- Email: [email protected]
Revising lysenin expression of earthworm coelomocytes
Developmental and Comparative Immunology 39 (2013) 214–218
Contents lists available at SciVerse ScienceDirect
Developmental and Comparative Immunology journal homepage: www.elsevier.com/locate/dci
Short communication
Revising lysenin expression of earthworm coelomocytes Balázs Opper a,b, András Bognár a, Diána Heidt a, Péter Németh a, Péter Engelmann a,⇑ a b
Department of Immunology and Biotechnology, Clinical Center, University of Pécs, Szigeti u. 12, H-7643 Pécs, Hungary Department of Anatomy, Faculty of Medicine, University of Pécs, Szigeti u. 12, H-7643 Pécs, Hungary
a r t i c l e
i n f o
Article history: Received 23 September 2012 Revised 20 November 2012 Accepted 20 November 2012 Available online 28 November 2012 Keywords: Innate immunity Coelomocytes Antimicrobial activity Monoclonal antibodies
a b s t r a c t Lysenin is a species-specific bioactive molecule of Eisenia andrei earthworms. This protein is a potent antimicrobial factor; however its cellular expression and induction against pathogens are still not fully understood. We developed a novel monoclonal antibody against lysenin and applied this molecular tool to characterize its production and antimicrobial function. We demonstrated by flow cytometry and immunocytochemistry that one subgroup of earthworm immune cells (so called coelomocytes), the chloragocytes expressed the highest amount of lysenin. Then, we compared lysenin expression with earlier established coelomocyte (EFCC) markers. In addition, we determined by immunohistology of earthworm tissues that lysenin production is only restricted to free-floating chloragocytes. Moreover, we observed that upon in vitro Staphylococcus aureus but not Escherichia coli challenged coelomocytes over-expressed and then secreted lysenin. These results indicate that among subpopulations of coelomocytes, lysenin is mainly produced by chloragocytes and its expression can be modulated by Gram-positive bacterial exposure. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Earthworm’s coelomic fluid exerts a wide spectrum of biological functions including immunity against environmental pathogens. Humoral factors of this compartment control the growth of commensal and pathogenic microorganisms. Coelomic fluid has been a subject of intensive research since many years in earthworms (Bilej et al., 2000; Kauschke et al., 2007; Valembois et al., 1986; Wang et al., 2010). The most extensively characterized factors are the unique fetidin/lysenin group of earthworm’s coelomic fluid. First, fetidin is described as a glycoprotein with antibacterial and haemolytic properties (Lassegues et al., 1997). In addition, another bioactive molecule, designated as lysenin, has been cloned (Sekizawa et al., 1997). Lysenin was described primarily as a smooth muscle contraction factor (Sekizawa et al., 1996a). Membrane biological studies proved that lysenin binds specifically to the sphingomyelin compartments of the cell membrane (Shogomori and Kobayashi, 2008). Initially, lysenin and fetidin were considered as separate molecules exhibiting similar molecular weights (Cooper and Roch, 2003). However, recently it is known that both lysenin and fetidin
Abbreviations: EFCC, Eisenia coelomocyte clusters; LBSS, Lumbricus balanced salt solution. ⇑ Corresponding author. Tel.: +36 72 536 288; fax: +36 72 536 289. E-mail address: [email protected] (P. Engelmann). 0145-305X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.dci.2012.11.006
belong to the same multimolecular family, that consist of four members such as lysenin, lysenin related protein 1, lysenin related protein 2 (fetidin), and lysenin related 3 (Bruhn et al., 2006). Lysenin/fetidin molecules were found abundantly in the coelomic fluid; however their exact production site and function in earthworms remained elusive. Chloragogenous tissue covers the gut of earthworms. Chloragocytes, thus forming this tissue, are considered as a functional homologue cell type for vertebrate hepatocytes (Jamieson, 1981). Chloragocytes can be separated into two distinctly localized cell populations such as the peripheral chloragocytes found on the gut wall, while the other one termed as central chloragocytes settled inside of the typhlosolis (Ohta et al., 2000). It is suggested that lysenin expression is coupled mainly with the central chloragocytes demonstrated by in situ hybridization and polyclonal antibody-based immunohistochemistry (Sekizawa et al., 1996b; Ohta et al., 2000). Previously we have developed a group of monoclonal antibodies (mAbs) against earthworm coelomocytes and with the aid of these molecular tools we characterized three subpopulations of freefloating coelomocytes (Engelmann et al., 2005). Furthermore, recently we paid more attention to characterize the functional status of coelomocytes (Engelmann et al., 2011). For this purpose we have developed a mAb against lysenin to characterize its cytotoxic nature. In addition, we aimed to revise lysenin expression profile in free-floating coelomocytes, its tissue localization and expression patterns upon bacterial challenge.
B. Opper et al. / Developmental and Comparative Immunology 39 (2013) 214–218
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2. Materials and methods
2.6. Statistical analysis
2.1. Earthworm culture Eisenia andrei earthworm species were maintained at room temperature and fed with manure complemented soil. Prior to coelomocyte isolations earthworms were placed onto moist tissue paper for depuration.
Statistical analysis was performed with Microcal Origin (Microcal Software Inc., Northhampton, MA USA). The results in the figures are representative values from four independent experiments. All results are presented as mean with standard error shown as error bar. The effect of treatments was analyzed by one way-ANOVA. p < 0.05 was denoted as statistically significant.
2.2. Preparation of mouse anti-lysenin monoclonal antibody
3. Results and discussion
Lysenin (PeptaNova GmbH, Sandhausen, Germany) were dissolved in distilled water at 1 mg/ml concentrations and used for immunizing 8 weeks old female BALB/c mice. Mice were injected with lysenin protein (10 lg) mixed in complete Freund’s adjuvant (CFA, Sigma–Aldrich Co., St. Louis, MO, USA) into each hind footpad followed with two intraperitoneal (i.p.) boosts in incomplete Freund’s adjuvant (IFA, Sigma) 3 weeks intervals. Finally, one booster was injected in mice selected, according to its antibody titer and isotype, 3 days prior to splenocyte/myeloma-cell fusion. Hybridomas were developed as we described earlier (Engelmann et al., 2005). Hybridoma supernatants were screened by enzyme linked immunosorbent assay (indirect ELISA, for technical details please see the Supplementary Material) using lysenin, coelomocyte lysate as target antigen, and bovine serum albumin (BSA, Sigma) as control antigen. Selected hybridomas were cloned three times by limiting dilution.
3.1. Lysenin expression of isolated coelomocytes
2.3. Extrusion of coelomocytes Coelomocytes were isolated as we described earlier (Engelmann et al., 2005) and living cell numbers were evaluated by trypan-blue exclusion method. Cell viability was above 95%. Coelomocytes of individual worms were analyzed further by immunocytochemistry, immunofluorescence, and flow cytometry. For technical details please see the SM.
2.4. In vitro bacterial challenge Isolated, pooled coelomocytes (6 106/ml) were incubated with heat-inactivated Staphylococcus aureus (OKI II2001) and Escherichia coli (ATCC 25922) at room temperature by end-over-end rotation for 6 h (Bacterial strains were obtained from Dr. Béla Kocsis MD, PhD, Department of Medical Microbiology and Immunology, Clinical Center, University of Pécs). Coelomocytes (106) and bacteria (107) were mixed in 1 ml final volume in 12 75 mm tubes (Falcon, BD Labware). Following phagocytosis, coelomocyte samples were washed in LBSS, centrifuged at 1000 rpm for 5 min (Engelmann et al., 2005). Then supernatant was kept at 80 °C for further analysis and the pellet was applied for the preparation of coelomocyte lysates.
2.5. Preparation of coelomocyte lysate Coelomocytes were lysed in RIPA buffer (50 mM Tris/HCl; pH 8.0, 150 mM NaCl, 1% (v/v) NP-40, 0.5% (w/v) Na-deoxycholate, 5 mM EDTA, 0.1% SDS) complemented with Protease Inhibitor Cocktail (Sigma) on ice for 15 min and then centrifuged by 13,000 rpm (15 min, 4 °C). Total protein concentrations of the coelomocyte lysates and supernatants were measured with BCA Reagent Kit (Pierce, Rockford, IL, USA). Immunoreactivity of supernatants and coelomocyte lysates were analyzed by ELISA and Western blot assays (further technical details can be obtained in SM).
Coelomocytes are multi-tasking cellular mediators of immune response in earthworms. In addition to their cellular immune functions (e.g. phagocytosis, encapsulation) they participate in the humoral immune mechanisms by producing haemolytical and antimicrobial factors (Bilej et al., 2000; Cooper et al., 2002). Most of these proteins belong to the fetidin/lysenin multiprotein family characterized mainly by membrane biologists. However, the exact role of lysenin in the immune response of earthworms is not so well understood (Kobayashi et al., 2004). Previously, we observed the cytotoxicity mediated by earthworm coelomocytes produced factors (Engelmann et al., 2011). Now we intend to uncover more details about the cytotoxic mechanisms (Macsik et al., in preparation). For this purpose we raised a monoclonal antibody against lysenin (a-EFCC5) and first we initiated to characterize its expression profile in coelomocytes. Antibody positive cells have large granular cytoplasm and relatively small nucleus revealed by immunocytochemistry (Fig. 1A). Immune positive reaction is localized mainly in the intracellular granules. This morphology marks one characteristic subgroup of coelomocytes, the free-floating chloragocytes (sometimes denoted as eleocytes). Other cell types with larger nucleus and smaller cytoplasm (effector coelomocytes, presumably hyaline amoebocytes) were negative for a-EFCC5 staining (Fig. 1A). Using the previously generated mAbs against coelomocyte subgroups we revealed co-expression with lysenin positive coelomocytes (Fig. S1). A-EFCC3 positive coelomocytes are tend to attach together and form aggregates, while EFCC5 positive cells are mainly solitary (Fig. 1B, Figs. S1 and S2). By means of flow cytometry, circulating coelomocytes of the body cavity has several subgroups (Engelmann et al., 2005; Fuller-Espie et al., 2010; Vernile et al., 2007). In our experiments, we could identify three physically distinct subpopulations of coelomocytes denoted as R1, R2, and R3 (Fig. 1C). R1 and R2 populations resemble effector coelomocytes such as hyaline and granular coelomocytes, while R3 subgroup is the highly granular and autofluorescent eleocyte population (Plytycz et al., 2006) (Fig. 1C). EFCC5 positivity could be observed in all three subgroups with various intensities compared with appropriate isotype control antibodies (Fig. 1D). R3 group majorly consisting eleocytes had the highest percentage (39.5 ± 10.2%) of EFCC5 antibody positive cells (Fig. 1D and Table S1). 3.2. Solitary free-floating chloragocytes are restricted for lysenin expression Chloragocytes are usually very abundant, highly granular coelomocyte subpopulation of the coelomic cavity. Their origin is a matter of debate, however most authors agreed that free-floating chloragocytes derived from the chloragogenous tissue of the gut (Jamieson, 1981). These cells are harboring nutrient factors such as glycogens, lipids and participate mainly in general metabolism. Moreover, they are able to produce haemoglobin and metalsequestering cysteine-rich proteins as well (Fischer, 1993; Morgan
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Fig. 1. Lysenin expression of coelomocytes using a-EFCC5 mAb. Isolated coelomocytes were stained with a-EFCC5 mAb, discrete granular reactions were observed mainly in the chloragocyte population (arrows, A). Note that not all eleocytes were positive for EFCC5 staining, because numerous negative eleocytes with brownish colored chloragosomes are clearly visible (asterisks) in the coelomocyte samples (A). While some smaller cells with peripherally shifted nucleus (arrowhead, A) were also positive for EFCC5, but large hyaline cells were negative for a-lysenin specific mAb (number sign, A). For double immunofluorescence analysis (B) lysenin positive cells were marked with anti-EFCC5 (arrows), while hyaline coelomocytes stained with FITC conjugated a-EFCC3 (number sign). Flow cytometry revealed the presence of three distinct subpopulations of earthworm coelomocytes (C). After staining with a-EFCC5-FITC antibody (solid line) the strongest positivity appeared in the R3 population compared with FITC conjugated isotype antibody (dashed line) (D). Cryostat cross-sections of earthworm tissues were stained with a-EFCC5 mAb to reveal lysenin expression in the whole organism. EFCC5 positive cells only appeared among free-floating coelomocytes (arrows) in the coelomic cavity (E, H). Some solitary EFCC5 positive coelomocytes were in the close proximity of other tissues (arrows) such the ventral nerve cord (F) and muscle layers (G). Chloragogenous tissue was consistently negative compared with control tissues (I and J). Figure abbreviations: bv, blood vessel; chl, chloragogenous tissue; g, gut; mu, muscle layers; n, nerve cord. Haematoxylin (A, E, G, I) and DAPI (B, F, H, J) were used for nuclear counter staining. Scale bars: 50 lm (B and E), 100 lm (A, F, G, H, I), 200 lm (J).
B. Opper et al. / Developmental and Comparative Immunology 39 (2013) 214–218
et al., 2004). Regarding to their role in immune mechanisms, freefloating chloragocytes are not phagocytic as other coelomocyte subgroups (Engelmann et al., 2005) but they are potential source of antimicrobial molecules (Cooper et al., 2002). Initial research data claim that lysenin is mainly produced by sessile chloragocytes located in the typhlosolis (so called central chloragocytes) revealed by in situ hybridization and immunohistochemistry (Ohta et al., 2000; Sekizawa et al., 1996b). In addition, fetidin (considered yet as a separate antimicrobial molecule) is also mentioned to be produced by coelomocytes; however it is not specified exactly which cell group is the source of this protein (Valembois et al., 1986; Lassegues et al., 1997). Our immunohistochemical and immunofluorescence staining of earthworm tissues evidently marked free-flotating chloragocytes for lysenin expression (Fig. 1E–H and Fig. S3). Lysenin (EFCC5) positive cells appeared in the group of coelomocytes of coelomic cavity (Fig. 1E and H) or solitary coelomocytes bound to nerve cord or muscle layers (Fig. 1F and G). Anti-lysenin pAb marked mainly the free-floating coelomocytes as well (Fig. S3A–D). Applied negative controls of immunohistochemical staining confirmed the observation of lysenin negative sessile chloragocytes (Fig. 1I and J). Sessile chloragocytes forming the chloragogenous tissue were negative for a-EFCC5 staining. This staining pattern was regardless to the applied polyclonal anti-lysenin or a-EFCC5 mAb. Indeed, we cannot rule out the possibility that central chloragocyte population express lysenin only at mRNA level and then free-floating matured chloragocytes are the protein-secreting ‘‘effector’’ cells, however Ohta et al. (2000) demonstrated that central chloragocytes express lysenin at protein levels by immunohistochemistry using polyclonal lysenin antiserum. Another explanation for these discrepancies could be considered by any seasonal or maturational fluctuation of lysenin expression. However till now no information is available about any switch in lysenin production during seasonal changes or maturation in the earthworms.
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to measure the released lysenin content from the supernatant of exposed coelomocytes. Lysenin expression of coelomocytes was significantly elevated after 6 h of S. aureus challenge compared with E. coli treatment (p = 0.0043). We observed non-significant elevation compared with untreated coelomocyte samples (p = 0.058). Moreover, E. coli treated samples had a significantly decreased lysenin expression (p = 0.042) compared with unchallenged coelomocytes (Fig. 2A). In addition, secreted lysenin showed a similar pattern that we noticed in the coelomocyte lysates (Fig. 2B). In the supernatant of S. aureus exposed coelomocytes we measured elevated lysenin concentration compared with unchallenged samples or E. coli treated coelomocytes (p = 0.018, p = 0.012; respectively). We have not observed significant decrease of lysenin expression in E. coli challenged samples compared with control coelomocytes (p = 0.55). Reduction of lysenin expression following E. coli treatment is in agreement with previous observations (Wang et al., 2010). Indeed, our in vitro approach is not comparable with the previously mentioned experiments where whole earthworms were exposed to various bacteria strains (Köhlerová et al., 2004; Wang et al., 2010). However, in these experiments also different approaches were used for bacteria administration (injection of worms and bacteria-spiked soil) that may explain the differences in the experimental read-outs. In our case we chose a not physiological but experimentally well controlled in vitro setup where we determined exactly the coelomocytes–bacteria ratio. In summary, we revised lysenin expression in earthworm coelomocytes and whole tissues, confirming that it is produced rather by free-floating chloragocytes of the coelomic cavity, and not by the sessile central chloragogenous tissue. For the functional point of view, we found that lysenin expression is increased by in vitro
3.3. Gram positive bacteria enhance lysenin production Another debated question is whether lysenin expression is altered by any microbial challenge. Initially, fetidin was considered as an inducible factor upon in vivo bacterial exposure (Lassegues et al., 1989). In contrast to the previous observations, in vivo treatment with E. coli and Bacillus subtilis bacteria strains did not modulate noticeable fluctuation of fetidin mRNA expression in the coelomocytes, however coelomic protein levels were significantly elevated (Köhlerová et al., 2004). In addition, a recent proteomic and transcriptomic approach showed that lysenin expression was decreased after E. coli O157:H7 bacteria strain challenge (Wang et al., 2010). Fluctuation of lysenin expression in general was noted when earthworms were removed from breeding soil and placed into experimental (OECD) soils (Brulle et al., 2011). After the cytochemical and histological localization of lysenin, we aimed to observe the lysenin expression of coelomocytes after bacterial challenge. First, we analyzed that if our anti-EFCC5 mAb recognizes lysenin from coelomocyte lysate and its size is identical with the protein used for immunization. Indeed, anti-EFCC5 mAb recognizes one discrete single band in coelomocyte lysates and its molecular size was identical to the lysenin protein detected parallel by Western blot. In addition, the anti-lysenin pAb recognized a protein band in the lysates similarly to the size of lysenin protein, however applying this antibody we observed much stronger background compared to the reaction of a-EFCC5 mAb (Fig. S3E). Next, coelomocytes were exposed in vitro to heat-inactivated E. coli and S. aureus bacteria strains for 6 h along with untreated controls. Lysenin expression was determined by Western blot from the coelomocyte lysate. Furthermore, sandwich ELISA was applied
Fig. 2. Lysenin expression is modulated by bacterial challenge. Lysenin expression of coelomocytes was measured after in vitro E. coli and S. aureus challenge (A and B). Western blot analysis of coelomocyte lysates (A) revealed that S. aureus treatment increased lysenin expression (⁄⁄p < 0.01, n = 4), while E. coli treatment inhibited lysenin expression (⁄p < 0.05, n = 4). Lysenin protein expression was normalized to a-a-tubulin. Moreover, secretion of lysenin was also observed by sandwich ELISA (B) from the supernatant of exposed coelomocytes. S. aureus treatment of coelomocytes induced significant lysenin secretion (⁄p < 0.05, n = 4). Figure abbreviations: A.U., arbitrary units.
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Gram-positive bacteria challenge which is in contrast with previous findings based on in vivo and mRNA studies, so it needs further investigations concentrating on the lysenin expression at protein level. Acknowledgements We gratefully acknowledge the financial support of Medical Faculty Research Foundation, University of Pécs (PTE-ÁOK-KA 34039/10-06). We thank to Mária Pápa, Márta Szelier for their great assistance and to Mariann Szabó for careful reading the manuscript and helping to improve it with useful advices. Earthworm species identification was performed with the aid of Kinga Futó and József Orbán (Dept. of Biophysics, University of Pécs). We also greatly appreciate the help of Ildikó Somogyi, Dóra Gunszt and László Molnár (Faculty of Sciences, University of Pécs) for maintaining and providing the experimental earthworm specimens. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.dci.2012.11.006. References Bilej, M., De Baetselier, P., Beschin, A., 2000. Antimicrobial defense of the earthworm. Folia Microbiol. 45, 283–300. Bruhn, H., Winkelmann, J., Andersen, C., Andrä, J., Leippe, M., 2006. Dissection of the mechanisms of cytolytic and antibacterial activity of lysenin, a defense protein of the annelid Eisenia fetida. Dev. Comp. Immunol. 30, 597–606. Brulle, F., Lemiére, S., Waterlot, C., Douay, F., Vandenbulcke, F., 2011. Gene expression analysis of 4 biomarker candidates in Eisenia fetida exposed to an environmental metallic trace elements gradient: a microcosm study. Sci. Total Environ. 409, 5470–5482. Cooper, E.L., Kauschke, E., Cossarizza, A., 2002. Digging for innate immunity since Darwin and Metchnikoff. Bioessays 24, 319–333. Cooper, E.L., Roch, P., 2003. Earthworm immunity: a model of immune competence. Pedobiologia 47, 676–688. Engelmann, P., Palinkas, L., Cooper, E.L., Németh, P., 2005. Monoclonal antibodies identify four distinct annelid leukocyte markers. Dev. Comp. Immunol. 29, 599– 614. Engelmann, P., Cooper, E.L., Opper, B., Németh, P., 2011. Earthworm innate immune system. In: Karaca, A. (Ed.), Biology of Earthworms. Springer Verlag, Heidelberg, pp. 229–245.
Fischer, E., 1993. The myelo-erytroid nature of the chloragogenous like tissues of the annelids: a review. Exp. Gerontol. 12, 69–74. Fuller-Espie, S., Nacarelli, T., Blake, E.L., Bearoff, F.M., 2010. The effect of oxidative stress and apoptosis in the earthworm Eisenia hortensis. Invertebr. Surviv. J. 7, 89–106. Jamieson, B.G.M., 1981. Chloragocytes. In: Jamieson, B.G.M. (Ed.), The Ultrastructure of the Oligochaeta. Academic Press, New York, pp. 96–118. Kauschke, E., Mohrig, W., Cooper, E.L., 2007. Coelomic fluid proteins as basic components of innate immunity in earthworms. Eur. J. Soil Biol. 43, S110–S115. Kobayashi, H., Ohta, N., Umeda, M., 2004. Biology of lysenin, a protein in the coelomic fluid of the earthworm Eisenia foetida. Int. Rev. Cytol. 236, 45–99. Köhlerová, P., Beschin, A., Šilerová, M., De Baetselier, P., Bilej, M., 2004. Effect of experimental microbial challenge on the expression of defense molecules in Eisenia foetida earthworms. Dev. Comp. Immunol. 28, 701–711. Lassegues, M., Milochau, A., Doignon, F., Du Pasquier, L., Valembois, P., 1997. Sequence and expression of an Eisenia fetida derived cDNA clone that encodes the 40 kDa fetidin antibacterial protein. Eur. J. Biochem. 246, 756–762. Lassegues, M., Roch, P., Valembois, P., 1989. Antibacterial activity of Eisenia fetida andrei coelomic fluid: specificity of the induced activity. J. Invertebr. Pathol. 54, 28–31. Morgan, A.J., Stürzenbaum, S.R., Winters, C., Grime, G.W., Aziz, N.A., Kille, P., 2004. Differential metallothionein expression in earthworm (Lumbricus rubellus) tissues. Ecotoxicol. Environ. Saf. 57, 11–19. Ohta, N., Shioda, S., Sekizawa, Y., Nakai, Y., Kobayashi, H., 2000. Sites of the expression of mRNA for lysenin a protein isolated from the coelomic fluid of the earthworm Eisenia foetida. Cell Tissue Res. 302, 263–270. Plytycz, B., Homa, J., Koziol, B., Rózanowska, M., Morgan, A.J., 2006. Riboflavin content in autofluorescent earthworm coelomocytes is species-specific. Folia Histochem. Cytobiol. 44, 275–280. Sekizawa, Y., Hagiwara, K., Nakajima, T., Kobayashi, H., 1996a. A novel protein, lysenin that causes contraction of the isolated rat aorta: its purification from the coelomic fluid of the earthworm Eisenia foetida. Biomed. Res. 17, 197–203. Sekizawa, Y., Ohta, N., Natori, S., Kobayashi, H., 1996b. Immunocytochemical localization of lysenin a novel protein isolated from the coelomic fluid of earthworm Eisenia foetida. Biomed. Res. 17, 327–330. Sekizawa, Y., Kubo, T., Kobayashi, H., Nakajima, T., Natori, S., 1997. Molecular cloning of lysenin, a novel protein in the earthworm Eisenia foetida that causes contraction of rat vascular smooth muscle. Gene 191, 97–102. Shogomori, H., Kobayashi, T., 2008. Lysenin: a sphingomyelin specific pore-forming protein. Biochim. Biophys. Acta 1780, 612–618. Valembois, P., Roch, P., Lasségues, M., 1986. Antibacterial molecules in annelids. In: Bréhelin, M. (Ed.), Immunity in Invertebrates. Springer-Verlag, Heidelberg, pp. 74–93. Vernile, P., Fornelli, F., Bari, G., Spagnuolo, M., Minervini, F., de Lillo, E., Ruggiero, P., 2007. Bioavailability and toxicity of pentachlorophenol in contaminated soil evaluated on coelomocytes of Eisenia andrei (Annelida: Lumbricidae). Toxicol. In Vitro 21, 302–327. Wang, X., Chang, L., Sun, Z., Zhang, Y., 2010. Comparative proteomic analysis of differentially expressed proteins in the earthworm Eisenia fetida during Escherichia coli O157:H7 stress. J. Proteome Res. 9, 6547–6560.
Contents lists available at SciVerse ScienceDirect
Developmental and Comparative Immunology journal homepage: www.elsevier.com/locate/dci
Short communication
Revising lysenin expression of earthworm coelomocytes Balázs Opper a,b, András Bognár a, Diána Heidt a, Péter Németh a, Péter Engelmann a,⇑ a b
Department of Immunology and Biotechnology, Clinical Center, University of Pécs, Szigeti u. 12, H-7643 Pécs, Hungary Department of Anatomy, Faculty of Medicine, University of Pécs, Szigeti u. 12, H-7643 Pécs, Hungary
a r t i c l e
i n f o
Article history: Received 23 September 2012 Revised 20 November 2012 Accepted 20 November 2012 Available online 28 November 2012 Keywords: Innate immunity Coelomocytes Antimicrobial activity Monoclonal antibodies
a b s t r a c t Lysenin is a species-specific bioactive molecule of Eisenia andrei earthworms. This protein is a potent antimicrobial factor; however its cellular expression and induction against pathogens are still not fully understood. We developed a novel monoclonal antibody against lysenin and applied this molecular tool to characterize its production and antimicrobial function. We demonstrated by flow cytometry and immunocytochemistry that one subgroup of earthworm immune cells (so called coelomocytes), the chloragocytes expressed the highest amount of lysenin. Then, we compared lysenin expression with earlier established coelomocyte (EFCC) markers. In addition, we determined by immunohistology of earthworm tissues that lysenin production is only restricted to free-floating chloragocytes. Moreover, we observed that upon in vitro Staphylococcus aureus but not Escherichia coli challenged coelomocytes over-expressed and then secreted lysenin. These results indicate that among subpopulations of coelomocytes, lysenin is mainly produced by chloragocytes and its expression can be modulated by Gram-positive bacterial exposure. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Earthworm’s coelomic fluid exerts a wide spectrum of biological functions including immunity against environmental pathogens. Humoral factors of this compartment control the growth of commensal and pathogenic microorganisms. Coelomic fluid has been a subject of intensive research since many years in earthworms (Bilej et al., 2000; Kauschke et al., 2007; Valembois et al., 1986; Wang et al., 2010). The most extensively characterized factors are the unique fetidin/lysenin group of earthworm’s coelomic fluid. First, fetidin is described as a glycoprotein with antibacterial and haemolytic properties (Lassegues et al., 1997). In addition, another bioactive molecule, designated as lysenin, has been cloned (Sekizawa et al., 1997). Lysenin was described primarily as a smooth muscle contraction factor (Sekizawa et al., 1996a). Membrane biological studies proved that lysenin binds specifically to the sphingomyelin compartments of the cell membrane (Shogomori and Kobayashi, 2008). Initially, lysenin and fetidin were considered as separate molecules exhibiting similar molecular weights (Cooper and Roch, 2003). However, recently it is known that both lysenin and fetidin
Abbreviations: EFCC, Eisenia coelomocyte clusters; LBSS, Lumbricus balanced salt solution. ⇑ Corresponding author. Tel.: +36 72 536 288; fax: +36 72 536 289. E-mail address: [email protected] (P. Engelmann). 0145-305X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.dci.2012.11.006
belong to the same multimolecular family, that consist of four members such as lysenin, lysenin related protein 1, lysenin related protein 2 (fetidin), and lysenin related 3 (Bruhn et al., 2006). Lysenin/fetidin molecules were found abundantly in the coelomic fluid; however their exact production site and function in earthworms remained elusive. Chloragogenous tissue covers the gut of earthworms. Chloragocytes, thus forming this tissue, are considered as a functional homologue cell type for vertebrate hepatocytes (Jamieson, 1981). Chloragocytes can be separated into two distinctly localized cell populations such as the peripheral chloragocytes found on the gut wall, while the other one termed as central chloragocytes settled inside of the typhlosolis (Ohta et al., 2000). It is suggested that lysenin expression is coupled mainly with the central chloragocytes demonstrated by in situ hybridization and polyclonal antibody-based immunohistochemistry (Sekizawa et al., 1996b; Ohta et al., 2000). Previously we have developed a group of monoclonal antibodies (mAbs) against earthworm coelomocytes and with the aid of these molecular tools we characterized three subpopulations of freefloating coelomocytes (Engelmann et al., 2005). Furthermore, recently we paid more attention to characterize the functional status of coelomocytes (Engelmann et al., 2011). For this purpose we have developed a mAb against lysenin to characterize its cytotoxic nature. In addition, we aimed to revise lysenin expression profile in free-floating coelomocytes, its tissue localization and expression patterns upon bacterial challenge.
B. Opper et al. / Developmental and Comparative Immunology 39 (2013) 214–218
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2. Materials and methods
2.6. Statistical analysis
2.1. Earthworm culture Eisenia andrei earthworm species were maintained at room temperature and fed with manure complemented soil. Prior to coelomocyte isolations earthworms were placed onto moist tissue paper for depuration.
Statistical analysis was performed with Microcal Origin (Microcal Software Inc., Northhampton, MA USA). The results in the figures are representative values from four independent experiments. All results are presented as mean with standard error shown as error bar. The effect of treatments was analyzed by one way-ANOVA. p < 0.05 was denoted as statistically significant.
2.2. Preparation of mouse anti-lysenin monoclonal antibody
3. Results and discussion
Lysenin (PeptaNova GmbH, Sandhausen, Germany) were dissolved in distilled water at 1 mg/ml concentrations and used for immunizing 8 weeks old female BALB/c mice. Mice were injected with lysenin protein (10 lg) mixed in complete Freund’s adjuvant (CFA, Sigma–Aldrich Co., St. Louis, MO, USA) into each hind footpad followed with two intraperitoneal (i.p.) boosts in incomplete Freund’s adjuvant (IFA, Sigma) 3 weeks intervals. Finally, one booster was injected in mice selected, according to its antibody titer and isotype, 3 days prior to splenocyte/myeloma-cell fusion. Hybridomas were developed as we described earlier (Engelmann et al., 2005). Hybridoma supernatants were screened by enzyme linked immunosorbent assay (indirect ELISA, for technical details please see the Supplementary Material) using lysenin, coelomocyte lysate as target antigen, and bovine serum albumin (BSA, Sigma) as control antigen. Selected hybridomas were cloned three times by limiting dilution.
3.1. Lysenin expression of isolated coelomocytes
2.3. Extrusion of coelomocytes Coelomocytes were isolated as we described earlier (Engelmann et al., 2005) and living cell numbers were evaluated by trypan-blue exclusion method. Cell viability was above 95%. Coelomocytes of individual worms were analyzed further by immunocytochemistry, immunofluorescence, and flow cytometry. For technical details please see the SM.
2.4. In vitro bacterial challenge Isolated, pooled coelomocytes (6 106/ml) were incubated with heat-inactivated Staphylococcus aureus (OKI II2001) and Escherichia coli (ATCC 25922) at room temperature by end-over-end rotation for 6 h (Bacterial strains were obtained from Dr. Béla Kocsis MD, PhD, Department of Medical Microbiology and Immunology, Clinical Center, University of Pécs). Coelomocytes (106) and bacteria (107) were mixed in 1 ml final volume in 12 75 mm tubes (Falcon, BD Labware). Following phagocytosis, coelomocyte samples were washed in LBSS, centrifuged at 1000 rpm for 5 min (Engelmann et al., 2005). Then supernatant was kept at 80 °C for further analysis and the pellet was applied for the preparation of coelomocyte lysates.
2.5. Preparation of coelomocyte lysate Coelomocytes were lysed in RIPA buffer (50 mM Tris/HCl; pH 8.0, 150 mM NaCl, 1% (v/v) NP-40, 0.5% (w/v) Na-deoxycholate, 5 mM EDTA, 0.1% SDS) complemented with Protease Inhibitor Cocktail (Sigma) on ice for 15 min and then centrifuged by 13,000 rpm (15 min, 4 °C). Total protein concentrations of the coelomocyte lysates and supernatants were measured with BCA Reagent Kit (Pierce, Rockford, IL, USA). Immunoreactivity of supernatants and coelomocyte lysates were analyzed by ELISA and Western blot assays (further technical details can be obtained in SM).
Coelomocytes are multi-tasking cellular mediators of immune response in earthworms. In addition to their cellular immune functions (e.g. phagocytosis, encapsulation) they participate in the humoral immune mechanisms by producing haemolytical and antimicrobial factors (Bilej et al., 2000; Cooper et al., 2002). Most of these proteins belong to the fetidin/lysenin multiprotein family characterized mainly by membrane biologists. However, the exact role of lysenin in the immune response of earthworms is not so well understood (Kobayashi et al., 2004). Previously, we observed the cytotoxicity mediated by earthworm coelomocytes produced factors (Engelmann et al., 2011). Now we intend to uncover more details about the cytotoxic mechanisms (Macsik et al., in preparation). For this purpose we raised a monoclonal antibody against lysenin (a-EFCC5) and first we initiated to characterize its expression profile in coelomocytes. Antibody positive cells have large granular cytoplasm and relatively small nucleus revealed by immunocytochemistry (Fig. 1A). Immune positive reaction is localized mainly in the intracellular granules. This morphology marks one characteristic subgroup of coelomocytes, the free-floating chloragocytes (sometimes denoted as eleocytes). Other cell types with larger nucleus and smaller cytoplasm (effector coelomocytes, presumably hyaline amoebocytes) were negative for a-EFCC5 staining (Fig. 1A). Using the previously generated mAbs against coelomocyte subgroups we revealed co-expression with lysenin positive coelomocytes (Fig. S1). A-EFCC3 positive coelomocytes are tend to attach together and form aggregates, while EFCC5 positive cells are mainly solitary (Fig. 1B, Figs. S1 and S2). By means of flow cytometry, circulating coelomocytes of the body cavity has several subgroups (Engelmann et al., 2005; Fuller-Espie et al., 2010; Vernile et al., 2007). In our experiments, we could identify three physically distinct subpopulations of coelomocytes denoted as R1, R2, and R3 (Fig. 1C). R1 and R2 populations resemble effector coelomocytes such as hyaline and granular coelomocytes, while R3 subgroup is the highly granular and autofluorescent eleocyte population (Plytycz et al., 2006) (Fig. 1C). EFCC5 positivity could be observed in all three subgroups with various intensities compared with appropriate isotype control antibodies (Fig. 1D). R3 group majorly consisting eleocytes had the highest percentage (39.5 ± 10.2%) of EFCC5 antibody positive cells (Fig. 1D and Table S1). 3.2. Solitary free-floating chloragocytes are restricted for lysenin expression Chloragocytes are usually very abundant, highly granular coelomocyte subpopulation of the coelomic cavity. Their origin is a matter of debate, however most authors agreed that free-floating chloragocytes derived from the chloragogenous tissue of the gut (Jamieson, 1981). These cells are harboring nutrient factors such as glycogens, lipids and participate mainly in general metabolism. Moreover, they are able to produce haemoglobin and metalsequestering cysteine-rich proteins as well (Fischer, 1993; Morgan
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Fig. 1. Lysenin expression of coelomocytes using a-EFCC5 mAb. Isolated coelomocytes were stained with a-EFCC5 mAb, discrete granular reactions were observed mainly in the chloragocyte population (arrows, A). Note that not all eleocytes were positive for EFCC5 staining, because numerous negative eleocytes with brownish colored chloragosomes are clearly visible (asterisks) in the coelomocyte samples (A). While some smaller cells with peripherally shifted nucleus (arrowhead, A) were also positive for EFCC5, but large hyaline cells were negative for a-lysenin specific mAb (number sign, A). For double immunofluorescence analysis (B) lysenin positive cells were marked with anti-EFCC5 (arrows), while hyaline coelomocytes stained with FITC conjugated a-EFCC3 (number sign). Flow cytometry revealed the presence of three distinct subpopulations of earthworm coelomocytes (C). After staining with a-EFCC5-FITC antibody (solid line) the strongest positivity appeared in the R3 population compared with FITC conjugated isotype antibody (dashed line) (D). Cryostat cross-sections of earthworm tissues were stained with a-EFCC5 mAb to reveal lysenin expression in the whole organism. EFCC5 positive cells only appeared among free-floating coelomocytes (arrows) in the coelomic cavity (E, H). Some solitary EFCC5 positive coelomocytes were in the close proximity of other tissues (arrows) such the ventral nerve cord (F) and muscle layers (G). Chloragogenous tissue was consistently negative compared with control tissues (I and J). Figure abbreviations: bv, blood vessel; chl, chloragogenous tissue; g, gut; mu, muscle layers; n, nerve cord. Haematoxylin (A, E, G, I) and DAPI (B, F, H, J) were used for nuclear counter staining. Scale bars: 50 lm (B and E), 100 lm (A, F, G, H, I), 200 lm (J).
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et al., 2004). Regarding to their role in immune mechanisms, freefloating chloragocytes are not phagocytic as other coelomocyte subgroups (Engelmann et al., 2005) but they are potential source of antimicrobial molecules (Cooper et al., 2002). Initial research data claim that lysenin is mainly produced by sessile chloragocytes located in the typhlosolis (so called central chloragocytes) revealed by in situ hybridization and immunohistochemistry (Ohta et al., 2000; Sekizawa et al., 1996b). In addition, fetidin (considered yet as a separate antimicrobial molecule) is also mentioned to be produced by coelomocytes; however it is not specified exactly which cell group is the source of this protein (Valembois et al., 1986; Lassegues et al., 1997). Our immunohistochemical and immunofluorescence staining of earthworm tissues evidently marked free-flotating chloragocytes for lysenin expression (Fig. 1E–H and Fig. S3). Lysenin (EFCC5) positive cells appeared in the group of coelomocytes of coelomic cavity (Fig. 1E and H) or solitary coelomocytes bound to nerve cord or muscle layers (Fig. 1F and G). Anti-lysenin pAb marked mainly the free-floating coelomocytes as well (Fig. S3A–D). Applied negative controls of immunohistochemical staining confirmed the observation of lysenin negative sessile chloragocytes (Fig. 1I and J). Sessile chloragocytes forming the chloragogenous tissue were negative for a-EFCC5 staining. This staining pattern was regardless to the applied polyclonal anti-lysenin or a-EFCC5 mAb. Indeed, we cannot rule out the possibility that central chloragocyte population express lysenin only at mRNA level and then free-floating matured chloragocytes are the protein-secreting ‘‘effector’’ cells, however Ohta et al. (2000) demonstrated that central chloragocytes express lysenin at protein levels by immunohistochemistry using polyclonal lysenin antiserum. Another explanation for these discrepancies could be considered by any seasonal or maturational fluctuation of lysenin expression. However till now no information is available about any switch in lysenin production during seasonal changes or maturation in the earthworms.
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to measure the released lysenin content from the supernatant of exposed coelomocytes. Lysenin expression of coelomocytes was significantly elevated after 6 h of S. aureus challenge compared with E. coli treatment (p = 0.0043). We observed non-significant elevation compared with untreated coelomocyte samples (p = 0.058). Moreover, E. coli treated samples had a significantly decreased lysenin expression (p = 0.042) compared with unchallenged coelomocytes (Fig. 2A). In addition, secreted lysenin showed a similar pattern that we noticed in the coelomocyte lysates (Fig. 2B). In the supernatant of S. aureus exposed coelomocytes we measured elevated lysenin concentration compared with unchallenged samples or E. coli treated coelomocytes (p = 0.018, p = 0.012; respectively). We have not observed significant decrease of lysenin expression in E. coli challenged samples compared with control coelomocytes (p = 0.55). Reduction of lysenin expression following E. coli treatment is in agreement with previous observations (Wang et al., 2010). Indeed, our in vitro approach is not comparable with the previously mentioned experiments where whole earthworms were exposed to various bacteria strains (Köhlerová et al., 2004; Wang et al., 2010). However, in these experiments also different approaches were used for bacteria administration (injection of worms and bacteria-spiked soil) that may explain the differences in the experimental read-outs. In our case we chose a not physiological but experimentally well controlled in vitro setup where we determined exactly the coelomocytes–bacteria ratio. In summary, we revised lysenin expression in earthworm coelomocytes and whole tissues, confirming that it is produced rather by free-floating chloragocytes of the coelomic cavity, and not by the sessile central chloragogenous tissue. For the functional point of view, we found that lysenin expression is increased by in vitro
3.3. Gram positive bacteria enhance lysenin production Another debated question is whether lysenin expression is altered by any microbial challenge. Initially, fetidin was considered as an inducible factor upon in vivo bacterial exposure (Lassegues et al., 1989). In contrast to the previous observations, in vivo treatment with E. coli and Bacillus subtilis bacteria strains did not modulate noticeable fluctuation of fetidin mRNA expression in the coelomocytes, however coelomic protein levels were significantly elevated (Köhlerová et al., 2004). In addition, a recent proteomic and transcriptomic approach showed that lysenin expression was decreased after E. coli O157:H7 bacteria strain challenge (Wang et al., 2010). Fluctuation of lysenin expression in general was noted when earthworms were removed from breeding soil and placed into experimental (OECD) soils (Brulle et al., 2011). After the cytochemical and histological localization of lysenin, we aimed to observe the lysenin expression of coelomocytes after bacterial challenge. First, we analyzed that if our anti-EFCC5 mAb recognizes lysenin from coelomocyte lysate and its size is identical with the protein used for immunization. Indeed, anti-EFCC5 mAb recognizes one discrete single band in coelomocyte lysates and its molecular size was identical to the lysenin protein detected parallel by Western blot. In addition, the anti-lysenin pAb recognized a protein band in the lysates similarly to the size of lysenin protein, however applying this antibody we observed much stronger background compared to the reaction of a-EFCC5 mAb (Fig. S3E). Next, coelomocytes were exposed in vitro to heat-inactivated E. coli and S. aureus bacteria strains for 6 h along with untreated controls. Lysenin expression was determined by Western blot from the coelomocyte lysate. Furthermore, sandwich ELISA was applied
Fig. 2. Lysenin expression is modulated by bacterial challenge. Lysenin expression of coelomocytes was measured after in vitro E. coli and S. aureus challenge (A and B). Western blot analysis of coelomocyte lysates (A) revealed that S. aureus treatment increased lysenin expression (⁄⁄p < 0.01, n = 4), while E. coli treatment inhibited lysenin expression (⁄p < 0.05, n = 4). Lysenin protein expression was normalized to a-a-tubulin. Moreover, secretion of lysenin was also observed by sandwich ELISA (B) from the supernatant of exposed coelomocytes. S. aureus treatment of coelomocytes induced significant lysenin secretion (⁄p < 0.05, n = 4). Figure abbreviations: A.U., arbitrary units.
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Gram-positive bacteria challenge which is in contrast with previous findings based on in vivo and mRNA studies, so it needs further investigations concentrating on the lysenin expression at protein level. Acknowledgements We gratefully acknowledge the financial support of Medical Faculty Research Foundation, University of Pécs (PTE-ÁOK-KA 34039/10-06). We thank to Mária Pápa, Márta Szelier for their great assistance and to Mariann Szabó for careful reading the manuscript and helping to improve it with useful advices. Earthworm species identification was performed with the aid of Kinga Futó and József Orbán (Dept. of Biophysics, University of Pécs). We also greatly appreciate the help of Ildikó Somogyi, Dóra Gunszt and László Molnár (Faculty of Sciences, University of Pécs) for maintaining and providing the experimental earthworm specimens. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.dci.2012.11.006. References Bilej, M., De Baetselier, P., Beschin, A., 2000. Antimicrobial defense of the earthworm. Folia Microbiol. 45, 283–300. Bruhn, H., Winkelmann, J., Andersen, C., Andrä, J., Leippe, M., 2006. Dissection of the mechanisms of cytolytic and antibacterial activity of lysenin, a defense protein of the annelid Eisenia fetida. Dev. Comp. Immunol. 30, 597–606. Brulle, F., Lemiére, S., Waterlot, C., Douay, F., Vandenbulcke, F., 2011. Gene expression analysis of 4 biomarker candidates in Eisenia fetida exposed to an environmental metallic trace elements gradient: a microcosm study. Sci. Total Environ. 409, 5470–5482. Cooper, E.L., Kauschke, E., Cossarizza, A., 2002. Digging for innate immunity since Darwin and Metchnikoff. Bioessays 24, 319–333. Cooper, E.L., Roch, P., 2003. Earthworm immunity: a model of immune competence. Pedobiologia 47, 676–688. Engelmann, P., Palinkas, L., Cooper, E.L., Németh, P., 2005. Monoclonal antibodies identify four distinct annelid leukocyte markers. Dev. Comp. Immunol. 29, 599– 614. Engelmann, P., Cooper, E.L., Opper, B., Németh, P., 2011. Earthworm innate immune system. In: Karaca, A. (Ed.), Biology of Earthworms. Springer Verlag, Heidelberg, pp. 229–245.
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