The influence of temperature and dose on antibacterial peptide response against lipopolysaccharide in the blue mussel,Mytilus edulis

Blue mussels (Mytilus edulis) were inoculated with two different doses of lipopolysaccharides (LPS) or phosphate-saline (PS) buffer under different temperature conditions (6 and 20 degrees C). The activity of the antibacterial peptide fraction, purified through reverse phase chromatography from mussel haemolyph, was compared at different time intervals after the inoculation. The activity was determined as the minimal peptide concentration that inhibited growth of the Gram-negative bacteria Escherichia coli D21, by using radial diffusion assay. The antibacterial activity for mussels inoculated with LPS changed over time, both at 6 and 20 degrees C, but those inoculated with PS-buffer did not. The response was enhanced within a time course of 3h. The higher temperature did increase the inhibitory activity and made the mussel respond at an earlier stage, in comparison to that at 6 degrees C. At 20 degrees C, mussels inoculated with 10 microg of LPS responded faster than those inoculated with 0.1 microg of LPS. In addition, cytotoxic effects of LPS on mussel haemocytes were investigated in vitro, using a colorimetric assay. The survival index (SI%) for haemocytes decreased with 76% at 6 degrees C but increased with 100% at 20 degrees C, irrespective of the dose of LPS. This indicated that LPS did not influence the viability of the haemocytes but the high temperature increased their metabolic state. Likely, antibacterial response was provoked by LPS in a dose-dependent manner and favoured by higher metabolic state of the haemocytes, elicited at higher temperature. These results provide important considerations for variability in the internal defence of mussels and consequently, also the retention of viable human pathogens in mussels.     

The influence of Zn on signaling pathways and attachment of Mytilus galloprovincialis haemocytes to extracellular matrix proteins

The present study investigates the cytotoxic mechanisms induced by zinc (Zn) in haemocytes of mussel Mytilus galloprovincialis. Haemocytes play a key role in the immune defence of mussels. Micromolar concentration of Zn (50 microM) play an important role in the elevation of pHi and increase in Na+ influx in haemocytes. The observed effects were inhibited by the Na+/H+ exchanger (NHE) inhibitor, ethyl-N-isopropyl-amiloride (EIPA). Furthermore, our results showed that Zn caused an increase in O(-)(2) production that was reversed after NHE inhibition. Phorbol ester (PMA) caused a significant rise both in pHi and Na+ influx as well as in O(-)(2) production. These effects were reversed by calphostin C. Our results indicated that Zn also enhanced haemocyte attachment to both BSA and laminin which was reversed by EIPA and calphostin C. The enhancement of haemocytes attachment to both BSA and laminin after Zn suggests that it is likely to play a signal role in cytoskeleton-dependent process of cell growth and migration in mussel M. galloprovincialis haemocytes. We conclude that Zn induces a signaling pathway with the involvement of NHE, PKC, O(-)(2) and alpha1- and beta-adrenergic receptors.     

Influence of 4-hydroxynonenal on chemiluminescence production by unstimulated and opsonized zymosan-stimulated human neutrophils

The lipid peroxidation product 4-hydroxy-2, 3-trans-nonenal (HNE) has a spectrum of biological effects on different cell types depending on the concentrations tested. In particular micromolar HNE concentrations stimulate neutrophil migration and polarization whereas higher doses inhibit. In our experimental conditions, fMet-Leu-Phe (fMLP) increased CL production of both unstimulated and zymosan-stimulated neutrophils, whereas cell stimulation with low HNE concentrations as well as zymosan addition to HNE incubated cells did not enhance light emission. In contrast 10(-4) M HNE reduced CL emission by unstimulated cells nearly to background values, completely depressed CL production by zymosan-stimulated cells and reduced phagocytosis. Cysteine was found to be able to counteract the HNE effect by about 70 per cent. The possibility that this aldehyde could exert its inhibitory effect through the alkylation of NADPH-oxidase SH-groups is postulated. Moreover, our present data on differences observed between fMLP and HNE indicate a different chemotactic mechanism induced by these two classes of compounds and lead to the conclusion that the local functional features of the attracted cells may be different.     

Factors influencing bactericidal activity of blue mussel (Mytilus edulis) haemocytes against Salmonella typhimurium

This study showed that in vitro survival of Salmonella typhimurium, after exposure to haemocytes of Mytilus edulis, was significantly affected by the lipopolysaccharides (LPS) structures expressed on the cell surface of the bacteria. Survival seemed to be affected by the surrounding temperature as well. Mussel haemocytes were in vitro exposed to mutants of S. typhimurium, expressing differences in O -antigen polysaccharide chains and core sugars of LPS on their cell surface. Surviving cells of the mutants were determined after incubation with the haemocytes at different temperatures, using a colorimetric assay. In addition, a complementary study on clearance of these mutants, inoculated into the adductor muscle of mussels, was performed at 6 and 20 degrees C. It was concluded that the survival index (SI%) measured in vitro for the mutant with complete LPS was significantly lower at 6 degrees C (c.15%) compared to that at 14 and 20 degrees C (c.70%). SI% for the other mutants was c.35-45% and was not affected by temperature. The in vivo study at 20 degrees C showed that during the first 24h, the clearance rate for the mutants with complete LPS was significantly higher than for the others. Thereafter all mutants, with exception for the most deficient, started to increase in numbers and caused death to the mussels. At 6 degrees C the mutants were slowly reduced and after 17 days, viable cells of the mutant with complete LPS were still detectable in the haemolymph. The study indicated that the mussel haemocytes responded in relation to the LPS of the mutants. However, more intact LPS also seemed to protect the bacteria from being killed. The higher temperatures favoured the growth of the mutants that managed to resist the haemocyte defence. Cell surface properties and temperature seem to affect the survival of bacteria in mussels, which consequently can affect risk assessments in regard to public health.     

The influence of age and sex on phagocyte chemiluminescence

The process of ageing is associated with increased susceptibility to infection. Phagocytes form the primary defence mechanism against infecting microorganisms, but the influence of ageing on phagocyte function remains controversial. In this study we have applied a microtitre plate phagocyte chemiluminescence (CL) assay suitable for clinical use to compare phagocyte oxidative metabolism in younger healthy subjects (age 20–60 years) and healthy older (60–70 years) subjects. Polymorphonuclear leukocytes (PMNL) and monocytes were stimulated using phorbol myristate acetate (PMA), serum opsonized zymosan (SOZ), and non-opsonized zymosan (ZYM) in the presence of both lucigenin and luminol. Monocytes showed a higher luminolenhanced CL response to PMA in males compared with females in the younger age group. No PMNL differences were observed between the sexes. Although no difference were found in relation to age when cells were stimulated with PMA and SOZ, significantly lower background (unstimulated) CL was obtained from PMNL with luminol. PMNL luminol-enhanced CL responses were also lower in response to ZYM. The findings suggest a reduced response of PMNL from older subjects to minimal stimulation. This could be related to abnormalities in the triggering of the respiratory burst or myeloperoxidase release due to ageing. The influence of age and sex should be taken into account in clinical studies of phagocyte CL.     

Change in the chemiluminescence reactivity pattern during in vitro differentiation of human monocytes to macrophages

We determined the luminol-enhanced chemiluminescence (CL) of fresh human monocytes and monocytes cultured for 1-14 days in vitro, within hydrophobic membranes, using a variety of stimuli known to trigger the respiratory burst of phagocytes. It was assured that CL emerged from an adherent subpopulation of mononuclear cells; polymorphonuclear leukocytes (PMN) contaminating mononuclear leukocytes (MNL) contributed little, if anything, to the CL response of MNL. Typical response patterns were established for fresh monocytes triggered by phorbol 12-myristate 13-acetate (PMA), zymosan, the Ca2+ ionophore A 23187, antibody-coated erythrocytes and Sendai virus. Differentiation in vitro into macrophages was associated with a general decrease in magnitude of the CL peak, in an overproportional decrease of the A23187 triggered response and in a complete loss of the response to Sendai virus--a loss which could not be prevented by addition of myeloperoxidase (MPO). In contrast to monocyte CL, macrophage CL was resistant to sodium azide, indicating its MPO-independent origin. Macrophage-type reactivity was obtained at day 4 of culture. Activation of macrophages with recombinant interferon-gamma for the last 2 days of culture was associated with a quantitative (approx. threefold) increase of the CL signal, although qualitatively the same reactivity pattern was obtained as with control macrophages. In contrast to luminol-dependent CL, the lucigenin-dependent CL response of macrophages was greater than that of monocytes, an increase which was particularly prominent for PMA stimulation.     

In vitro effects of LPS, IL-2, PDGF and CRF on haemocytes of Mytilus galloprovincialis Lmk

The cells in charge of the innate immune response in the marine mussel Mytilus galloprovincialis Lmk. are the haemocytes. These cells respond in different ways to agents such as lipopolysaccharide (LPS), interleukin-2 (IL-2), platelet-derived growth factor (PDGF) and corticotropin releasing factor (CRF). After stimulation of the haemocytes, the expression of molecules reactive with monoclonal antibodies raised to the alpha chain of the IL-2 receptor, present in their membrane, differed depending on the agent used. The same happened with regard to the levels of dopamine, adrenaline and noradrenaline released to the medium by the haemocytes. It should also be noted that no catecholamine release was detected and the level of expression of IL-2Ralpha showed no significant variation in cultured cells that had not been treated with inducers. These facts would indicate that most haemocytes were in the same starting condition at the moment that the stimulation was performed. Therefore, cultured haemocytes can be a highly reliable model in the study of the innate immune system.     

Factors affecting the chemiluminescent response of fish phagocytes

The effects of several factors on the phagocytic activity of cells isolated from the pronephros of striped bass, Morone saxatilis , were measured using a chemiluminescence (CL) assay. The CL responses of phagocytes to varying concentrations of bacteria, phorbol myristate acetate (PMA), and zymosan were shown to be dose-dependent. Incubation of phagocytes with PMA resulted in a decrease in cell numbers related to the concentration of PMA used in the assay. Opsonization of Aeromonas hydrophila with normal pooled bass serum decreased the number of colony forming units present in suspension while enhancing the CL response by striped bass phagocytes. Opsonization of zymosan also resulted in an enhanced CL response. Aeromonas hydrophila and Aerococcus viridans killed by heat treatment, incubation with formalin, or exposure to UV radiation elicited little or no CL when incubated with phagocytes.     

Effects of temperature on baseline and genotoxicant-induced DNA damage in haemocytes of Dreissena polymorpha

The potential application of the Comet assay for monitoring genotoxicity in the freshwater mussel Dreissena polymorpha was explored and a preliminary investigation was undertaken of the baseline levels of DNA damage in mussel haemocytes of animals kept at different temperatures. In addition, in vitro cell sensitivity against genotoxicants was assessed in relation to increasing temperatures. The mussels were kept at four different constant temperatures (4, 18, 28 and 37 degrees C) for 15 h. The haemocytes withdrawn were treated in vitro with melphalan, as a model genotoxic compound, or sodium hypochlorite, a common water disinfectant capable of producing mutagenic/carcinogenic by-products, at the established temperatures for 1h. The data obtained in vivo, in cells directly withdrawn from the mussels showed a significant (P<0.001, Student's t test) inter-individual variability, probably due to genetic and epigenetic factors and an increasing amount of DNA damage at increasing temperature. Mussel haemocytes showed a clear dose-response effect after in vitro melphalan treatment. Hypochlorite treatment also significantly increased DNA migration: the damage was temperature dependent, with a similar increase at 4 and 28 degrees C and a minimum level at 18 degrees C. This study demonstrates the potential application of the Comet assay to haemocytes of D. polymorpha. However, these findings suggest that temperature could alter both DNA damage baseline levels in untreated animals and cell sensitivity towards environmental pollutants in in vitro conditions. Therefore, more information is needed about seasonal variations and the natural background levels of DNA damage in mussels living in the wild, before they are used for the monitoring of genotoxic effects in aquatic environments.     

Flow cytometric analysis of crayfish haemocytes activated by lipopolysaccharides

Lipopolysaccharides (LPS) from Gram-negative bacteria are strong stimulators of white river crayfish, Procambarus zonangulus, haemocytes in vitro. Following haemocyte treatment with LPS and with LPS from rough mutant R5 (LPS Rc) from Salmonella minnesota, flow cytometric analysis revealed a conspicuous and reproducible decrease in cell size as compared to control haemocytes. These LPS molecules also caused a reduction in haemocyte viability as assessed by flow cytometry with the fluorescent dyes calcein-AM and ethidium homodimer. The onset of cell size reduction was gradual and occurred prior to cell death. Haemocytes treated with LPS from S. minnesota without the Lipid A moiety (detoxified LPS) decreased in size without a reduction of viability. The action of LPS on crayfish haemocytes appeared to be related to the activation of the prophenoloxidase system because phenoloxidase (PO)-specific activity in the supernatants from control and detoxified LPS-treated cells was significantly lower than that from LPS and LPS-Rc treated cells (P相似文献   

Validation of different chemilumigenic substrates for detecting extracellular generation of reactive oxygen species by phagocytes and endothelial cells

Chemiluminescence is a widely used tool to detect extracellular generation of reactive oxygen species (ROS). In the present study we tested four different chemilumigenic substrates (CLS)—luminol, isoluminol, lucigenin and pholasin—to detect extracellular CL in different cell types: polymorphonuclear leukocytes (PMN); DMSO‐differentiated HL‐60 cells; murine macrophages (RAW 264.7); and TNFα‐stimulated human endothelial cells (HUVEC). Extracellular ROS production was calculated by subtracting intracellular CL response in the presence of superoxide dismutase and catalase from the overall CL response in the absence of enzymes. CL varied considerably in dependence on the CLS and the stimulus used to evoke ROS generation. Luminol (oxidized LDL and zymosan stimulation) and isoluminol (FMLP and PMA stimulation) were the most effective CLS for PMN. Using 5 µmol/L lucigenin as CLS, small but consistent CL responses could be obtained in macrophages stimulated with PMA, zymosan or oxidized LDL. FMLP‐stimulated extracellular CL in H‐60 cells, HUVEC and macrophages was detected with the greatest sensitivity by pholasin. Our results demonstrate that none of the investigated CLS consistently yielded the highest CL quantum, either in different cell types with one stimulating agent or by different stimulating agents in one cell type. To get the highest CL quantum in experimental studies, we recommend optimizing the CLS depending on the cell type and the ROS‐generating stimulus used. Copyright © 2003 John Wiley & Sons, Ltd.     

Characterization of mussel gill cells in vivo and in vitro

Mussel gill cells are attractive models in ecotoxicological studies because gills are the first uptake site for many toxicants in the aquatic environment; gill cells are thus often affected by exposure to pollutants. Our aim was to characterize mussel gill cells in vivo and in vitro by using morphological, histochemical and functional end-points. In paraffin sections stained with haematoxylin–eosin, three zones were distinguished in the long central gill filaments: frontal, intermediate and abfrontal. Various types of ciliated cells were present in the frontal zone, and both ciliated and non-ciliated cells were found in the abfrontal zone. The intermediate zone was comprised of flattened endothelial cells. Lipofuscin granules occurred in the three zones in variable amounts, depending on the specimen. Haemocytes were found in the haemolymph sinus of gill filaments. Mucocytes were identified in both frontal and abfrontal zones by means of periodic acid Schiff-alcian blue (PAS-AB) staining. In cryostat sections, succinate dehydrogenase (SDH) activity was mainly found in ciliated cells, whereas neutral lipids and acid-phosphatase-reactive lysosomes were present in all portions of the gill filament, mostly being related to lipofuscin granules. In mussels exposed to 5-bromo-2-deoxyuridine in vivo, proliferating cells were scattered throughout the gill filament. Gill cells (typically 2×10⁷ cells/ml per mussel; 95% viability) were isolated by dissociation with dispase. Gill cell suspensions were heterogeneous: 58% were ciliated epithelial cells (positive for SDH), 42% were non-ciliated cells (including epithelial cells and haemocytes), 2.3% were mucocytes (positive for PAS-AB) and 4.25% were haemocytes (able to phagocytose neutral red-stained zymosan). Gill cell cultures were maintained up to 18 days without changing the culture medium, viability decreasing below 50% at day 18. Primary cultures of mussel gill cells might therefore be useful models for the in vitro assessment of xenobiotic impacts on coastal and estuarine ecosystems.This work was funded by the Spanish Ministry of Science and Technology (project AMB99-0324), by the Basque Government through the Cooperation Fund Aquitaine/Euskadi 2001, by the University of the Basque Country through a grant to Consolidated Research Groups and by the European Commission (BEEP project, contract no. EVK3-CT2000-00025). Amagoia Gómez-Mendikute is the recipient of a predoctoral fellowship from the Spanish Ministry of Education and Culture.     

In vitro production of superoxide and nitric oxide (as nitrite and nitrate) by Mytilus galloprovincialis haemocytes upon incubation with PMA or laminarin or during yeast phagocytosis

The phagocytic process is one of the most important elements of the self-defence system in mammals as well as in molluscs. In mammalian phagocytes, superoxide participates in the innate defence system by combining with nitric oxide to generate peroxynitrite, a strong oxidant that possesses highly cytotoxic properties against bacteria. To evidence a role of nitric oxide in the self-defence system of the marine bivalve Mytilus galloprovincialis similar to the role observed in the mammalian defence system, we measured the generation of superoxide and nitrite/nitrate (the stable end products of nitric oxide) upon in vitro stimulation of M. galloprovincialis haemocytes with PMA, laminarin, LPS and by phagocytosis of Saccharomyces cerevisiae (yeast cells). We show that stimulation with PMA, laminarin and yeast cell phagocytosis promotes superoxide and nitrite/nitrate generation from M. galloprovincialis haemocytes. Inhibitors of NADPH oxidase and inhibitors of NO synthase decreased the nitrite/nitrate levels generated by M. galloprovincialis haemocytes showing that both NADPH oxidase and NO synthase pathways are involved in the self-defence system of M. galloprovincialis.     

LPS-stimulated release of prostaglandin E and thromboxane B2 from the U937 cell line

Human monocytes are known to metabolize arachidonic acid (AA) and to release prostaglandins upon stimulation. Previous data indicate that in vitro maturation and differentiation of monocytes result in alteration of this property with greatly diminished response to stimulators of release of prostaglandin E (PGE) and thromboxane B2 (TxB2) occurring after cells have been cultured. To further study the effects of differentiation on human monocyte AA metabolism, a model system was established based upon the human histiocytic cell line U937. Among tested stimulants, which included opsonized zymosan, complement fragment C3b, phorbol myristate acetate (PMA), calcium ionophore A23187, and concanavalin A, it was found that Escherichia coli lipopolysaccharide (LPS) was unique in that it stimulated increased release of TxB2 from U937 cells. The effect of the phorbol ester PMA, a compound commonly used to induce differentiation of U937, on the ability of U937 to respond to LPS was examined. Following 48 hr of treatment with PMA, U937 became capable of releasing both PGE and TxB2 in response to small doses of LPS. As previously observed for human monocytes, the release of PGE was delayed for several hours following stimulation and failed to reach maximal cumulative levels in culture until 24-48 hr following stimulation. In contrast to human monocytes, PMA-induced U937 were capable of maintaining their responsiveness to LPS for several days. Thus, the U937 cell line provides a useful model for study of the effects of differentiation of human mononuclear phagocytes on their ability to metabolize AA, and for the effects of LPS on histiocytic tumor cell prostaglandin release.     

Different effects of exogenous horseradish peroxidase on luminol-amplified chemiluminescence induced by soluble and particulate stimuli in human neutrophils

The chemiluminescence (CL) technique with scavengers for superoxide anion (superoxide dismutase) and hydrogen peroxide (catalase) was used to characterize the generation of reactive oxygen species (ROS) inside and outside the human neutrophil after stimulation with both soluble (formyl-methionyl-leucyl-phenylalanine, FMLP) and particulate (urate crystals, zymosan, oxidized LDL) stimuli. Depending on the stimulus used, ROS generation differed in composition and absolute amounts. The ratio between extracellularly and intracellularly produced ROS ranged from 0.3 (zymosan) to 4.2 (FMLP). While enhancing substantially FMLP-stimulated CL, horseradish peroxidase inhibited CL induced by particulate stimuli by 40–80%. Furthermore, an azide-insensitive and therefore peroxidase-independent part of CL was found in FMLP-, LDL- and zymosan-stimulated cells. The results indicate that different agonists may lead through distinct chemical pathways to neutrophil luminol-amplified light generation. © 1998 John Wiley & Sons, Ltd.     

Inhibition of chemiluminescent response of olive flounder Paralichthys olivaceus phagocytes by the scuticociliate parasite Uronema marinum

Experiments were conducted to evaluate the in vitro capacity of the scuticociliatian parasite Uronema marinum to inhibit chemiluminescence (CL) of olive flounder Paralichthys olivaceus phagocytes. Luminol-enhanced CL was used to measure the production of reactive oxygen intermediates (ROIs) generated by respiratory bursts of phagocytes using zymosan as a stimulant. Cytotoxic and antioxidative activities of excretory-secretory (ES) products of the parasite were evaluated as well. Live U. marinum and its ES products had a negative and dose-dependent effect on luminol-enhanced CL responses of zymosan-stimulated phagocytes of olive flounder. After CL assay, the number of phagocytes showing viability was significantly reduced in the cells incubated with live U. marinum at ratios of 2:1 and 1:1 phagocytes:ciliates or ES products with 0.3 mg protein ml(-1) compared to controls. Lysis of phagocytes by exposure to ES products was observed also. ES products from U. marinum showed considerably high activities of superoxide dismutase (SOD) and catalase. The results of this study suggest that U. marinum can protect itself against host's phagocytes mediated oxidative damage by destroying phagocytes and scavenging ROIs.     

Effects of the pathogenic Vibrio tapetis on defence factors of susceptible and non-susceptible bivalve species: I. Haemocyte changes following in vitro challenge

In microbial infections, the interaction between microorganisms and phagocytic cells is a crucial determinant in the outcome of the disease process. We used flow cytometry to study the in vitro interactions between Vibrio tapetis, the bacterium responsible for Brown Ring Disease (BRD) in the Manila clam Ruditapes philippinarum, and haemocytes from three bivalve species: the Manila clam (susceptible to BRD), the hard clam Mercenaria mercenaria and the eastern oyster Crassostrea virginica (both non-susceptible to BRD). Results demonstrated that V. tapetis cells and extracellular products elicit major changes in the haemocytes of R. philippinarum, including decreased viability and phagocytic activity, and altered size and internal structure. V. tapetis was able to kill haemocytes from M. mercenaria and C. virginica but to a far lesser extent than those of R. philippinarum. These results suggest that disease resistance is not solely dependent on a host activity against the pathogen, but is also a function and magnitude of the injury to the host cell by a given pathogen.     

Modulation of mitogen-activated protein kinases (MAPK) activity in response to different immune stimuli in haemocytes of the common periwinkle Littorina littorea

The modulation of mitogen-activated protein kinase (MAPK) activity in haemocytes of the common periwinkle (Littorina littorea) in response to immune challenges by lipopolysaccharide from Echerichia coli (LPS), mannan from baker's yeast Saccharomyces cerevisiae and secretory-excretory products (SEP) of trematodes Himasthla elongata (Echinostomatidae) or after the treatment with phorbol ester (PMA) has been studied by Western blotting using affinity purified rabbit polyclonal antibodies. Exposure of the cells in suspension to PMA, LPS and mannan triggered an activation of p38 and ERK2. The JNK-mediated cascade was modulated differently by the elicitors examined. PMA treatment caused a transient activation of the JNK54 isoform, LPS exposure resulted in a decrease in activity of JNK46, and mannan had no effect on JNK phosphorylation status. Incubation of periwinkle haemocytes in culture medium containing trematode SEP did not affect the activity of any MAPK.     

Production of nitric oxide by mussel (Mytilus galloprovincialis) hemocytes and effect of exogenous nitric oxide on phagocytic functions

In the present study, the ability of mussel (Mytilus galloprovincialis) hemocytes to produce nitric oxide (NO) in response to phorbol myristate acetate (PMA) was determined using the Griess reaction. Significant NO production was found in these cells in response to PMA. This stimulation was reversed in the presence of the NO synthase inhibitor, N(G)-methyl-L-arginine (L-NMMA). Moreover, the effect of the pre-incubation of hemocytes with NO was also determined on phagocytic immune functions of mussel hemocytes using two NO donors, glycerin trinitrate (GTN) and S-nitroso-N-acetyl-penicillamine (SNAP). In the case of GTN, a visible cytotoxic effect of the compound at the higher doses was observed. Those GTN concentrations that did not have a negative effect on hemocyte viability did not produce sufficient NO to significantly alter the chemiluminescent response to zymosan in all cases, nor the ability of hemocytes to phagocytose bacteria (Escherichia coli). SNAP, however, did not affect cell viability at either of the concentrations used and produced NO levels up to 13-fold higher than controls after 2 h of incubation. In this case, NO exogenously produced by SNAP significantly inhibited the chemiluminescent response of mussel hemocytes, whereas it did not have a significant effect on the capability of these cells to phagocytose bacteria.     

Phorbol ester-stimulated superoxide production by murine bone marrow-derived macrophages requires preexposure to cytokines

Murine resident peritoneal macrophages (RPM) generate superoxide (O2-) in response to stimulation with PMA or zymosan. Murine bone marrow-derived macrophages (BMM) generate O2- in response to zymosan but not PMA. However, the ability to generate O2- in response to PMA could be induced in BMM by pre-exposing the cells to certain cytokines, including granulocyte-macrophage CSF (GM-CSF), tumor necrosis factor-alpha (TNF-alpha), IFN-gamma, and, to a lesser extent, IL-1 alpha. Bacterial LPS also induced the ability to respond to PMA. These same agents were also shown to prime RPM for enhanced PMA-induced respiratory burst. In contrast to GM-CSF, CSF-1 did not enhance the ability of BMM or RPM to generate O2- in response to PMA. Pretreatment with GM-CSF or TNF-alpha did not significantly affect the zymosan-induced release of O2- by BMM. These results suggest that unprimed BMM have a deficiency in the PMA-dependent signaling pathway that is corrected by exposure to selected cytokines. The results also raise the possibility that the basal ability of tissue macrophages to generate a respiratory burst in response to PMA may be a reflection of in vivo exposure to cytokines.