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Am J Physiol Regul Integr Comp Physiol 291: R664-R673, 2006. First published April 6, 2006; doi:10.1152/ajpregu.00139.2006
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INFLAMMATION AND CYTOKINES

Immunomodulation by 17beta-estradiol in bivalve hemocytes

Laura Canesi,1 Caterina Ciacci,1 Lucia Cecilia Lorusso,1 Michele Betti,1 Tiziana Guarnieri,2 Simona Tavolari,2 and Gabriella Gallo3

1Istituto di Scienze Fisiologiche, Università "Carlo Bo" di Urbino, Urbino; 2Cellular Physiology Unit, Department of Experimental Evolutionary Biology, Alma Mater Studiorum, University of Bologna, Bologna; and 3Dipartimento di Biologia, Sezione di Fisiologia, Università di Genova, Genoa, Italy

Submitted 28 February 2006 ; accepted in final form 29 March 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In mammals, estrogens have dose- and cell-type-specific effects on immune cells and may act as pro- and anti-inflammatory stimuli, depending on the setting. In the bivalve mollusc Mytilus, the natural estrogen 17beta-estradiol (E2) has been shown to affect neuroimmune functions. We have investigated the immunomodulatory role of E2 in Mytilus hemocytes, the cells responsible for the innate immune response. E2 at 5–25 nM rapidly stimulated phagocytosis and oxyradical production in vitro; higher concentrations of E2 inhibited phagocytosis. E2-induced oxidative burst was prevented by the nitric oxide (NO) synthase inhibitor NG-monomethyl-L-arginine and superoxide dismutase, indicating involvement of NO and O2–; NO production was confirmed by nitrite accumulation. The effects of E2 were prevented by the antiestrogen tamoxifen and by specific kinase inhibitors, indicating a receptor-mediated mechanism and involvement of p38 MAPK and PKC. E2 induced rapid and transient increases in the phosphorylation state of PKC, as well as of a aCREB-like (cAMP responsive element binding protein) transcription factor, as indicated by Western blot analysis with specific anti-phospho-antibodies. Localization of estrogen receptor-{alpha}- and -beta-like proteins in hemocytes was investigated by immunofluorescence confocal microscopy. The effects of E2 on immune function were also investigated in vivo at 6 and 24 h in hemocytes of E2-injected mussels. E2 significantly affected hemocyte lysosomal membrane stability, phagocytosis, and extracellular release of hydrolytic enzymes: lower concentrations of E2 resulted in immunostimulation, and higher concentrations were inhibitory. Our data indicate that the physiological role of E2 in immunomodulation is conserved from invertebrates to mammals.

estrogen; innate immunity; kinase-mediated cell signaling; Mytilus

IN MAMMALS, ESTROGENS EXERT a broad spectrum of activities on a wide variety of cells and tissues, including the immune system. Estrogens have dose- and cell-type-specific effects on immune cells and may act as pro- and anti-inflammatory stimuli, depending on the setting (5, 22, 24, 43). 17beta-Estradiol (E2) can modulate the function of neutrophil granulocytes, monocytes, and macrophages; these cell types have been reported to express intracellular and membrane estrogen receptors (ERs), and their response to E2 can be mediated by nuclear, classical or "genomic" pathways, as well as by rapid, "nongenomic" mechanisms of action (2, 3, 24, 45). These latter mechanisms may be initiated at membrane or cytosolic locations (29, 39, 49) and can result in direct local effects (e.g., modification or ion fluxes) and regulation of gene transcription secondary to activation of cytosolic kinase cascades (5, 22, 24, 29, 43).

Among invertebrates, estrogens have been identified in bivalve molluscs (25, 34, 40, 43), in which their role has been mainly investigated in the control of gametogenesis (20, 21, 30, 33, 34). However, there is evidence that estrogen can represent an important signaling molecule that is involved in roles other than reproduction in these organisms. In neural tissues of the marine mussel Mytilus spp., E2 has been shown to downregulate ganglionic microglial cells after surgical insult, which normally stimulates their egress from the tissue (46), as well as after activation of N-formylmethionyl-leucyl-phenylalanine (48). The effects of E2 on microglial cells were mediated by rapid induction of nitric oxide (NO) release by nervous tissue and were antagonized by classical antiestrogens. These data indicated a receptor-mediated event in Mytilus pedal ganglia (46), where a fragment of ER-beta with 100% sequence identity to the human receptor was identified (48).

In Mytilus, E2 was also shown to affect the digestive cells and circulating hemocytes, in particular at the level of lysosomal function (6, 32). In bivalves, hemocytes are responsible for cell-mediated immunity through phagocytosis and various cytotoxic reactions (e.g., lysosomal enzyme and antimicrobial peptide release and production of reactive oxygen intermediates) (13, 41). We previously showed that addition of E2 (in the low nanomolar range) to hemocyte monolayers induced a moderate increase in cytosolic Ca2+ concentration ([Ca2+]), destabilization of lysosomal membranes, morphological changes, hydrolytic enzyme release, and stimulated the bactericidal activity to Escherichia coli (12); all these effects were rapid, occurring from seconds to minutes from E2 addition, and were prevented by the antiestrogen tamoxifen. The effects of E2 on mussel hemocytes were affected by components of tyrosine kinase-mediated cell signaling (12), in particular, the stress-activated p38 MAPK and STAT-like (signal transducers and activators of transcription) proteins, which play a key role in activation of these cells (8–10).

To clarify the effects and mechanisms of action of E2 in the immune response of mussel hemocytes, the effects of the hormone on phagocytosis and oxyradical production were evaluated in vitro. To investigate estrogen signaling, we used specific kinase inhibitors and evaluated the phosphorylation state of PKC and the transcription factor cAMP response element binding protein (CREB) by electrophoresis and Western blot analysis with specific anti-phospho-antibodies. Intracellular localization of ER-like proteins was evaluated by immunofluorescence confocal microscopy. Finally, the possible immunomodulatory role of E2 in vivo was investigated in hemocytes collected from mussels injected with different concentrations of the hormone.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals. Mussels (Mytilus galloprovincialis Lam.; 4–5 cm long) were obtained from SEA (Gabicce Mare, Italy) and kept for 1–3 days in static tanks containing artificial seawater (ASW; 1 l/mussel) at 16°C. The seawater was changed daily.

Hemolymph collection, preparation of hemocyte monolayers, and hemocyte treatments. A sterile 1-ml syringe with an 18-gauge 0.5-in. needle was used to extract hemolymph from the posterior adductor muscle of 10–12 mussels for each experiment. The needle was removed, and hemolymph was filtered through sterile gauze and pooled in 50-ml Falcon tubes at 4°C. Hemolymph serum was obtained by centrifugation of whole hemolymph at 200 g for 10 min, and the supernatant was sterilized through a 0.22-µm-pore filter. Hemocyte monolayers were prepared as previously described (10). Briefly, aliquots of 0.5 ml of hemolymph, corresponding to ~1–2 x 106 cells, were seeded onto glass coverslips (40 x 22 mm), placed in plastic culture dishes, and incubated at 16°C for 30 min to allow for cell attachment. Nonadherent hemocytes were subsequently removed by gentle washing of the preparations with sterilized ASW. Hemocyte monolayers were added with 1.5 ml of hemolymph serum and kept before use at 16°C.

Hemocytes were incubated at 16°C with E2 (from 10 mM stock solutions in ethanol suitably diluted in ASW). Untreated and control vehicle hemocyte samples were run in parallel. In experiments with antiestrogens, tamoxifen (100 nM final concentration, from a 10 mM stock solution in ethanol) was added 10 min before addition of E2. In experiments with kinase inhibitors, before addition of E2, hemocyte monolayers were pretreated for 20 min with 20 µM SB-203580 (for p38 MAPK) or 2.5 µM GF-109203X (for PKC) as previously described (10, 15). For each experiment, control hemocyte samples were run in parallel. Triplicate preparations were made for each sample. All incubations were carried out at 16°C.

Phagocytosis assay. Phagocytosis of neutral red-stained zymosan by hemocyte monolayers was used to assess the phagocytic ability of hemocytes according to the method of Pipe et al. (36) with slight modifications (4). Neutral red-stained zymosan in 0.05 M Tris·HCl buffer [Tris-buffered saline (TBS), pH 7.8, with 2% NaCl (to maintain osmotic conditions)] was added to each monolayer at a concentration of ~1:50 hemocytes-zymosan diluted in ASW in the presence or absence of E2 and allowed to incubate for 60 min. Monolayers were then washed three times with TBS, fixed with Baker's formol calcium [4% (vol/vol) formaldehyde, 2% NaCl, and 1% calcium acetate] for 30 min, and mounted in Kaiser's medium for microscopic examination with an Olympus Vanox optical microscope. For each slide, the percentage of phagocytic hemocytes was calculated from ≥200 cells. The effect of E2 on phagocytosis was also compared with that induced by 200 nM human recombinant tumor necrosis factor-{alpha} (4).

Oxidative burst. Extracellular oxyradical production by mussel hemocytes was measured by the reduction of cytochrome c (36) with slight modifications. Hemolymph was extracted into an equal volume of TBS (0.05 M Tris·HCl buffer, pH 7.6, containing 2% NaCl). Aliquots (500 µl) of hemocyte suspension in triplicate were incubated with 500 µl of cytochrome c solution (75 µM ferricytochrome c in TBS), with or without E2. Cytochrome c in TBS was used as a blank. The samples were read at 550 nm from 0 to 60 min, and the results are expressed as change in optical density (OD) per milligram of protein. Intracellular oxyradicals were also detected by nitro blue tetrazolium (NBT) assay (36) as described elsewhere (1). Aliquots (500 µl) of hemocyte suspension in triplicate were diluted 1:1 with TBS added with 2% NaCl and incubated for 60 min with an equal volume of 0.1% NBT solution in the presence or absence of E2 at room temperature in the dark. The samples were centrifuged (180 g, 5 min), and the pellet was resuspended in 70% methanol for fixation. The samples were centrifuged again, the supernatant was discarded, and the pellet was air-dried. The pellet was resuspended in 1.1 ml of extraction medium (6 ml of 2 M KOH + 7 ml DMSO) to dissolve the insoluble formazan that had formed, mixed by vortexing, and centrifuged at 3,500 g for 20 min at 10°C. Supernatants were read at 620 nm, and the results were expressed as OD at 620 nm per milligram of protein. Experiments were carried out also in the presence of 1 mM NG-monomethyl-L-arginine (L-NMMA) or superoxide dismutase (SOD) or after preincubation of the sample with tamoxifen or kinase inhibitors. The effects of E2 on extra- and intracellular oxyradical production were compared with those induced by 10 µg/ml phorbol 12-myristate 13-acetate (PMA) in the same experimental conditions.

Nitrite production. NO production by mussel hemocytes was evaluated as described previously (51) by the Griess reaction, which quantifies the nitrite (NO2–) content of supernatants. Aliquots of hemocyte suspensions (1.5 ml) were incubated at 16°C with 25 nM E2 or vehicle (ethanol) for 0–5 h in the presence and absence of tamoxifen or inhibitors (L-NMMA, SB-203580, and GF-109203X). Every 30 min, the samples were immediately frozen and stored at –80°C until use. Before analysis, the samples were thawed and centrifuged (12,000 g for 30 min at 4°C), and the supernatants were analyzed for NO2– content. Aliquots (200 µl) in triplicate were incubated for 10 min in the dark with 200 µl of 1% (wt/vol) sulfanilamide in 5% H3PO4 and 200 µl of 0.1% (wt/vol) N-(1-naphthyl)-ethylenediamine dihydrochloride. The samples were read at 540 nm, and the molar concentration of NO2– in the sample was calculated from standard curves generated using known concentrations of sodium nitrite. The effect of E2 on NO2– accumulation was compared with that induced by PMA (50 µg/ml) in the same experimental conditions.

Lysosomal membrane stability. Lysosomal membrane stability in mussel hemocytes was evaluated by the neutral red retention time assay as previously described in in vitro (9, 12, 14, 15) and in vivo (11) experiments by Lowe et al. (31). Hemocyte monolayers on glass slides were incubated with 30 µl of a neutral red solution (final concentration 40 µg/ml from a stock solution of neutral red at 20 mg/ml DMSO); after 15 min the excess dye was washed out, 30 µl of ASW were added, and the slides were sealed with a coverslip. In in vitro experiments, hemocyte monolayers were incubated with E2 for 30 min before addition of neutral red (12). Every 15 min, hemocytes were examined under an optical microscope, and the percentage of cells showing loss of the dye from lysosomes in each field was evaluated. For each time point, 10 fields, each containing 8–10 cells, were randomly observed. The end point of the assay was defined as the time at which 50% of the cells showed lysosomal leaking (i.e., the cytosol stains red and the cells are rounded). The effect of E2 on lysosomal membrane stability was compared with that induced by hemocyte incubation with E. coli (strain MG155) under the experimental conditions previously described (9).

Immunolocalization of ER-like receptors. Immunolocalization of ER-like receptors in hemocytes was evaluated by confocal laser scanning microscopy (CLSM). Hemocyte monolayers, seeded on polylysine-coated glass coverslips, were fixed for 30 min at room temperature in 4% paraformaldheyde dissolved in 5 mM PBS, pH 7.4, with 2% NaCl. All subsequent steps were carried out at room temperature in the presence of PBS with NaCl. The cells were washed three times with PBS, permeabilized for 10 min with 0.2% Triton X-100 in PBS, and treated for 30 min with 2% BSA in PBS (PBS-BSA) to minimize possible unspecific binding of the antiserum. Hemocytes were then incubated with human polyclonal anti-ER-{alpha} (anti-rabbit) or anti-ER-beta (anti-goat) antibodies (1:50 dilution in PBS-BSA) for 60 min. The cells were washed three times in PBS-BSA and incubated for 60 min with donkey anti-rabbit IgG-rhodamine-labeled (Santa Cruz Biotechnology) or rabbit anti-goat IgG-Cy3-labeled (Santa Cruz Biotechnology) as secondary antibodies (1:250 dilution in PBS-BSA), respectively, following the manufacturer's instructions. The cells were washed three times in PBS, and coverslips were mounted on glass slides using 5% glycerol-1,4-diazabicyclo[2.2.2]octane in a 90% glycerol-10% PBS (10x) antifading mixture.

The slides were mounted on the stage of a Leica inverted microscope, which is part of a CLSM system (model TCS SL, Leica). The hemocytes were observed using a x63 immersion objective (1.32 NA). Illumination was provided by an argon ion laser filter. A tetramethylrhodamine isothiocyanate wide filter (excitation at 543 nm) and a Cy3 filter (excitation at 520 nm) were used to visualize ER-{alpha}- and ER-beta-like immunoreactivity. Neutral density filters and reduced laser powers were utilized to reduce photobleaching nearly to zero. To focus hemocytes, samples were viewed under reduced transmitted light illumination; then, in confocal fluorescence mode, 10 video frames, from 500-nm adjacent sections, were averaged. The confocal images (1,024 x 1,024 bit resolution) were viewed on a high-resolution monitor and saved to disk.

Electrophoresis and Western blot analysis. Levels of phosphorylated PKC and CREB in whole cell extracts from hemocyte monolayers were determined using phosphospecific antibodies as previously described (9, 11, 14). Supernatants from each culture dish were discarded, and each hemocyte monolayer was lysed with 1 ml of ice-cold lysis buffer [50 mM Tris·HCl, pH 7.8, 0.25 M sucrose, 1% (wt/vol) SDS, 1 µg/ml pepstatin, 10 µg/ml leupeptin, 2 mM sodium orthovanadate, 10 mM NaF, 5 mM EDTA, 5 mM N-ethylmaleimide, 40 µg/ml PMSF, and 0.1% Nonidet P-40] and sonicated for 45 s at 50 W. The samples were boiled for 4 min and then centrifuged for 10 min at 14,000 g to remove insoluble debris. Supernatants were mixed 1:1 (vol/vol) with sample buffer (0.5 M Tris·HCl, pH 6.8, 2% SDS, 10% glycerol, 4% 2-mercaptoethanol, and 0.05% bromophenol blue), and the samples (normalized for protein content before they were loaded to 30 µg of protein) were resolved by 10% (for PKC) or 12% (for CREB) SDS-PAGE (27). Prestained molecular mass markers were run on adjacent lanes. The gels were electroblotted and stained with Coomassie blue (53). The blots were probed with human recombinant anti-pan-phospho-PKC (1:1,000 dilution), anti-phospho-PKC-{alpha}/betaII (1:1,000 dilution), or anti-phospho-CREB (1:1,000 dilution) as primary antibodies and horseradish-peroxidase-conjugated goat anti-rabbit IgG (1:3,000 dilution) as secondary antibody. Nitrocellulose membranes were stripped for 30 min at 50°C with stripping buffer (62.5 mM Tris·HCl, pH 6.7, containing 10 mM beta-mercaptoethanol and 2% SDS) and reprobed with anti-actin antibodies (1:1,000 dilution) as loading control (9, 14) or with anti-CREB (1:1,000 dilution) (11). Immune complexes were visualized using an enhanced chemiluminescence Western blot analysis system (Amersham Pharmacia) following the manufacturer's specifications. Western blot films were digitized (Chemidoc; Bio-Rad), and band ODs were quantified using a computerized imaging system (QuantityOne). Relative ODs (arbitrary units) were normalized for the control band in each series.

In vivo E2 exposure experiments. Mussels were injected with different concentrations of E2: 50 µl of 0.1, 0.5, and 2 µM E2 solutions containing 5, 25, and 100 pmol, respectively, of E2 (from a 10 mM stock solution in ethanol diluted in ASW) were injected into the posterior adductor muscle of groups of 10–12 mussels with use of a sterile 0.1-ml syringe as previously described (11). This range of concentrations was chosen because the average volume of hemolymph that can be withdrawn from mussels of this size (4–5 cm) is 0.6–1 ml. Experiments were repeated three times. For each experiment, a parallel set of control mussels were injected with 50 µl of a solution of ASW containing an equal amount of ethanol (≤0.005%). The mussels were then placed in plastic tanks containing ASW at 16°C (0.5 l/mussel). At 6 and 24 h after injection, hemolymph was withdrawn using a sterile syringe, and hemocytes were utilized for determination of lysosomal membrane stability, phagocytosis, and hydrolytic enzyme release. Lysosomal enzyme release was evaluated by measurement of lysozyme activity in the extracellular medium (12) as described previously (18), and data were expressed as lysozyme equivalents per milligram of protein per milliliter with hen egg white lysozyme as a standard.

Data analysis. Values are means ± SD of at least three experiments in triplicate. Data from densitometric analysis of Western blots are means ± SD of three independent experiments. Statistical analysis was performed by using Mann-Whitney's U-test, with significance at P ≤ 0.05.

Chemicals. All reagents were of analytic grade. E2 and anti-beta-actin (rabbit polyclonal) antibody were obtained from Sigma (St. Louis, MO); rabbit polyclonal anti-pan-phospho-PKC (Ser660), anti-phospho-PKC-{alpha}/betaII (Thr638/641), anti-phospho-CREB (Ser133), and anti-CREB from New England Biolabs (Beverly, MA); anti-ER-{alpha} (catalog no. H-184) and anti-ER-beta (catalog no. N-19) antibodies from Santa Cruz Biotechnology (Santa Cruz, CA); and prestained low- and high-molecular-mass markers from Bio-Rad (Hercules, CA). All other reagents were purchased from Sigma.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of E2 on hemocyte functional parameters. The effects of E2 on hemocyte phagocytosis are reported in Fig. 1. A significant stimulation was observed at 5 and 25 nM E2 (+26 and +37%, respectively, P ≤ 0.05), whereas higher concentrations of E2 (50 nM) inhibited the phagocytic process (–32%, P ≤ 0.05; Fig. 1A). E2-stimulated phagocytosis was prevented by cell pretreatment with the antiestrogen tamoxifen and with the specific p38 MAPK inhibitor SB-302580; a similar effect was observed with the PKC inhibitor GF-109203X (Fig. 1B). Neither inhibitor, at the concentration tested, showed cytotoxic effects in mussel hemocytes or affected lysosomal membrane stability (10, 15) and phagocytosis (4) in control cells.


Figure 1
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  		Fig. 1. Effects of 17beta-estradiol (E2) on phagocytosis of neutral red (NR)-conjugated zymosan particles. A and B: effects of 5–50 nM and 25 nM E2, respectively, on hemocytes preincubated with the antiestrogen tamoxifen (100 nM, TAM/E2) or the specific kinase inhibitors SB-203580 (20 µM, SB/E2) and GF-109203X (2.5 µM, GF/E2). C, control. Effect of 200 nM TNF-{alpha} is shown as positive control. Values are means ± SD of 4 experiments in triplicate. *P ≤ 0.05.

 
The effects of E2 on oxyradical production by mussel hemocytes are reported in Fig. 2. As shown in Fig. 2A, 25 nM E2 induced a rapid and time-dependent increase in extracellular oxyradical production, evaluated spectrophotometrically as cytochrome c reduction, reaching a maximal twofold increase with respect to control at 60 min (P ≤ 0.05); the effect of E2 was prevented by hemocyte pretreatment with tamoxifen. A significant increase was also observed with 5 nM E2 (+50%, P ≤ 0.05). As shown in Fig. 2B, addition of the O2– scavenger SOD, which significantly decreased the basal level of oxyradical production in control hemocytes, did not affect E2-induced cytochrome c reduction; on the other hand, the NO synthase (NOS) inhibitor L-NMMA prevented the effect of E2. Similar results were obtained with SB-302580 and GF-109203X (Fig. 2B).


Figure 2
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  		Fig. 2. Effect of E2 on oxyradical and nitrite (NO2–) production by mussel hemocytes. A and B: extracellular oxyradical production, evaluated as cytochrome c reduction. Effect of PMA (10 µg/ml, 60 min) is shown as positive control. OD, optical density. A: time course of cytochrome c reduction in control hemocytes and hemocytes treated with 5 and 25 nM E2 (E) and effect of tamoxifen (Tam/E). B: effects of cell pretreatment with different inhibitors on cytochrome c reduction in control and 25 nM E2-treated hemocytes at 60 min. SB, SB-203580; GF, GF-109203X. SOD (300 mU/ml) and NG-monomethyl-L-arginine (L-NMMA, 1 mM) were utilized as described in MATERIALS AND METHODS. C: intracellular oxyradical production, evaluated as nitro blue tetrazolium (NBT) reduction, in hemocytes incubated with 25 nM E2 for 60 min in the absence and presence of inhibitors. D: nitric oxide (NO) production, evaluated as NO2– concentration by the Griess reaction, in control hemocytes and hemocytes incubated with 25 nM E2 for 4 h. Effect of E2 on NO production was also evaluated in the presence of different inhibitors. NO production induced by PMA (50 µg/ml, 4 h) is shown as positive control. Values are means ± SD of 3 experiments in triplicate. *P ≤ 0.05, E2 or PMA vs. C. °P ≤ 0.05, inhibitors/E2 vs. E2 alone.

 
As shown in Fig. 2C, 25 nM E2 induced a twofold increase in intracellular oxyradical production compared with control (P ≤ 0.05), as evaluated by the NBT reduction assay. The effect was abolished by SOD and L-NMMA. Moreover, E2-induced NBT reduction was prevented by tamoxifen, as well as by SB-302580 and GF-109203X.

NO production in hemocyte supernatants was evaluated by the Griess reaction, which measures the concentration of the stable NO2– product. In control hemocytes, little NO2– production was observed over 0–6 h; addition of 25 nM E2 induced a significant increase in NO2– concentration from 30 min to 4 h (not shown); although a large variability in the time course of E2-induced NO production was observed among different hemocyte pools, in all experiments, maximal NO2– concentration was observed at 4 h (Fig. 2D). The effect was prevented by cell pretreatment with L-NMMA, as well as with tamoxifen and p38 MAPK and PKC inhibitors. Higher concentrations of E2 (50 nM) did not affect oxyradical production (data not shown).

We previously showed that E2-induced destabilization of lysosomal membranes in mussel hemocytes was mediated by p38 MAPK activation (12). The effect of E2 on lysosomal membrane stability was prevented by cell pretreatment with the PKC inhibitor GF-109203X (Fig. 3).


Figure 3
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  		Fig. 3. Effect of hemocyte pretreatment with the PKC inhibitor GF-109203X (20 min, 2.5 µM) on lysosomal membrane destabilization induced by 25 nM E2. Lysosomal membrane destabilization induced by hemocyte incubation with E. coli is shown as positive control. Values are means ± SD of 4 experiments in triplicate. *P ≤ 0.05.

 
Immunofluorescence microscopy of ER-like receptors. Intracellular localization of ER-like receptors in hemocyte monolayers was investigated by immunofluorescence labeling and CLSM utilizing anti-ER-{alpha} and anti-ER-beta antibodies (Fig. 4). Prominent ER-{alpha} immunoreactivity was observed in the nuclei; cytoplasmic staining was also observed, except in large intracellular vacuoles (Fig. 4A). ER-beta immunoreactivity was extranuclear; a strong signal was observed in the perinuclear region, and punctate cytoplasmic staining indicated association with small intracellular organelles (Fig. 4B).


Figure 4
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  		Fig. 4. Confocal laser scanning microscopic images of estrogen receptor (ER)-{alpha} (A) and ER-beta (B) immunoreactivity in mussel hemocytes. Note presence of ER-{alpha}-like immunoreactivity in nucleus as well as cytoplasm, but not in large intracellular vacuoles. ER-beta-like immunoreactivity is mainly localized in perinuclear region and cytoplasm, where it appears to be associated with small vacuoles. Scale bars, 10 µm.

 
Effect on PKC phosphorylation. The effect of E2 on hemocyte PKC phosphorylation was first evaluated by Western blotting with anti-pan-phospho-PKC antibodies as previously described (9, 14) (Fig. 5). The antibody, which detects {alpha}-, betaI-, betaII-, {delta}-, {epsilon}-, and {eta}- isoforms of mammalian PKC only when phosphorylated at a COOH-terminal residue homologous to Ser660 of human PKC-betaII, recognizes two phosphorylated protein bands of ~70 and 75 kDa, respectively, in mussel hemocytes, as in protein extracts from mammalian cells (9). As shown in Fig. 5A, 25 nM E2 induced a rapid and transient increase in phosphorylation of the 75-kDa protein band; densitometric band analysis (inset) revealed that the effect was maximal at 5 min (~5-fold compared with control, P ≤ 0.05). On the other hand, phosphorylation of the 70-kDa protein band was decreased. The 75-kDa PKC isoform was previously shown to correspond to PKC-{alpha} and -beta isoforms, as evaluated with a specific anti-phospho-PKC-{alpha}/betaII antibody directed toward phosphorylated Thr638/641 residues (14). As shown in Fig. 5B, E2 induced an increase in phosphorylation of the PKC-{alpha}/betaII protein band (up to 3-fold compared with control at 5 min, P ≤ 0.05). An anti-actin blot is shown as loading control (Fig. 5C). As previously demonstrated (9), no significant changes in PKC phosphorylation were observed in control hemocytes (not shown).


Figure 5
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  		Fig. 5. Effect of incubation of mussel hemocytes with 25 nM E2 for 5–60 min on PKC phosphorylation. Protein extracts from control and E2-treated hemocytes were subjected to 10% SDS-PAGE followed by Western blot analysis using polyclonal phosphospecific antibodies to anti-pan-phospho-PKC (A) and anti-phospho-PKC-{alpha}/betaII (B). C: anti-actin blot (loading control). Bands were detected using enhanced chemiluminescence reagents. Results are representative of 3 independent experiments. p-PKC, phosphorylated PKC. Insets: densitometric analysis of blots from 3 independent experiments. Values are means ± SD. *P ≤ 0.05. Relative increases in band ODs (arbitrary units) were normalized for control band in each series.

 
Effect on CREB phosphorylation. The effect of E2 on CREB phosphorylation was evaluated as previously described (11) using specific antibodies directed toward the phosphorylated Ser133 of CREB (Fig. 6). E2 induced a rapid increase in CREB phosphorylation compared with control (Fig. 6A), whereas the level of total, unphosphorylated CREB did not change (Fig. 6B). Densitometric band analysis revealed a maximal effect at 5 min (4-fold compared with control, P ≤ 0.05).


Figure 6
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  		Fig. 6. Effect of incubation of mussel hemocytes with E2 (25 nM) for different periods of time (5–60 min) on CREB phosphorylation. Protein extracts from control and E2-treated hemocytes were subjected to 12% SDS-PAGE followed by Western blot analysis using polyclonal phosphospecific antibodies to CREB (p-CREB; A), stripped and reprobed with antibodies directed towards the unphosphorylated (total) form (CREB; B). Bands were detected using enhanced chemiluminescence reagents (see MATERIALS AND METHODS). Results are representative of 3 independent experiments. C, control. Inset: densitometric analysis of blots from three independent experiments (means ± SD). P/Tot, ratio between the phosphorylated and total protein band. *P ≤ 0.05. Relative increases in band optical densities (arbitrary units) were normalized for control band in each series.

 
In vivo effects of E2 on hemocyte function. The possible in vivo effects of exogenous E2 on hemocyte function were investigated in mussels injected with different amounts of E2. Hemocyte parameters were evaluated in cells collected from mussels injected with 5, 25, and 100 pmol of E2 after 6 and 24 h, and the results were compared with those obtained in hemocytes from vehicle-injected mussels (Fig. 7). E2 induced a significant and concentration-dependent decrease in lysosomal membrane stability at 6 and 24 h of exposure (Fig. 7A). A particularly large lysosomal membrane destabilization (≥50%) was observed with 25 and 100 pmol of E2 (nominal concentration in hemolymph). A small but significant increase in phagocytic activity was observed only after 6 h with the lowest E2 concentration (+16% compared with control, P ≤ 0.05; Fig. 7B). On the other hand, higher concentrations inhibited phagocytosis; with 100 pmol of E2, the effect was observed after only 6 h (–23% compared with control, P ≤ 0.05), and with 25 and 100 pmol of E2 the effect was observed after 24 h (–17 and –37%, respectively, P ≤ 0.05).


Figure 7
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  		Fig. 7. Effects of in vivo exposure to 0–100 nM E2 on hemocyte functional parameters: lysosomal membrane stability (A), phagocytosis (B), and hydrolytic enzyme release (C). Mussels were injected with 5, 25, and 100 pmol of E2 or vehicle, and hemocytes collected 6 and 24 h after injection were analyzed. NRR, neutral red retention. Values are means ± SD of 3 experiments in triplicate. *P ≤ 0.05.

 
E2 also stimulated extracellular enzyme release, evaluated as lysozyme activity in the extracellular medium (Fig. 7C). The lowest concentration induced a large increase in lysozyme release 24 h after injection (+231% compared with control, P ≤ 0.05); higher concentrations induced smaller but significant increases at 6 and 24 h (+70 and +31% at 25 pmol of E2 and +35 and +23% at 100 pmol of E2, respectively, P ≤ 0.05).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results demonstrate that E2 in vitro can rapidly affect phagocytosis and oxyradical production in Mytilus hemocytes; the effects were prevented by the antiestrogen tamoxifen. The results confirm and expand previous observations indicating that E2, in the same experimental conditions, induced tamoxifen-sensitive lysosomal membrane destabilization, morphological changes, degranulation of hydrolytic enzymes, and stimulation of bactericidal activity (12).

E2 significantly stimulated the phagocytic process in a narrow concentration range (5–25 nM), whereas higher concentrations (50 nM) were inhibitory. Along with increased phagocytosis, stimulation of the oxidative burst was observed, as indicated by measurement of extra- and intracellular oxyradical production by standard methods for bivalve hemocytes (54). The extent of stimulation was similar to that observed after challenge of Mytilus hemocytes with PMA (1; present study) or laminarin (1). Addition of SOD prevented only E2-stimulated intracellular oxyradical production, indicating an increase in O2– generation associated with increased phagocytic activity. Moreover, extra- and intracellular oxyradical production was prevented by addition of the NOS inhibitor L-NMMA, which prevents NO synthesis in molluscan hemocytes (44). A similar effect was induced by nitro-L-arginine methyl ester (data not shown). NO production was confirmed by the significant increase in NO2– concentration, with a maximum after 4 h of E2 addition. In the same experimental conditions, a similar, although smaller, effect was induced by PMA (50 µg/ml). The effect was prevented by tamoxifen and L-NMMA, suggesting that, in Mytilus hemocytes, E2 induces NO synthesis through a receptor-mediated event, as previously demonstrated in ganglionic tissue (46). Because in bivalve molluscs phagocytosis and oxyradical production through the action of NADPH oxidase and NOS represent important nonspecific defense mechanisms (1, 23, 35, 44, 50), the results demonstrate that E2 can act as an immunostimulant on mussel hemocytes. In invertebrates, NO can be produced rapidly and after a latent period through constitutive (cNOS) and inducible (iNOS) NOS isoforms, with cNOS modulating basal NO actions that are further enhanced by iNOS-derived NO (44). If we consider that the NO2– determination usually does not include cNOS-derived NO release and the time course of NO production in response to E2, our data support the hypothesis that rapid E2 signaling subsequently leads to increased NO production, probably through disinhibition of an iNOS form in the hemocyte (44).

In vertebrate phagocytes, the oxidative burst is mediated by several intracellular pathways, including MAPKs, and Ca2+-dependent and Ca2+-independent PKC isoforms, leading to NADPH oxidase activation (52). E2-stimulated phagocytosis and oxyradical (including NO) production were prevented by SB-302580, confirming that, as previously demonstrated, the stress-activated p38 MAPK represents a target for the action of E2 in these cells (12). In particular, 5 and 25 nM E2 induced a rapid (from 5 min) and transient increase in the level of phosphorylated p38 (12). The p38 MAPK inhibitor prevented the effect of E2 on lysosomal membrane stability (12), a sensitive parameter of in vitro exposure to E2 in Mytilus cells (6, 32). In mussel hemocytes, the effects of E2 on all the functional parameters tested, including lysosomal membrane stability, were prevented by GF-109203X, and PKC and, in particular, a PKC-{alpha}/betaII-like isoform (14) were rapidly and transiently phosphorylated in response to E2. In the same conditions, E2 induced a small but significant increase in cytosolic [Ca2+], indicating that [Ca2+]-mediated cell signaling may participate in activation of PKC-like classical isoforms (12). PKC is involved in rapid estrogen signaling through ER-dependent and -independent mechanisms (19, 50). Because activation of p38 MAPK and PKC has been shown to play a key role in the response of Mytilus hemocytes to bacterial challenge (9, 10), overall, the results demonstrate that estrogen signaling through p38 MAPK and PKC occurs in invertebrate cells and participates in immunostimulation.

E2 also induced a transient increase in the phosphorylation of a CREB-like protein previously identified in mussel hemocytes (11). CREB, a transcription factor that is activated by serine/threonine kinases, is involved in the cross talk between estrogen, ERs, and cytosolic kinase cascades, including MAPKs (17, 28). CREB-like proteins have been identified in the nervous system of gastropod molluscs, where they play a role in neuronal plasticity and learning (42). In mussel hemocytes, in vivo exposure to the xenoestrogen bisphenol A significantly decreased phosphorylation of CREB at Ser133 (11); on the other hand, large CREB phosphorylation is rapidly induced by bacterial challenge with E. coli (unpublished results). The present results demonstrate that E2 signaling in mussel hemocytes, as in mammalian cells, involves CREB-like proteins.

Overall, the results further support the hypothesis that rapid activation of kinase pathways represents a significant mode of action by the natural estrogen E2 in Mytilus hemocytes in vitro. In Mytilus ganglia, the effects of the membrane-impermeant E2-BSA conjugate were similar to those of free E2, suggesting an E2-mediated signaling pathway at the cell surface coupled to NO production (46). In hemocytes, E2-BSA, similar to E2, induced lysosomal destabilization and MAPK and STAT activation; however, part of the E2-BSA conjugate was rapidly internalized by the cells and, therefore, seemed unsuitable to demonstrate the extracellular action of the hormone (12). E2-BSA stimulated the oxidative burst, but it induced a slight decrease (10–15%), rather than an increase, in phagocytosis of zymosan particles (data not shown).

The results obtained with the antiestrogen tamoxifen indicate a possible role for ER-like receptors in mediating the rapid effects of E2. We previously demonstrated the presence of ~70- and 49-kDa immunoreactive ER-{alpha}- and ER-beta-like proteins, respectively, by electrophoresis and Western blot analysis of soluble hemocyte protein extracts with use of antibodies directed against a specific region in the NH2-terminal domain of ER-{alpha} and ER-beta, respectively, which is the highly divergent domain between the two mammalian forms (12). The results of confocal microscopy utilizing the same antibodies confirm the presence of ER-like proteins in control hemocytes. ER-{alpha} immunoreactivity showed a prominent nuclear localization and a diffuse signal in the cytoplasm; on the other hand, ER-beta-like proteins were observed at extranuclear sites, with most cells showing a strong signal in the perinuclear region. A distinct immunostaining was also observed in cytoplasmic organelles: large vacuoles showed a characteristic absence of immunoreactivity to ER-{alpha} antibodies, whereas punctate ER-beta immunostaining indicated association with smaller vacuoles. In Mytilus spp., granular hemocytes, the dominant cell type in the hemolymph, are characterized by a low nucleus-to-cytoplasm ratio, high phagocytic activity, and capacity for oxyradical production (54). The distinct association of the two ER-like proteins with intracellular granules may reflect the heterogeneity of these organelles in terms of enzyme composition (lysosomal hydrolytic enzymes and oxidases) (16, 37) and the possible role of lysosomal compartments in ER degradation (39). E2-induced localization of ER in perinuclear lysosomes was first demonstrated by Szego (49) and others (Ref. 39 and references quoted therein), supporting a role for lysosomes in nucleocytoplasmic communication in the response to estradiol. Moreover, recent studies indicated that, in different cell types, perinuclear localization of ER-beta isoforms was due to association of the ER with the mitochondria (Ref. 7 and references quoted therein). Our results, although preliminary, suggest that, in mussel hemocytes, ER localization in lysosomal compartments is highly likely; however, experiments are needed to identify the nature of ER-positive vacuoles utilizing lysosomal and mitochondrial markers in control and E2-treated hemocytes.

The possibility that E2 may affect the hemocyte function in vivo at longer exposure times was investigated in mussels injected with different concentrations of E2. In hemocytes from control and E2-injected mussels, functional parameters that have been shown to be affected by E2 in vitro, i.e., lysosomal membrane stability, extracellular lysozyme release, and phagocytosis (12; present study), were evaluated. In mussels injected with E2, a concentration-dependent decrease in lysosomal membrane stability was observed compared with control 6 and 24 h after injection. These results demonstrate that in vivo the lysosomal function represents a sensitive target for the action of the hormone in mussel cells, as previously shown in vitro (6, 12, 32). At the lowest concentration tested (5 pmol), E2 induced a large increase in extracellular release of lysosomal hydrolytic enzymes, comparable to that induced by E. coli (38). The smaller effect observed at higher concentrations may be due to unspecific degranulation associated with cell damage, as indicated by the large decrease in lysosomal membrane stability observed in these conditions. Similarly, the lowest concentration of E2 stimulated phagocytosis only 6 h after injection, whereas at longer exposure times, and with higher concentrations, a decrease in phagocytosis was observed.

The effects and mechanisms of action of E2 at longer incubation times in vivo may be more complex than those involved in the rapid effects on cell signaling observed in vitro: they may be mediated by membrane and intracellular signaling pathways, with or without involvement of ER-like receptors, or reflect the integrations of actions at different receptor pools, as in mammalian cells (29). In addition to the direct effect on the hemocytes, exposure to E2 can also affect the activity of steroid-metabolizing enzymes in other mussel tissues (gonad and digestive gland) that metabolize exogenous E2 to an esterified form (25), thereby affecting the concentration of free E2 in the hemolymph; E2 exposure may also affect the levels of endogenous modulators that have been shown to affect the immune function in bivalves (4, 26, 47).

Steroid concentrations reported in the literature in bivalve tissues widely differed (from picograms to nanograms) depending on the species, tissue, sexual maturation stage, and analytic technique (21). The only available data on estrogen content in the hemolymph are those reported by Stefano et al. (46) and Zhu et al. (55) in Mytilus edulis. Although the concentration of free E2 was not evaluated in the present study at different times after injection, preliminary data, obtained utilizing a commercial competitive chemiluminescent enzyme immunoassay kit (Immulite 2000 Estradiol), indicate that a only a small fraction of the injected E2 was retained in hemolymph serum (unpublished observations). Further research utilizing E2 dissolved in seawater and measurements of E2 isoforms in the hemolymph with appropriate analytic techniques are needed to clarify the effects of exogenous E2 on mussel immune function in vivo.

In mammalian immunocytes, estrogens induce dose- and cell-type-specific responses, thus resulting in pro- or anti-inflammatory effects (2, 5, 22, 24, 43, 45). Overall, the results indicate that, in Mytilus hemolymph, changes in the concentrations of E2 in a narrow range may elicit immunostimulation through activation of the signaling pathways involved in the immune response. Higher concentrations can downregulate the hemocyte function, probably through impairment of the mechanism involved in controlled membrane fusion events during the phagocytic process leading to lysosomal membrane destabilization (present study; 6), lysosomal enlargement, and increased protein degradation (6). In bivalve immunocytes, the lysosomal vacuolar system, which plays a key role in different aspects of the immune function, seems to represent a significant target for the action of E2.

Synthetic estrogens, such as 17{alpha}-ethynylestradiol, diethylstilbestrol, and mestranol, showed similar effects, although at much higher concentrations (15; unpublished data). In mussels, the effects of the natural estrogen E2 on the immune function may be relevant in physiological conditions. Overall, our data demonstrate that, in bivalve molluscs, E2 can modulate the immune function in vitro and in vivo and support the hypothesis that the role of estrogens in nonreproductive functions is conserved in evolution.


   FOOTNOTES
 

Address for reprint requests and other correspondence: L. Canesi, Dipartimento di Biologia, Sezione di Fisiologia, Corso Europa 26, Università di Genova, 16132 Genova, Italy (e-mail: Laura.Canesi{at}unige.it)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Arumugam M, Romestand B, Torreilles J, and Roch P. 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. Exp J Cell Biol 79: 513–519, 2000.
  2. Azenabor AA and Chaudhry AU. 17beta-Estradiol induces L-type Ca2+ channel activation and regulates redox function in macrophages. J Reprod Immunol 59: 17–28, 2003.[CrossRef][Web of Science][Medline]
  3. Benten WPM, Lieberherr M, Giese G, and Wunderlich F. Estradiol binding to cell surface receptors. Endocrinology 142: 1669–1677, 2001.[Abstract/Free Full Text]
  4. Betti M, Ciacci C, Lorusso LC, Canonico B, Falcioni T, Gallo G, and Canesi L. Effects of tumor necrosis factor-{alpha} (TNF-{alpha}) on Mytilus hemocytes: role of stress-activated MAP kinases. Biol Cell 98: 233–244, 2006.[CrossRef][Web of Science][Medline]
  5. Bruce-Keller A, Keeling JL, Keller JN, Huang FF, Camondola S, and Mattson MP. Anti-inflammatory effects of estrogen on microglial activation. Endocrinology 141: 3646–3656, 2000.[Abstract/Free Full Text]
  6. Burlando B, Marchi B, Panfoli I, and Viarengo A. Essential role of Ca2+-dependent phospholipase A2 in estradiol-induced lysosome activation. Am J Physiol Cell Physiol 283: C1461–C1468, 2002.[Abstract/Free Full Text]
  7. Cammarata PR, Flynn J, Gottipati S, Chu S, Dimitrijevich S, Younes M, Skliris G, and Murphy LC. Differential expression and comparative subcellular localization of estrogen receptor-beta isoforms in virally transformed and normal cultured human lens epithelial cells. Exp Eye Res 81: 165–175, 2005.[CrossRef][Web of Science][Medline]
  8. Canesi L, Betti M, Ciacci C, Citterio B, Pruzzo C, and Gallo G. Tyrosine kinase mediated cell signaling in the activation of Mytilus hemocytes: possible role of STAT proteins. Biol Cell 95: 608–618, 2003.
  9. Canesi L, Betti M, Ciacci C, Lorusso LC, Gallo G, and Pruzzo C. Interactions between Mytilus hemocytes and different strains of Escherichia coli and Vibrio cholerae O1 El Tor: role of kinase-mediated signaling. Cell Microbiol 7: 667–674, 2005.[CrossRef][Web of Science][Medline]
  10. Canesi L, Betti M, Ciacci C, Scarpato A, Citterio B, Pruzzo C, and Gallo G. Signaling pathways involved in the physiological response of mussel hemocytes to bacterial challenge: the role of stress-activated p38 MAPK. Dev Comp Immunol 26: 325–334, 2002.[CrossRef][Web of Science][Medline]
  11. Canesi L, Betti M, Lorusso LC, Ciacci C, and Gallo G. "In vivo" effects of bisphenol A in Mytilus hemocytes: modulation of kinase-mediated signalling pathways. Aquat Toxicol (Amst) 71: 73–84, 2005.
  12. Canesi L, Ciacci C, Betti M, Lorusso LC, Marchi B, Burattini S, Falcieri E, and Gallo G. Rapid effect of 17beta-estradiol on cell signaling and function of Mytilus hemocytes. Gen Comp Endocrinol 136: 58–71, 2004.[CrossRef][Web of Science][Medline]
  13. Canesi L, Gavioli M, Pruzzo C, and Gallo G. Bacteria-hemocyte interactions and phagocytosis in marine bivalves. Microsc Res Tech 57: 469–476, 2002.[CrossRef][Web of Science][Medline]
  14. Canesi L, Lorusso LC, Ciacci C, Betti M, and Gallo G. Effects of the brominated flame retardant tetrabromobisphenol-A (TBBPA) on cell signaling and function of Mytilus hemocytes: involvement of MAP kinases and protein kinase C. Aquat Toxicol (Amst) 75: 277–287, 2005.
  15. Canesi L, Lorusso LC, Ciacci C, Betti M, and Gallo G. Environmental estrogens can affect the function of mussel hemocytes through rapid modulation of kinase pathways. Gen Comp Endocrinol 138: 58–69, 2004.[CrossRef][Web of Science][Medline]
  16. Carballal MJ, Lòpez C, Azevedo C, and Villalba A. Enzymes involved in defense functions of hemocytes of mussel Mytilus galloprovincialis. J Invertebr Pathol 70: 96–105, 1997.[CrossRef][Web of Science][Medline]
  17. Carlstrom L, Ke Z, Unnerstall JR, and Cohen RS. Estrogen modulation of the cyclic AMP response element binding protein pathway. Effects of long-term and acute treatments. Neuroendocrinology 74: 227–243, 2001.[CrossRef][Web of Science][Medline]
  18. Chu FLE and La Peyre JF. Effect of environmental factors and parasitism on hemolymph lysozyme and protein of American oysters (Crassostrea virginica). J Invertebr Pathol 54: 224–232, 1989.
  19. Devidze N, Pfaff DW, and Kow LM. Potentiation of genomic actions of estrogen by membrane actions in mcf-7 cells and the involvement of protein kinase C activation. Endocrine 27: 253–258, 2005.[CrossRef][Web of Science][Medline]
  20. Gagnè F, Blaise C, Salazar M, Salazar S, and Hansen PD. Evaluation of estrogenic effects of municipal effluents to the freshwater mussel Elliptio complanata. Comp Biochem Physiol 128C: 213–225, 2001.[CrossRef]
  21. Gauthier-Clerc S, Pellerin J, and Amiard JC. Estradiol-17beta and testosterone concentrations in male and female Mya arenaria (mollusca bivalvia) during the reproductive cycle. Gen Comp Endocrinol 145: 133–139, 2006.[CrossRef][Web of Science][Medline]
  22. Ghisletti S, Meda C, Maggi A, and Vegeto E. 17beta-Estradiol inhibits inflammatory gene expression by controlling NF-{kappa}B intracellular localization. Mol Cell Biol 25: 2957–2968, 2005.[Abstract/Free Full Text]
  23. Gourdon J, Guerin MC, Torreilles J, and Roch P. Nitric oxide generation by hemocytes of the mussel Mytilus galloprovincialis. Nitric Oxide 5: 1–6, 2001.[CrossRef][Web of Science][Medline]
  24. Guo Z, Krucken J, Benten PM, and Wuderlich F. Estradiol-induced nongenomic calcium signaling regulates genotropic signaling in macrophages. J Biol Chem 277: 7044–7050, 2002.[Abstract/Free Full Text]
  25. Janer G, Lavado R, Thibaut R, and Porte C. Effects of 17beta-estradiol exposure in the mussel Mytilus galloprovincialis: a possible regulating role for steroid acyltransferases. Aquat Toxicol (Amst) 75: 32–42, 2005.
  26. Lacoste A, Malham SK, Cueff A, and Poulet SA. Noradrenaline modulates oyster hemocyte phagocytosis via a beta-adrenergic receptor-cAMP signaling pathway. Gen Comp Endocrinol 122: 252–259, 2001.[CrossRef][Web of Science][Medline]
  27. Laemmli UK. Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227: 680–685, 1970.[CrossRef][Medline]
  28. Lazennec G, Thomas JA, and Katzenellenbogen BS. Involvement of cyclic AMP response element binding protein (CREB) and estrogen receptor phosphorylation in the synergistic activation of the estrogen receptor by estradiol and protein kinase activators. J Steroid Biochem Mol Biol 77: 193–203, 2001.[CrossRef][Web of Science][Medline]
  29. Levin ER. Integration of the extranuclear and nuclear actions of estrogen. Mol Endocrinol 19: 1951–1959, 2005.[Abstract/Free Full Text]
  30. Li Q, Osada M, Suzuki M, and Mori K. Changes in vitellin during oogenesis and effect of estradiol-17beta on vitellogenesis in the Pacific oyster Crassostrea gigas. Invertebr Reprod Dev 33: 87–93, 1998.
  31. Lowe DM, Fossato VU, and Depledge MH. Contaminant-induced lysosomal membrane damage in blood cells of mussels Mytilus galloprovincialis from the Venice Lagoon: an in vitro study. Mar Ecol Prog Ser 129: 189–196, 1995.
  32. Moore MN, Lowe DM, and Fieth PEM. Responses of lysosomes in the digestive cells of the common mussel Mytilus edulis, to sex steroids and cortisol. Cell Tissue Res 188: 1–9, 1978.
  33. Osada M, Takamura T, Sato H, and Mori K. Vitellogenin synthesis in the ovary of scallop Patinopecten yessoensis: control by estradiol-17beta and the central nervous system. J Exp Zool 299A: 172–179, 2003.
  34. Osada M, Tawarayama H, and Mori K. Estrogens in relation to gonadal development of the Japanese scallop, Patinopecten yessoensis: gonadal profile and immunolocalisation of P450 aromatase and estrogen. Comp Biochem Physiol 139C: 123–128, 2004.[CrossRef]
  35. Ottaviani E, Paemen LR, Cadet P, and Stefano GB. Evidence for nitric oxide production and utilization as a bactericidal agent by invertebrate immunocytes. Eur J Pharmacol 248: 319–324, 1993.[Web of Science][Medline]
  36. Pipe R, Coles JA, and Farley SR. Assays for measuring immune response in the mussel Mytilus edulis. In: Techniques in Fish Immunology. Immunology and Pathology of Aquatic Invertebrates, edited by Stolen J, Fletcher TC, Smith SA, Zelikoff JT, Kaattari SL, Anderson RS, Söderhäll K, and Weeks-Perkins BA. Fair Haven, NJ: SOS, 1995, vol. 4, p. 93–100.
  37. Pipe RK. Hydrolytic enzymes associated with granular haemocytes of the marine mussel Mytilus. Histochem J 22: 595–603, 1990.[CrossRef][Web of Science][Medline]
  38. Pruzzo C, Gallo G, and Canesi L. Persistence of vibrios in marine bivalves: the role of interactions with hemolymph components. Environ Microbiol 7: 761–772, 2005.
  39. Qualmann B, Kessels MM, Thole HH, and Sierrata WD. A hormone pulse induces transient changes in the subcellular distribution and leads to lysosomal accumulation of the estrogen receptor-{alpha} in target tissues. Eur J Cell Biol 79: 383–393, 2000.[CrossRef][Web of Science][Medline]
  40. Reis-Henriques MA, Le Guellec D, Remy-Martin JP, and Adessi GL. Studies on endogenous steroids from the marine mollusc Mytilus edulis L. by gas chromatography and mass spectrometry. Comp Biochem Physiol 95B: 303–309, 1990.[CrossRef]
  41. Renwrantz L. Internal defence system of Mytilus edulis. In: Neurobiology of Mytilus edulis, edited by Stefano GB. Manchester, UK: Manchester University Press, 1990, p. 256–275.
  42. Ribeiro MJ, Serfozo Z, Papp A, Kemenes J, O'Shea M, Yin JC, Benjamin PR, and Kemenes G. Cyclic AMP response element-binding protein (CREB)-like proteins in a molluscan brain: cellular localization and learning-induced phosphorylation. Eur J Neurosci 18: 1223–1234, 2003.[CrossRef][Web of Science][Medline]
  43. Soucy G, Boivin G, Labrie F, and Rivest S. Estradiol is required for a proper immune response to bacterial and viral pathogens in the female brain. J Immunol 174: 6391–6198, 2005.[Abstract/Free Full Text]
  44. Stefano GB and Ottaviani E. The biochemical substrate of nitric oxide signaling is present in primitive non-cognitive organisms. Brain Res 924: 82–89, 2002.[CrossRef][Web of Science][Medline]
  45. Stefano GB, Cadet P, Breton C, Goumon Y, Prevot V, Deissant JP, Beauvillain JC, Roumier AS, Welters I, and Salzet M. Estradiol-stimulated nitric oxide release in human granulocytes is dependent on intracellular calcium transients: evidence for a cell surface estrogen receptor. Blood 95: 3951–3958, 2000.[Abstract/Free Full Text]
  46. Stefano GB, Cadet P, Mantione K, Cho JJ, Jones D, and Zhu W. Estrogen signalling at the cell surface coupled to nitric oxide release in Mytilus edulis nervous system. Endocrinology 144: 1234–1240, 2003.[Abstract/Free Full Text]
  47. Stefano GB, Leung MK, Bilfinger TV, and Scharrer B. Effect of prolonged exposure to morphine on responsiveness of human and invertebrate immunocytes to stimulatory molecules. J Neuroimmunol 63: 175–181, 1995.[CrossRef][Web of Science][Medline]
  48. Stefano GB, Zhu W, Mantione K, Jones D, Salamon E, Cho JJ, and Cadet P. 17beta-Estradiol down regulates ganglionic microglial cells via nitric oxide release: presence of an estrogen receptor beta transcript. Neuroendocrinol Lett 24: 130–136, 2003.
  49. Szego CM. Lysosomal function in nucleocytoplasmic communication. In: Lysosomes in Biology and Pathology, edited by Dingle JT and Dean R. Amsterdam: Elsevier, 1975, vol. 4, p. 385–477.
  50. Sylvia VL, Hughes T, Dean DD, Boyan BD, and Schwartz Z. 17beta-Estradiol regulation of protein kinase C activity in chondrocytes is sex-dependent and involves non-genomic mechanisms. J Cell Physiol 176: 435–444, 1998.[CrossRef][Web of Science][Medline]
  51. Tafalla C, Novoa B, and Figueras A. Production of nitric oxide by mussel (Mytilus galloprovincialis) hemocytes and effects on phagocytic functions. Comp Biochem Physiol 132B: 423–431, 2002.[CrossRef]
  52. Tan SL and Parker J. Emerging and diverse roles of protein kinase C in immune cell signaling. Biochem J 376: 545–552, 2003.[CrossRef][Web of Science][Medline]
  53. Towbin H, Stahelin T, and Gordon J. Electrophoretic transfer of protein from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76: 4350–4354, 1979.[Abstract/Free Full Text]
  54. Wootton EC, Dyrynda EA, and Ratcliffe NA. Bivalve immunity: comparisons between the marine mussel (Mytilus edulis), the edible cockle (Cerastoderma edule) and the razor-shell (Ensis siliqua). Fish Shellfish Immunol 15: 195–210, 2003.[CrossRef][Web of Science][Medline]
  55. Zhu W, Mantione K, Jones D, Salamon E, Cho JJ, Cadet P, and Stefano GB. The presence of 17beta-estradiol in Mytilus edulis gonadal tissues: evidence for estradiol isoforms. Neuroendocrinol Lett 24: 137–140, 2003.[Medline]




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