular signal regulated kinase 1/2 (ERK1/2) pathways. Moreover, andrographolide reduces IFN-gamma and IL-2 production induced by concanavalin A in murine T-cell. Nevertheless, the mechanisms involved in the decrease of cytokine production are unknown. In the present study, we determined that andrographolide reduced IL-2 production in Jurkat cells stimulated with phorbol myristate acetate and ionomycin (PMA/Ionomycin). We then showed that andrographolide reduced NFAT luciferase activity and interfered with its nuclear distribution, with these effects being linked to an increase in c-jun-N-terminal kinase (JNK) phosphorylation. Additionally, reduction of NF-kappaB activity in Jurkat cells treated with andrographolide was observed. Using Western blotting, we demonstrated that andrographolide decreased ERK1 and ERK5 phosphorylation induced by anti-CD3 or PMA/Ionomycin. Andrographolide did not affect cell viability at concentration of 10 and 50 muM; however, our results suggest that andrographolide increase early apoptosis at 100 muM. We concluded that andrographolide can exert immunomodulatory effects by interfering with NFAT activation and ERK1 and ERK5 phosphorylation in T-cells.

Eimeria bovis-triggered neutrophil extracellular trap formation is CD11b-, ERK 1/2-, p38 MAP kinase- and SOCE-dependent

Eimeria bovis is an important coccidian parasite that causes high economic losses in the cattle industry. We recently showed that polymorphonuclear neutrophils (PMN) react upon E. bovis sporozoite exposure by neutrophil extracellular trap (NET) formation. We focused here on the molecular mechanisms that are involved in this process. The sporozoite encounter led to an enhanced surface expression of neutrophil CD11b suggesting a potential role of this receptor in E. bovis-mediated NETosis. Antibody-mediated blockage of CD11b confirmed this assumption and led to a significantly decreased sporozoite-triggered NET. In addition, E. bovis-induced NETosis was found to be Ca2+-dependent since the inhibition of store-operated calcium entry (SOCE) significantly diminished NET. Furthermore, NADPH oxidase, neutrophil elastase (NE) and myeloperoxidase (MPO) were confirmed as key molecules in sporozoite-triggered NETosis, as inhibition thereof blocked parasite-triggered NET. PMN degranulation analyses revealed a significant release of matrix metalloprotease-9 containing granules upon sporozoite exposure. We further show a significantly enhanced phosphorylation of ERK1/2 and p38 MAPK in sporozoite-exposed PMN indicating a key role of this signaling pathway in E. bovis-mediated NETosis. Accordingly, ERK 1/2 and p38 MAPK inhibition led to a significant decrease in NET formation. Finally, we demonstrate that sporozoite-induced NETosis is neither a stage-, species-, nor host-specific process.

Store-operated calcium entry mediates intracellular alkalinization, ERK1/2, and Akt/PKB phosphorylation in bovine neutrophils

Neutrophil’s responses to G protein‐coupled chemoattractants are highly dependent on store‐operated calcium (Ca2+) entry (SOCE). Platelet‐activating factor (PAF), a primary chemoattractant, simultaneously increases cytosolic‐free Ca2+, intracellular pH (pHi), ERK1/2, and Akt/protein kinase B (PKB) phosphorylation. In this study, we looked at the efficacy of several putative SOCE inhibitors and whether SOCE mediates intracellular alkalinization, ERK1/2, and Akt/PKB phosphorylation in bovine neutrophils. We demonstrated that the absence of external Ca2+ and the presence of EGTA reduced the intracellular alkalinization and ERK1/2 phosphorylation induced by PAF, apparently via SOCE influx inhibition. Next, we tested the efficacy of several putative SOCE inhibitors such as 2‐aminoethoxydiphenyl borate (2‐APB), capsaicin, flufenamic acid, 1‐{β‐[3‐(4‐methoxy‐phenyl)propoxy]‐4‐methoxyphenethyl}‐1H‐imidazole hydrochloride (SK&F 96365), and N‐(4‐[3,5‐bis(trifluoromethyl)‐1H‐pyrazol‐1‐yl]phenyl)‐4‐methyl‐1,2,3‐thiadiazole‐5‐carboxamide (BTP2) on Ca2+ entry induced by PAF or thapsigargin. 2‐APB was the most potent SOCE inhibitor, followed by capsaicin and flufenamic acid. Conversely, SK&F 96365 reduced an intracellular calcium ([Ca2+]i) peak but SOCE partially. BTP2 did not show an inhibitory effect on [Ca2+]i following PAF stimuli. 2‐APB strongly reduced the pHi recovery, whereas the effect of flufenamic acid and SK&F 96365 was partial. Capsaicin and BTP2 did not affect the pHi changes induced by PAF. Finally, we observed that 2‐APB reduced the ERK1/2 and Akt phosphorylation completely, whereas the inhibition with flufenamic acid was partial. The results suggest that 2‐APB is the most potent SOCE inhibitor and support a key role of SOCE in pH alkalinization and PI‐3K–ERK1/2 pathway control. Finally, 2‐APB could be an important tool to characterize Ca2+ signaling in neutrophils.

Calcium influx, a new potential therapeutic target in the control of neutrophil-dependent inflammatory diseases in bovines

Neutrophils are the first line of defense against pathogens in bovines; however, they are also one of the most aggressive cells during the inflammatory process, causing injury in surrounding tissues. At present, anti-inflammatory drugs are limited in acute diseases, such as pneumonia, mastitis and endometritis, because neutrophils are mostly insensitive. One of the earliest events during neutrophil activation is the increase in intracellular calcium concentration. The calcium movement is attributed to the release from intracellular stores and influx through the calcium channels in the plasma membrane, a process called store operated calcium entry (SOCE). Recently, several calcium influx blockers have been shown to have strong effects on bovine neutrophils, and this suggests that the manipulation of this pathway can be useful in the control of neutrophil functions during acute inflammatory processes. In this paper, we will review the role of calcium influx as a potential anti-inflammatory target and summarize the most recent evidences for this in bovine neutrophils.

Oleic acid induces intracellular calcium mobilization, MAPK phosphorylation, superoxide production and granule release in bovine neutrophils

Oleic acid (OA) is a nonesterified fatty acid that is released into the blood during lipomobilization at the time of calving in cows, a period where increased risk of infection and acute inflammation is observed. These data suggest potential OA-mediated regulation of innate immune responses. In the present study, we assessed the effects of OA on intracellular calcium release, ERK1/2 phosphorylation, superoxide production, CD11b expression and matrix metalloproteinase-9 (MMP-9) release in bovine neutrophils. Furthermore, the presence of GPR40, an OA receptor, was assessed by RT-PCR, immunoblotting and confocal microscopy. OA induced, in a dose-dependent manner, intracellular calcium mobilization, superoxide production and CD11b expression in bovine neutrophils; these effects were reduced by the intracellular chelating agent BAPTA-AM. OA also induced ERK2 phosphorylation and MMP-9 release. RT-PCR analysis detected mRNA expression of a bovine ortholog of the GPR40 receptor. Using a polyclonal antibody against human GPR40, we detected a protein of 31kDa by immunoblotting that was localized predominately in the plasma membrane. The selective agonist of GPR40, GW9508, induced intracellular calcium mobilization and ERK2 phosphorylation. In conclusion, OA can modulate bovine neutrophil responses in an intracellular calcium-dependent manner; furthermore, these responses could be induced by GPR40 activation.

2-Aminoethoxydiphenyl borate (2-APB) reduces respiratory burst, MMP-9 release and CD11b expression, and increases l-selectin shedding in bovine neutrophils

This study describes the effect of 2-aminoethoxydiphenyl borate (2-APB), a putative store-operated calcium (Ca(2+)) entry (SOCE) inhibitor, on reactive oxygen species (ROS) production, matrix metalloproteinase 9 (MMP-9) release, CD11b and l-selectin (CD62L) expression, size changes and apoptosis in bovine neutrophils stimulated with platelet-activating factor (PAF). It was observed that doses ⩾1μM 2-APB significantly reduced ROS production, whereas 50 and 100μM 2-APB reduced MMP-9 release induced by PAF. Moreover, concentrations ⩾10μM 2-APB reduced CD11b expression and increased l-selectin shedding. PAF induced size changes in neutrophils, and this effect was inhibited by 2-APB. From this work it is possible to conclude that 2-APB at concentrations that inhibit SOCE responses was able to inhibit ROS and MMP-9 release and CD11b expression, and increase l-selectin shedding, suggesting that the Ca(2+) channel involved in SOCE is a potential target for the development of new anti-inflammatory drugs in cattle.

Platelet-activating factor increases pH(i) in bovine neutrophils through the PI3K–ERK1/2 pathway

Platelet‐activating factor (PAF) is known to stimulate a variety of neutrophil activities, including chemotaxis, phagocytosis, degranulation, reactive oxygen species production and intracellular pH increase. The purpose of this study was to investigate the effect of PAF on pH(i), specifically if these changes in pH are the result of phosphatidylinositol 3‐kinase (PI3K) and mitogen‐activated protein kinase (MAPK) pathway activation in bovine neutrophils. PAF caused intracellular alkalinization in 2′,7′‐bis‐(2‐carboxyethyl)‐5‐(and‐6)‐carboxyfluorescein acetoxymethyl ester‐loaded bovine neutrophils. This phenomenon seems to be mediated by amiloride‐sensitive Na+/H+ exchange, and is inhibited by WEB2086 (a selective PAF receptor antagonist), genistein (a tyrosine kinase inhibitor), wortmannin and LY294002 (PI3K inhibitors), and PD98059 and UO126 (MEK inhibitors). PAF 100 nM induced an increase in tyrosine phosphorylation of proteins 62, 44 and 21 kDa with a maximum response at 2 min of incubation. Unlike human neutrophils, bovine neutrophils are strongly stimulated by PAF via phosphorylation of ERK1/2 (extracellular‐signal‐regulated protein kinase) with an EC50 of 30 and 13 nM, respectively. PAF MAPK activation was also inhibited by WEB2086, pertussis toxin (PTX), genistein, wortmannin, LY294002, PD98059 and UO126 in bovine neutrophils. The ERK1/2 activation is dependent on PI3K pathway, because protein kinase B was phosphorylated by PAF and inhibited by wortmannin and LY294002, but not by U0126. Our results suggest that PAF induces intracellular alkalinization via PI3K–MAPK activation. This effect is upstream regulated by PAF receptor, PTX‐sensitive G protein, tyrosine kinase, PI3K and MEK1/2 in bovine neutrophils.

Propionate induces the release of granules from bovine neutrophils

Short-chain fatty acids (SCFA) are produced by bacterial fermentation in the rumen of cattle and are the primary energy source in ruminants. Propionate is one of the main SCFA and it can exert multiple effects on the inflammatory process and neutrophil function via calcium (Ca(2+)) release, reactive oxygen species, and intracellular pH changes. However, currently no evidence has shown whether propionate can induce granule release from bovine neutrophils. The purpose of this study was to analyze the effect of propionate on granule release and to evaluate the expression of two G-protein coupled receptors-GPR41 and GPR43-that are activated by propionate. Neutrophil degranulation was assessed by quantifying the release of the neutrophil enzymes myeloperoxidase (MPO), lactoferrin, and matrix metalloprotease-9 (MMP-9) as markers of azurophil, specific granules, and gelatinase granules, respectively. Isolated bovine neutrophils were treated with millimolar concentrations of propionate (0.3, 3 and 30mM), and the cell-free supernatants were recovered. The stimulation of neutrophils with 0.3mM propionate induced the release of lactoferrin and MMP-9 as revealed by ELISA and gelatin zymography, respectively. Propionate at 30mM induced the release of MPO as demonstrated using an enzymatic assay. The role of intracellular Ca(2+) influx and the signaling pathways that may regulate the propionate effect on granules release were also determined. Reverse transcription (RT)-PCR and real-time PCR were performed to analyze the expression of GPR41 and GPR43 mRNA in bovine neutrophils. Both mRNA were detected, whereas the expression of GPR43 was higher than that of GPR41, and the synthetic agonists for this receptor, phenylacetamides 1 and 2, caused an increase in intracellular Ca(2+), lactoferrin, and MMP-9 release. These results support that propionate-induced granule release is mediated by intracellular Ca(2+) influx and activation of extracellular signal-regulated kinase ERK 1/2. We also propose a potential role of GPR43 in propionate-induced granule release from bovine neutrophils that may be involved in regulatory effects of propionate in the innate immune response in cattle.

Andrographolide a New Potential Drug for the Long Term Treatment of Rheumatoid Arthritis Disease

Andrographis paniculata, (Burm. f.) Wall. ex Nees, a herbaceous plant belonging to the Family Acanthaceae, is one of the most commonly used medicinal plants in the traditional systems of Unani and Ayurvedic medicines. It grows in hedge rows throughout the plains of India and is also cultivated in gardens. It also grows in many other Asian countries and is used as a tradition‐ al herbal medicine in China, Hong Kong, the Philippines, Malaysia, Indonesia, and Thailand. It is an annual plant of 1-3 ft high, also known as the “king of bitters”, being the aerial parts most commonly used. A. paniculata have shown a broad range of pharmacological effects such as in‐ hibition of replication of the HIV virus, prevention of common cold, and antimalarial, antidiar‐ rheal, antibacterial, antihyperglycemic effects, suppression of various cancer cells, and principally anti-inflammatory properties. Andrographolide is the major labdane diterpenoid isolated from A. paniculata and exhibits anti-inflammatory and anticancer activities, either in vi‐ tro or in vivo experimental models of inflammation and cancer. Several immunomodulatory re‐ sponses of andrographolide have been observed in in vitro studies, such as reduction of iNOS, COX-2, NO, PGE2, TNF-alpha and IL-12 in macrophages and microglia. In neutrophils is able to reduce the radical oxygen species production, and Mac-1, IL-8 and COX-2 expression. In T cells, andrographolide inhibits the expression of IL-2, IFNγ and IL-6, reducing the humoral and cellu‐ lar adaptive immune response. Andrographolide was able to reduce the dendritic cells matura‐ tion and their ability to present antigens to T cells. Andrographolide administered in rodents reduced the Th2 cytokine IL-4, IL-5, IL-13 and serum immunoglobulin in an ovalbumin in‐ duced asthma model. A reduction of T cells response also has been observed in experimental au‐ toimmune encephalomyelitis and systemic lupus erythematosus mouse model. Several of immunomodulatory responses have been associated to the inhibition of Nuclear Factor-κB