Signal transduction of the prophenoloxidase activating system of prawn haemocytes triggered by CpG oligodeoxynucleotides

Intracellular phenoloxidase (PO) activity in haemocyte lysate supernatant (HLS) of giant freshwater prawn (Macrobrachium rosenbergii) was shown to be enhanced by CpG oligodeoxynucleotide (ODN) 2006, but not by so-ODN13. When haemocytes were treated in vitro with 50 microg/ml of ODN2006 for 30 min, the increases in both intra- and extracellular stimulated PO activity (POS) and extracellular total PO activity (POT) and the reduction of POT suggest that the PO activity of haemocytes is enhanced by ODN2006 stimulation, but new prophenoloxidase (proPO) is not synthesised. In an attempt to determine which signal transduction pathway is involved in the activation of the proPO system, haemocytes were separately treated with activators or inhibitors of specific signalling components. The results show that there was an increase in both intra- and extracellular POT of haemocytes treated with sodium fluoride (a G-protein activator); the addition of phosphokinase A (PKA)-activating 8-bromo-cAMP to haemocytes only increased intracellular POT, and the addition of either phorbol-12-myristate-13-acetate (PMA; a phosphokinase C (PKC) activator) or caffeine (a phosphodiesterase inhibitor) only increased extracellular POT. When PMA-stimulated haemocytes were treated with chelerythrine (a PKC inhibitor), the induced extracellular POT was significantly reduced. Furthermore, the study of ODN2006-stimulated haemocytes treated with chelerythrine or palmitoyl-DL-carnitine (a PKC inhibitor) showed that the enhancement effects of ODN2006 on the intra- and extracellular POS and extracellular POT were significantly decreased. ODN-stimulated haemocytes treated with genistein (an inhibitor of protein tyrosine kinase) showed a further increase in extracellular POT, but the other PO activities remained the same as those of the ODN-stimulated group. These results suggest that the activation of the proPO system of prawn haemocytes, including degranulation and PO activity, is induced by ODN2006 via a PKC-activating signalling pathway, but negatively regulated via the tyrosine kinase pathway.     

The effect of two CpG oligodeoxynucleotides with different sequences on haemocytic immune responses of giant freshwater prawn,Macrobrachium rosenbergii

A previous study has demonstrated that the intrahaemocytic phenoloxidase (PO) activity of the prawn Macrobrachium rosenbergii was enhanced by CpG oligodeoxynucleotide (ODN) 2006, but not by os-ODN13. The study described herein determined the binding characteristics of the two ODNs to haemocytes. Treating haemocytes with FAM-ODN or FAM-ODN plus different concentrations of the same ODN revealed that both ODNs specifically bound to haemocytes. Results from haemocytes treated with FAM-ODN2006 plus ROX-os-ODN13 in a competitive assay indicated that about 91% of haemocytes were simultaneously bound by the two ODNs and that the remaining haemocytes were only bound by ODN2006; moreover, ODN2006 binding to haemocytes was stronger than that of os-ODN13. To clarify the interactive effect of the two ODNs on haemocytic function, mRNA levels of haemocytic genes from single or double ODN-injected prawns were determined by semi-quantitative RT-PCR. The expression of either the prophenoloxidase (proPO) or peroxinectin (pon) genes was elevated by ODN2006 but reduced by os-ODN13; furthermore, ODN2006-increased proPO expression was abated following treatment with os-ODN13. In comparison with ODN-injected prawns alone, the NF-κB inhibitor PDTC reduced the proPO mRNA levels induced by ODN2006, but it elevated proPO expression inhibited by os-ODN13. These results support the notion that os-ODN13 may be able to neutralise or negate the enhancing effect of ODN2006 on proPO expression via the NF-κB signalling pathway as well as an unknown signalling pathway.     

Unmethylated CpG motifs mimicking bacterial DNA triggers the local and systemic innate immune parameters and expression of immune-relevant genes in gilthead seabream

Unmethylated CpG motifs present in bacterial DNA are recognized by leucocyte receptors triggering an immune response. We have evaluated herein the immunomodulatory actions of a CpG motif in an important commercial fish, the gilthead seabream. Thus, 1, 3 and 7days after intraperitoneal injection of the CpG motif the seabream immune parameters and gene expression profile were evaluated. Firstly, humoral innate immune responses were unaffected by CpG ODN 1668. On the other hand, ODN injection significantly enhanced the number of peritoneal leucocytes (PELs) 1day after injection and increased the main innate immune parameters of PELs and HKLs (head-kidney leucocytes). Thus, injection of ODN 1668 significantly increased respiratory burst, peroxidase, cytotoxic and phagocytic activities, with variations in increment and time. The cytotoxic activity of HKLs was the most increased (up to 4.2-fold). Moreover, the expression profile of immune-relevant genes in head-kidney was affected, with substantial up-regulation of TLR9, IL-1beta, Mx, TGFbeta and Gal8 gene expression. These results demonstrate that unmethylated CpG motifs prime the fish immune response with promising applications for aquaculture.     

Immunoprotective activity and safety of a respiratory syncytial virus vaccine: mucosal delivery of fusion glycoprotein with a CpG oligodeoxynucleotide adjuvant

CpG oligodeoxynucleotides (ODN) were identified that stimulated immunoglobulin production and cell proliferation in cotton rat cells in vitro. Three of these ODN were used as a mucosal adjuvant in the noses of cotton rats immunized via this route with respiratory syncytial virus fusion (F) protein. The CpG ODN markedly increased the cotton rat humoral neutralizing-antibody response to respiratory syncytial virus. Such immunized animals had a marked reduction in the production of infectious virus after a live-virus challenge. Animals immunized with the combination of F protein and CpG developed enhanced pulmonary pathology consisting of alveolitis and interstitial pneumonitis after a live-virus challenge. Similar enhanced disease has been seen in cotton rats and children immunized with formalin-inactivated respiratory syncytial virus.     

Differentiation of HL-60 promyelocytic leukemia cells monitored by flow cytometric measurement of nitro blue tetrazolium (NBT) reduction

Reduction of nitro blue tetrazolium (NBT) to insoluble blue formazan granules occurs during the stimulus-induced respiratory burst of mature granulocytes and is routinely used as an indicator of the extent of granulocytic differentiation of HL-60 acute promyelocytic leukemia cells. In the present study, the differentiation of HL-60 leukemia cells induced by dimethylsulfoxide (DMSO) or retinoic acid was monitored by flow cytometric (FCM) measurement of forward and 90 degree light scatter of NBT treated cells. Two-parameter correlated analysis permitted a distinction between cells with increased forward and decreased 90 degree light scatter (NBT-), and cells with decreased forward and increased 90 degree light scatter (NBT+). Fixation of NBT treated cells with 1% paraformaldehyde facilitated flow cytometric analysis, and allowed differences in NBT reduction to be quantitated. DMSO-induced cells expressed an all-or-none reduction of NBT to formazan, compared with retinoic acid treated cells that exhibited a graded response. Three parameter flow cytometric analysis of HL-60 leukemia cells stained with propidium iodide in combination with NBT allowed the determination of the cell cycle distribution of NBT-treated cells.     

Stimulation of innate immune responses by CpG oligodeoxynucleotide in newborn lambs can reduce bovine herpesvirus-1 shedding

Stimulation of the innate immune system is potentially very important in neonates who have an immature adaptive immune system and vaccination cannot be used to reduce the risk of infection. CpG oligodeoxynucleotide (ODN) can stimulate innate immune responses in newborn chickens and mice, but similar studies are lacking in other mammalian species. We have shown previously that CpG ODN can both stimulate an acute-phase immune response and induce the antiviral effector molecule, 2'5'-A synthetase, in adult sheep. Therefore, the immunostimulatory activity of A class and B class CpG ODN was evaluated in newborn lambs, and the capacity of CpG ODN-induced responses to reduce viral shedding was evaluated following aerosol challenge with the respiratory pathogen, bovine herpesvirus-1 (BHV-1). In vitro CpG ODN stimulation of peripheral blood mononuclear cells (PBMC) isolated from newborn lambs (3-5 days old) and adult sheep induced equivalent CpG-specific proliferative responses and interferon-alpha (IFN-alpha) secretion. CpG ODN-induced IFN-alpha secretion by neonatal PBMCs was, however, significantly (p < 0.01) enhanced 6 days after subcutaneous (s.c.) injection of 100 microg/kg CpG ODN 2007. Newborn lambs injected s.c. with B class CpG ODN 2007 or the inverted GpC control ODN formulated in 30% Emulsigen (MVP Laboratories, Ralston, NE) displayed CpG ODN-specific increases in body temperature (p < 0.0001), serum 2'5'-A synthetase activity (p = 0.0015), and serum haptoglobin (p = 0.07). CpG ODN-treated lambs also displayed a transient reduction in viral shedding on day 2 postinfection (p < 0.05), which correlated (p < 0.03) with serum 2'5'-A synthetase levels on the day of viral challenge. These observations confirmed that CpG ODNs effectively activate innate immune responses in newborn lambs and CpG ODN-induced antiviral responses correlated with a reduction in viral shedding.     

A study on the protective effects of CpG oligodeoxynucleotide-induced mucosal immunity against lung injury in a mouse acute respiratory distress syndrome model

This study aims to determine the feasibility of using oligodeoxynucleotides with unmethylated cytosine-guanine dinucleotide sequences (CpG ODN) as an immunity protection strategy for a mouse model of acute respiratory distress syndrome (ARDS). This is a prospective laboratory animal investigation. Twenty-week-old BALB/c mice in Animal research laboratory were randomized into groups. An ARDS model was induced in mice using lipopolysaccharides (LPSs). CpG ODN was intranasally and transrectally immunized before or after the 3rd and 7th days of establishing the ARDS model. Mice were euthanized on Day 7 after the second immunization. Then, retroorbital bleeding was carried out and the chest was rapidly opened to collect the trachea and tissues from both lungs for testing. CpG ODN significantly improved the pathologic impairment in mice lung, especially after the intranasal administration of 50 μg. This resulted in the least severe lung tissue injury. Furthermore, interleukin-6 (IL-6) and IL-8 concentrations were lower, which was second to mice treated with the rectal administration of 20 µg CpG ODN. In contrast, the nasal and rectal administration of CpG ODN in BALB/c mice before LPS immunization did not appear to exhibit any significant protective effects. The intranasal administration of CpG ODN may be a potential treatment approach to ARDS. More studies are needed to further determine the protective mechanism of CpG ODN.     

Protein kinase C beta is required for human monocyte chemotaxis to MCP-1

Monocyte chemoattractant protein 1 (MCP-1) is important in attracting monocytes to sites of inflammation. Using predominantly pharmacological approaches, prior studies have indicated that serine/threonine kinases are involved in the MCP-1-induced signaling pathways. We report here that there is substantial inhibition of MCP-1-stimulated chemotaxis of human monocytes treated with inhibitors selective for the subset of serine/threonine kinases, protein kinase C (PKC). Selective inhibitors of PKC such as GF109203X and Calphostin C both caused approximately 80% inhibition of chemotaxis. Because these pharmacological inhibitors do not specifically inhibit individual PKC isoforms, we chose to use antisense oligodeoxyribonucleotides (ODN) to specifically reduce PKC isoform expression, first by inhibiting expression of the conventional PKC family, and next by using specific antisense ODN for PKCalpha and PKCbeta. Conventional PKC-antisense ODN treatment completely and significantly inhibited monocyte chemotaxis to MCP-1, whereas sense-control ODN caused no significant inhibition. PKCbeta-antisense ODN caused 89.2% inhibition of chemotaxis at its highest dose. In contrast, PKCbeta-sense ODN and PKCalpha-antisense and -sense ODN were without effect. Further studies evaluating the calcium response that is triggered upon MCP-1 interaction with its receptor, CCR2, indicate that this response is not altered by antisense or sense ODN treatment, thus supporting our hypothesis that PKCbeta is critical for post-receptor signal transduction downstream of the immediate calcium signal. These data contribute to our developing understanding of the signal transduction pathways involved in the chemotactic response of human monocytes to MCP-1 and uniquely identify the requirement for the PKCbeta isoform in this important process.     

A protective role of locally administered immunostimulatory CpG oligodeoxynucleotide in a mouse model of genital herpes infection

Unmethylated CpG dinucleotides in bacterial DNA or synthetic oligodeoxynucleotides (ODNs) are known as potent activators of the immune system and inducers of several Th1-associated immunomodulatory cytokines. We therefore investigated whether such a CpG-containing ODN (CpG ODN) given mucosally in the female genital tract could enhance innate immunity and protect against genital herpes infection. Groups of C57BL/6 mice were treated intravaginally with either CpG ODN or a non-CpG ODN control in the absence of any antigen either 2 days before or 4 h after an intravaginal challenge with a normally lethal dose of herpes simplex virus type 2 (HSV-2). Mice treated with CpG ODN exhibited significantly decreased titers of HSV-2 in their vaginal fluids compared with non-CpG ODN-treated mice. Furthermore, CpG ODN pretreatment significantly protected against development of disease and death compared to non-CpG ODN pretreatment. Most strikingly, CpG ODN conferred protection against disease and death even when given after the viral challenge. The CpG ODN-induced protection was associated with a rapid production of gamma interferon (IFN-gamma), interleukin-12 (IL-12), IL-18, and RANTES in the genital tract mucosa following CpG ODN treatment. The observed protection appeared to be dependent on IFN-gamma, IL-12, IL-18, and T cells, as CpG ODN pretreatment did not confer any significant protection in mice deficient in IFN-gamma, IL-12, IL-18, or T cells. Further, a complete protective immunity to reinfection was elicited in CpG ODN-treated, HSV-2-challenged mice, suggesting a role for mucosally administered CpG ODN in inducing the development of an acquired immune response in addition to its potent stimulation of innate immunity.     

Activation of Toll‐like receptor 9 inhibits lipopolysaccharide‐induced receptor activator of nuclear factor kappa‐ B ligand expression in rat B lymphocytes

B lymphocytes express multiple TLRs that regulate their cytokine production. We investigated the effect of TLR4 and TLR9 activation on receptor activator of NF‐κB ligand (RANKL) expression by rat spleen B cells. Splenocytes or purified spleen B cells from Rowett rats were cultured with TLR4 ligand Escherichia coli LPS and/or TLR9 ligand CpG‐oligodeoxynucleotide (CpG‐ODN) for 2 days. RANKL mRNA expression and the percentage of RANKL‐positive B cells were increased in rat splenocytes challenged by E. coli LPS alone. The increases were less pronounced when cells were treated with both CpG‐ODN and E. coli LPS. Microarray analysis showed that expressions of multiple cyclin‐dependent kinase (CDK) pathway‐related genes were up‐regulated only in cells treated with both E. coli LPS and CpG‐ODN. This study suggests that CpG‐ODN inhibits LPS‐induced RANKL expression in rat B cells via regulation of the CDK pathway.     

Response of nitric oxide production to CpG oligodeoxynucleotides in turkey and chicken peripheral blood monocytes

We evaluated the innate immune response to various synthetic CpG-containing oligodeoxynucleotides (CpG ODNs) by measuring nitric oxide production in the peripheral blood monocytes from turkey poults. The results indicate that the presence of the CpG dinucleotide in ODNs was a prerequisite for activation of turkey monocytes and induction of nitric oxide (NO) synthesis. CpG motifs and sequence structure of the ODNs were also found to influence stimulatory activity greatly. The most potent CpG ODN to induce NO synthesis in turkey monocytes was human-specific CpG ODN M362, followed by CpG ODN 2006 (human), CpG ODN#17 (chicken) and CpG ODN 1826 (mouse). The optimal CpG motif for NO induction was GTCGTT. Phosphorothioate modification of CpG ODNs also significantly increased stimulatory activity. Compared with chicken monocytes, turkey monocytes appeared to be less sensitive to CpG motif variation, whereas chicken monocytes were found to respond more strictly to human-specific CpG ODNs or ODNs that contain GTCGTT motifs.     

Regulation of the respiratory burst by cyclic 3',5'-AMP, an association with inhibition of arachidonic acid release.

The mechanism of cAMP regulation of the respiratory burst was studied with HL-60 cells that had been DMSO-differentiated to a neutrophil-like cell. To evaluate the effects of known cAMP concentrations, cells were permeabilized with streptolysin-O. Chemotactic peptide (FMLP)-stimulated NADPH oxidase activity was inhibited by cAMP at concentrations higher than 3 microM. Because intracellular calcium was buffered, inhibitory actions of cAMP were not mediated by modulation of calcium concentration. Effects of cAMP on chemotactic peptide signal transduction mediated by phospholipase C, phospholipase D, and phospholipase A2 were then determined. Neither inositol phosphate generation (phospholipase C) nor phosphatidylethanol generation (phospholipase D activity in presence of 1.6% ethanol) induced by FMLP were significantly affected by cAMP. In contrast, cAMP potently inhibited FMLP-induced arachidonic acid mobilization (phospholipase A2). NADPH oxidase activity induced by exogenous arachidonic acid was not inhibited by cAMP. These results indicate that cAMP-mediated inhibition of arachidonic acid mobilization may be important in regulation of the respiratory burst.     

Modulation of N-type calcium channel activity by G-proteins and protein kinase C

N-type voltage-gated calcium channel activity in rat superior cervical ganglion neurons is modulated by a variety of pathways. Activation of heterotrimeric G-proteins reduces whole-cell current amplitude, whereas phosphorylation by protein kinase C leads to an increase in current amplitude. It has been proposed that these two distinct pathways converge on the channel's pore-forming alpha(1B) subunit, such that the actions of one pathway can preclude those of the other. In this study, we have characterized further the actions of PKC on whole-cell barium currents in neonatal rat superior cervical ganglion neurons. We first examined whether the effects of G-protein-mediated inhibition and phosphorylation by PKC are mutually exclusive. G-proteins were activated by including 0.4 mM GTP or 0.1 mM GTP-gamma-S in the pipette, and PKC was activated by bath application of 500 nM phorbol 12-myristate 13-acetate (PMA). We found that activated PKC was unable to reverse GTP-gamma-S-induced inhibition unless prepulses were applied, indicating that reversal of inhibition by phosphorylation appears to occur only after dissociation of the G-protein from the channel. Once inhibition was relieved, activation of PKC was sufficient to prevent reinhibition of current by G-proteins, indicating that under phosphorylating conditions, channels are resistant to G-protein-mediated modulation. We then examined what effect, if any, phosphorylation by PKC has on N-type barium currents beyond antagonizing G-protein-mediated inhibition. We found that, although G-protein activation significantly affected peak current amplitude, fast inactivation, holding-potential-dependent inactivation, and voltage-dependent activation, when G-protein activation was minimized by dialysis of the cytoplasm with 0.1 mM GDP-beta-S, these parameters were not affected by bath application of PMA. These results indicate that, under our recording conditions, phosphorylation by PKC has no effect on whole-cell N-type currents, other than preventing inhibition by G-proteins.     

NOC/oFQ PKC-dependent superoxide generation contributes to hypoxic-ischemic impairment of NMDA cerebrovasodilation

This study determined whether nociceptin/orphanin FQ (NOC/oFQ) generates superoxide anion (O(2)(-)) in a protein kinase C (PKC)-dependent manner and whether such production contributes to hypoxic-ischemic (H-I) impairment of N-methyl-D-aspartate (NMDA)-induced pial artery dilation in newborn pigs equipped with closed cranial windows. Superoxide dismutase (SOD)-inhibitable nitroblue tetrazolium (NBT) reduction was an index of O(2)(-) generation. Under non-H-I conditions, topical NOC/oFQ (10(-10) M, concentration present in cerebrospinal fluid after I or H-I) increased SOD-inhibitable NBT reduction from 1 +/- 1 to 20 +/- 3 pmol/mm(2). PKC inhibitors staurosporine and chelerythrine (10(-7) M) blunted NBT reduction (1 +/- 1 to 7 +/- 2 pmol/mm(2) for chelerythrine), whereas the NOC/oFQ receptor antagonist [F/G]NOC/oFQ (1-13)-NH(2) (10(-6) M) blocked NBT reduction. [F/G]NOC/oFQ(1-13)-NH(2) and staurosporine also blunted the NBT reduction observed after I or H-I. NMDA (10(-8), 10(-6) M)-induced pial artery dilation was reversed to vasoconstriction after H-I. The NOC/oFQ antagonist staurosporine and free radical scavengers partially prevented this impaired dilation (sham: 9 +/- 1 and 16 +/- 1; H-I: -5 and -10 +/- 1; H-I staurosporine pretreated: 3 +/- 1 and 6 +/- 1%). These data show that NOC/oFQ increased O(2)(-) production in a PKC-dependent manner and contributed to this production after insult and that NOC/oFQ contributed to impaired NMDA-induced pial artery dilation after H-I, suggesting, therefore, that PKC-dependent O(2)(-) generation by NOC/oFQ links NOC/oFQ release to impaired NMDA dilation after H-I.     

The expression of prophenoloxidase mRNA in red swamp crayfish, Procambarus clarkii, when it was challenged

The expression of the prophenoloxidase (proPO) gene was investigated in nine tissues of red swamp crayfish Procambarus clarkii, by real-time PCR after challenges by CpG oligodeoxynucleotide (ODN), Aeromonas hydrophila and white spot syndrome virus (WSSV). The results can be summarized as follows: (i) the expression level of the proPO gene in haemocytes was highest among nine studied tissues before the challenge; (ii) the expression of proPO increased in all studied tissues after stimulation by CpG ODN and WSSV, and also increased in all tissues, except the ovary, after the A. hydrophila challenge; (iii) the whole expression profiles were different, suggesting that different immune mechanisms may exist for crayfish that are resistant to WSSV and A. hydrophila, although the expression in haemocytes was similar before and after the WSSV and A. hydrophila challenges.     

In vitro superoxide activity in the haemolymph of the West Indian leaf cockroach,Blaberus discoidalis

The respiratory burst is an NADPH oxidase-driven reduction of molecular oxygen to superoxide, which can occur in phagocytic cells as part of an antimicrobial defence, and is well documented among the vertebrates. This paper describes a process resembling the respiratory burst, which occurs in the haemolymph and haemocytes of the cockroach, Blaberus discoidalis. The in vitro reduction of nitroblue tetrazolium by superoxide to formazan was measured spectrophotometrically in B. discoidalis haemolymph in response to various immune elicitors. Nitroblue tetrazolium reduction was partly impeded in the presence of superoxide dismutase, a specific antioxidant which converts superoxide to hydrogen peroxide, as well as by chemicals known to inhibit the respiratory burst in vertebrates (trifluoperazine, diphenylene iodonium, and N-ethylmaleimide). This suggests the generation of superoxide anions by haemolymph as part of an immune response. Furthermore, formazan staining of elicitor-treated haemocytes was observed microscopically, with less intense staining in the presence of superoxide dismutase. Finally, respiratory burst inhibitors and superoxide dismutase enhanced the growth of E. coli incubated in whole haemolymph, implying a role for haemolymph-derived superoxide in antibacterial defence.     

Formulation with CpG oligodeoxynucleotides prevents induction of pulmonary immunopathology following priming with formalin-inactivated or commercial killed bovine respiratory syncytial virus vaccine

Commercial killed bovine respiratory syncytial virus (K-BRSV) and formalin-inactivated BRSV (FI-BRSV) tend to induce Th2-type immune responses, which may not be protective and may even be detrimental during subsequent exposure to the virus. In this study we assessed the ability of CpG oligodeoxynucleotides (ODNs) to aid in the generation of effective and protective BRSV-specific immune responses. Mice were immunized subcutaneously with FI-BRSV formulated with CpG ODN, Emulsigen (Em), CpG ODN and Em, or non-CpG ODN and Em. Two additional groups were immunized with K-BRSV or K-BRSV and CpG ODN. After two vaccinations, the mice were challenged with BRSV. FI-BRSV induced Th2-biased immune responses characterized by production of serum immunoglobulin G1 (IgG1) and IgE, as well as interleukin-4 (IL-4), by in vitro-restimulated splenocytes. Formulation of FI-BRSV with CpG ODN, but not with non-CpG ODN, enhanced serum IgG2a and IFN-gamma production by splenocytes, whereas serum IgE was reduced. Although the immune response induced by K-BRSV was not as strongly Th2 biased, the addition of CpG ODN to this commercial vaccine also resulted in a more Th1-type response. Furthermore, the addition of CpG ODN to the BRSV vaccine formulations resulted in enhanced neutralizing antibody responses. Significant production of IL-5, eotaxin, and eosinophilia was observed in the lungs of FI-BRSV- and K-BRSV-immunized mice. However, IL-5 and eotaxin levels, as well as the number of eosinophils, were decreased in the mice vaccinated with the CpG ODN-formulated vaccines. Finally, when formulated with CpG ODN, both FI-BRSV and K-BRSV significantly reduced virus production after challenge with BRSV.     

Conventional protein kinase C isoenzymes undergo dephosphorylation in neutrophil-like HL-60 cells treated by chelerythrine or sanguinarine

The quaternary benzo[c]phenanthridine alkaloid chelerythrine is widely used as an inhibitor of protein kinase C (PKC). However, in biological systems chelerythrine interacts with an array of proteins. In this study, we examined the effects of chelerythrine and sanguinarine on conventional PKCs (cPKCs) and PKC upstream kinase, phosphoinositide-dependent protein kinase 1 (PDK1), under complete inhibition conditions of PKC-dependent oxidative burst. In neutrophil-like HL-60 cells, sanguinarine and chelerythrine inhibited N-formyl-Met-Leu-Phe, phorbol 12-myristate 13-acetate (PMA)-, and A23187-induced oxidative burst with IC₅₀ values not exceeding 4.6 μmol/L, but the inhibition of PMA-stimulated cPKC activity in intact cells required at least fivefold higher alkaloid concentrations. At concentrations below 10 μmol/L, sanguinarine and chelerythrine prevented phosphorylation of ∼80 kDa protein and sequestered ∼60 kDa phosphoprotein in cytosol. Moreover, neither sanguinarine nor chelerythrine impaired PMA-stimulated translocation of autophosphorylated PKCα/βII isoenzymes, but both alkaloids induced dephosphorylation of the turn motif in PKCα/βII. The dephosphorylation did not occur in unstimulated cells and it was not accompanied by PKC degradation. Furthermore, cell treatment with sanguinarine or chelerythrine resulted in phosphorylation of ∼70 kDa protein by PDK1. We conclude that PKC-dependent cellular events are affected by chelerythrine primarily by multiple protein interactions rather than by inhibition of PKC activity.     

Intracellular free calcium regulates the onset of the respiratory burst of human neutrophils activated by phorbol myristate acetate.

Thapsigargin was used to study the regulation of different static calcium level ([Ca2+]i) on the respiratory hurst of human neutrophils stimulated with phorbol myristate acetate (PMA). The result showed that the onset time of the respiratory hurst was obviously reduced by elevation of static [Ca2+]i but is still much longer than that stimulated with N-formylmethionylleucylphenylalanine (fMLP). To find the reason, the onset times of the respiratory burst stimulated with fMLP, 1,2-dioctanoyl-sn-glycerol (DiC8), and PMA were determined at different static [Ca2+]i. It turns out that although DiC8 was unable to induce the respiratory burst at low [Ca2+], the onset time of DiC8-stimulated response at high [Ca2+]i was almost the same as that stimulated with fMLP. The study revealed that the fast onset of the fMLP-stimulated respiratory burst in comparison with PMA-stimulated response is not only due to the transient rise of [Ca2+]i, but is also due to the higher efficiency of diacylglycerol (DAG) in activating protein kinase c (PKC). The determining step in governing the onset of a respiratory burst is the activation of PKC.     

Activation of toll-like receptor-9 induces matrix metalloproteinase-9 expression through Akt and tumor necrosis factor-alpha signaling

CpG oligodeoxunucleotide (ODN) plays an important role in immune cell function. The present study examined whether temporal control of toll-like receptor (TLR)-9 by CpG ODN can regulate the expression of matrix metalloproteinase-9 (MMP-9). CpG ODN induced the release of tumor necrosis factor (TNF)-alpha and the expression of TNF receptor (TNFR)-II, but not of TNFR-I, in a time-dependent manner and stimulated significant, though delayed, MMP-9 expression. The endosomal acidification inhibitors, chloroquine or bafilomycin A, inhibited CpG ODN-induced TNF-alpha, TNFR-II, and MMP-9 expression. CpG ODN induced the phosphorylation of Akt, and the inhibition of Akt by LY294002 suppressed CpG ODN-induced TNF-alpha, TNFR-II, and MMP-9 expressions. Moreover, neutralizing TNF-alpha antibody significantly suppressed CpG ODN-induced MMP-9 expression, suggesting the involvement of TNF-alpha. These observations suggest that CpG ODN may play important roles in macrophage activation by regulating the expression of MMP-9 via a TLR-9/Akt/TNF-alpha-dependent signaling pathway.