Folia Microbiol. 49 (3), 315–319 (2004)
http://www.biomed.cas.cz/mbu/folia/
Silkworm (Bombyx mori) Hemocytes Do Not Produce Reactive Oxygen Metabolites As a Part of Defense Mechanisms P. HYRŠLa, M. ÍŽb, L. KUBALAb, A. LOJEKb* aDepartment of Comparative Animal Physiology and General Zoology, Faculty of Science, Masaryk University, 611 37 Brno, Czechia
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[email protected] bInstitute of Biophysics, Academy of Sciences of the Czech Republic, 612 65 Brno, Czechia
Received 6 October 2003 Revised version 22 December 2003
ABSTRACT. To investigate whether hemocytes of Bombyx mori (Lepidoptera) larvae produce reactive oxygen species (ROS) as part of the oxidative killing of invading pathogens, the production of ROS was measured as a luminol- and lucigenin-enhanced chemiluminescence of unstimulated or stimulated (zymosan particles, phorbol myristate acetate, calcium ionophore, rice starch or Xenorhabdus nematophila) hemolymph. No detectable ROS production was found. The spontaneous and activated ROS production measured with hemocytes, i.e. under the conditions when the antioxidative potential of hemolymph plasma was eliminated, was again undetectable. Likewise, ROS production by isolated hemocytes was observed by spectrophotometric (NBT test, cytochrome c assay) and fluorimetric (using dihydrorhodamine and hydroethidine probes) methods. Hence none of the experimental approaches used indicated the production of ROS by hemocytes of B. mori larvae as part of their immune response.
Hemocytes are basic to the invertebrate innate immune system that is divided into cellular and humoral defense responses. The most common types of hemocytes reported in the literature are prohemocytes, plasmatocytes, granulocytes, spherulocytes and enocytes (Yamashita and Iwabuchi 2001; Lavine and Strand 2002). These have been identified by their morphology and histochemical and functional reactions (e.g., Gardiner and Strand 1999). Four basic types of hemocyte immune reactions have been described: phagocytosis, encapsulation, nodulation and coagulation. These activities are always connected with a particular type of hemocytes. During phagocytosis, plasmatocytes and granulocytes are mainly activated while other types of hemocytes have mostly no possibility to phagocytose. Mechanisms participating in the recognition of foreign material are still under study (Lavine and Strand 2002). The production of reactive oxygen species (ROS) seems to be an important microbicidal factor in both invertebrate hemocytes and vertebrate phagocytes since an increase in ROS production by activated hemocytes of some invertebrates such as Bivalvia, Clitellata, Malacostraca, Arachnida, Echinoidea or Ascidiacea has been reported (e.g., Nakamura et al. 1985; Ito et al. 1992; Bell and Smith 1993; Valembois and Lassegues 1995; Lambert and Nicolas 1998; Ordas et al. 2000; Pereira et al. 2001; Azumi et al. 2002). However, reports about similar mechanisms in insects are controversial. The aim of the present study was to investigate whether stimulated hemocytes of Bombyx mori can produce ROS as part of oxidative killing invading pathogens. Understanding of the defense mechanisms of B. mori has a high significance for providing a better insight into the evolution of animal immune systems; it also has a great impact on silk production sericulture. MATERIALS AND METHODS Sample preparation. The hemolymph of the Japan polyvoltinne NO2 × CO2 hybrid of the silkworm, Bombyx mori LINNÉ 1758 (Lepidoptera, Bombycidae), was obtained from larvae of the 5th instar. Larvae were reared on mulberry leaves (Morus alba) ad libitum. The sex was not determined. After the collection of the hemolymph from the first-pair proleg (approximately 150 μL per larva), phenylthiourea was added to protect the hemolymph from melanization. (It was verified that this agent does not have a significant effect *
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on the parameters studied.) To obtain isolated hemocytes, hemolymph was centrifuged (200 g, 10 min) and washed twice with saline (15 mmol/L NaCl). Hemocyte counts were then determined microscopically. Hemocytes were resuspended in the saline with ions (15 mmol/L NaCl + 0.4 mmol/L MgSO4·7H2O + 0.5 mmol/L CaCl2·2H2O). Hemocyte viability (as assessed by eosin exclusion test) in collected hemolymph was ≥98 %, after isolation of hemocytes it was ≥84 %. The ability of hemocytes to phagocytose was unchanged after the sample preparation, as verified microscopically using inert 1-μm microspheric hydrophilic particles (MSHP kit; Artim, Czechia). Heparinized (sodium heparin, 50 U/mL) human blood was obtained from the cubital vein of healthy volunteers after overnight fasting. Human blood phagocytes were isolated according to íž and Lojek (1997). Hemocyte activators. Zymosan particles (ZP; Zymosan A from Saccharomyces cerevisiae, Sigma, USA; final concentration of 0.25 mg/mL reaction mixture), phorbol myristate acetate (PMA; Sigma, USA; 0.81 μmol/L), calcium ionophore (Ca-I A23187; Sigma, USA; 9.55 μmol/L), rice starch (0.1 % amylum oryzae in saline; Lachema, Czechia) or Xenorhabdus nematophila CCM 7081 (50 μmol/L bacterial suspension resuspended in saline to obtain cell concentration of 240/nL, i.e. 2.4 × 108 cells per mL) were used as activators of ROS production. Chemiluminescence (CL) assay. The modification of the method of Lojek et al. (2002) was used. Briefly, the hemolymph (0.3, 3, 30, 50 μL) and one of the activators of phagocytic cells were mixed with luminol or lucigenin dissolved in borate buffer (pH = 9) (both luminophores Sigma, USA). The final concentration of the luminophores was always 1 mmol/L. The total volume in the cuvettes was adjusted to 500 μL using saline. Chemiluminescence was measured for 1 h using Luminometer 1251 (Bio-Orbit, Finland) at 20, 25 or 37 °C. Results are expressed in mV. The same experiments were performed with isolated hemocytes (0.1, 0.5, 1.0 or 1.5 × 106 per measuring cuvette). All measurements were repeated six times. The generation of ROS by phagocytes isolated from human whole blood (106 cells per measuring cuvette, 37 °C) activated with bacteria or PMA was used as a positive control. Spectrophotometry. Nitroblue tetrazolium (NBT) test. After stimulation with PMA, ZP or the bacterium Xenorhabdus nematophila, NBT (Fluka, Germany; 0.2 mg/mL) was added to react with ROS produced by activated hemocytes or human blood phagocytes (1.5 × 106 cells). Cells were then lysed using Triton X-100 (Fluka, Germany) in 0.1 mol/L HCl (1 : 9) and absorbance was measured using ELISA-reader Rainbow (Tecan, Austria) at 570 nm. Reduction of cytochrome c. Cytochrome c (Sigma, USA) was added to a final concentration of 10 mg/mL to PMA or ZP activated hemocytes (1.5 × 106 cells). Absorbance was measured by Elisa-reader Rainbow at 550 nm (Benov et al. 1998). Fluorimetry. ROS were also detected using dihydrorhodamine-123 (DHR-123; Molecular Probes, USA; 1 μmol/L) or hydroethidine (HE; Molecular Probes; 1 μmol/L) after PMA or ZP stimulation of hemocytes according to Vowells et al. (1995) and Rothe and Valet (1990) with modifications. Hemocytes (1.5 × 106 cells) were incubated with DHR-123 (10 μmol/L) or HE (10 μmol/L) in microwell plates for 15 min. The activators PMA or ZP were then added for 30 min and the resulting fluorescence was measured using Fluorostar Galaxy fluorimeter (BMG Labtechnologies, Germany) at an excitation wavelength of 485 nm and emission wavelengths 520 nm for DHR-123 and 612 nm for HE. Total radical-trapping antioxidative potential (TRAP) assay. The TRAP measurement was done according to Slavíková et al. (1998). Peroxyl radicals produced at a constant rate by thermal decomposition of 2,2´-azo-bis-2-amidinopropane hydrochloride (ABAP; Polyscience, USA) were monitored by luminolenhanced CL. The reaction was initiated by mixing 490 μL of phosphate-buffered saline, 50 μL of 10 mmol/L luminol in 100 mmol/L borate buffer (pH 10) and 50 μL of ABAP. This mixture was incubated (37 °C) in the temperature-controlled sample carousel of Luminometer 1251 (Bio-Orbit, Finland) for 10 min. During this period a steady state of the CL signal was reached. Then 10 μL of the sample was added directly to the cuvette, and the sample was measured for further time interval (τ ) until 50 % recovery of the original steady state CL signal was achieved. 6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox; 8 nmol/L; Aldrich Chemicals, USA), a water-soluble analog of tocopherol, was used as a reference inhibitor instead of the sample. The stoichiometric factor of trolox (the number of peroxyl radicals trapped per added molecule of antioxidant) is 2. The TRAP value for each sample measured was obtained from the equation: TRAP = 2 ctrolox τsample / Vsample τtrolox where c is concentration, V is volume, and τ is time interval. The results are expressed as nmol of peroxyl radicals trapped by 1 mL of sample.
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RESULTS Hemolymph chemiluminescence measured by luminol-enhanced CL showed that the spontaneous generation of ROS by unstimulated hemolymph remained at the background level. Significant changes in ROS production were not observed even after stimulation with the different activators PMA, ZP, Ca-I, rice starch or X. nematophila. There were no changes in background CL activity when different volumes of hemolymph at various temperatures (20, 25, 37 °C) were analyzed. The same results were obtained when lucigenin was used as luminophore. Testing of the TRAP of the hemolymph showed that it is significantly higher in comparison with human blood plasma (5.97 ± 2.26 μmol/mL and 1.00 ± 0.13 μmol/mL, respectively). The CL reactions for hemolymph, human blood plasma and the reference antioxidant trolox are shown in Fig. 1. To eliminate the influence of the antioxidative potential of hemolymph plasma on the analyses of ROS production, isolated hemocytes were used in subsequent experiments.
Fig. 1. TRAP of Bombyx mori hemolymph in comparison with human blood plasma shown as a CL (mV) reaction kinetics; ABAP was used as a source of peroxyl radicals and trolox as a reference antioxidant; diamonds – human plasma, squares – ABAP, triangles – trolox, circles – hemolymph.
Chemiluminescence analysis of isolated hemocytes. Spontaneous as well as activated generation of ROS by hemocytes remained at the background level in previous experiments with whole hemolymph. Luminol-enhanced CL was not changed even when horseradish peroxidase (HRP; EC 1.11.1.7, Sigma, USA; 10 U/mL of hemocyte suspension) was used to enhance the CL reaction. The same results were obtained when lucigenin was used. To validate the CL method, leukocytes isolated from human peripheral blood were used as a positive control. While spontaneous CL of human phagocytes also remained at the background level, activated CL significantly increased indicating ROS production. The comparison of CL activity of human phagocytes and hemocytes of B. mori activated with PMA or X. nematophila is shown in Fig. 2.
Fig. 2. Kinetics of ROS production by hemocytes of Bombyx mori or human blood phagocytes measured as chemiluminescence (mV); luminol was used as a luminophore and PMA or bacterium (Xenorhabdus nematophila) as activators; squares – human blood phagocytes–bacteria, triangles – human blood phagocytes–PMA, circles – silkworm hemocytes–bacteria or PMA.
Spectrophotometry confirmed the results obtained by the chemiluminescence technique. No superoxide anion production by hemocytes activated by PMA, ZP or X. nematophila was observed using a NBT test or the test based on the reduction of cytochrome c (the values were below the detection limit). Similar results were obtained by fluorimetry; DHR-123 and HE assays (specific for hydrogen peroxide and superoxide anion, respectively) proved no ROS production in unstimulated as well as in activated (PMA, ZP, Ca-I, rice starch) hemocytes of B. mori.
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DISCUSSION Various types of soluble and particulate activators with different modes of action were used. X. nematophila was used as one of the particulate activators, because it is highly pathogenic to insects (a 50 % insect mortality has been reported after direct infection with fewer than 20 bacteria per larva; Akhurst and Dunphy 1993). ZP and rice starch were used as other nonspecific particulate ligands of surface receptors. On the other hand, both PMA (the structural analog of diacylglycerol) and Ca-I (which facilitates the movement of calcium across cellular membranes) by-pass cell membranes without receptor binding and they activate protein kinase C with consecutive phosphorylation of endogenous proteins and activation of NADPH dehydrogenase (EC 1.6.99.1), the key enzyme of an oxidative burst (Drábiková et al. 2000). ROS generated during the oxidative burst (mainly superoxide anion, hydroxyl radical and hydrogen peroxide) damage important biological macromolecules (lipids, proteins and nucleic acids) of the invading pathogen. Moreover, ROS produced by phagocytes can react with nitric oxide, which results in the production of other reactive metabolites such as peroxynitrite. These reactions are well understood in vertebrates (e.g., Marnila et al. 1995; Kubala et al. 1996; Lojek et al. 2002) but there is a growing body of evidence to suggest that ROS are also of importance in the immunity of invertebrates including Bivalvia (e.g., Nakamura et al. 1985; Lambert and Nicolas 1998; Ordas et al. 2000), Clitellata (Valembois and Lassegues 1995), Malacostraca (Bell and Smith 1993), Arachnida (Pereira et al. 2001), Echinoidea (Ito et al. 1992) or Ascidiacea (Azumi et al. 2002). Nevertheless, the role of NADPH dehydrogenase and ROS in the immune reaction of insects remains controversial (Whitten and Ratcliffe 1999). Using several experimental approaches (luminometry, spectrophotometry, fluorimetry), we found no increase in ROS production in the hemolymph of B. mori. Our results correspond well with the data of Mazet et al. (1994) who were unable to detect ROS production by CL in the washed hemocytes of Spodoptera exigua (Lepidoptera) after exposing them to opsonized bacterial and fungal preparations and PMA. Research conducted by Anderson et al. (1973) showed that the hemocytes of Blaberus craniifer (Blattodea) did not reduce NBT in response to zymosan. In contrast, Arakawa (1994, 1995a,b) found considerable superoxide generation in hemolymph supernatants from larvae of Pseudaletia separata (Lepidoptera) – whole hemolymph, however, was not assayed. Whitten and Ratcliffe (1999) provided evidence for the existence of an immune response resembling the respiratory burst in the hemolymph and hemocytes of the cockroach Blaberus discoidalis (Blattodea). Our NBT test results showing the lack of ROS activity differ from those of Glupov et al. (2001) who observed ROS activity in Galleria mellonella (Lepidoptera) hemocytes. The high antioxidant potential of some hemolymph plasma constituents could be the reason for the absence of any CL reaction in the whole hemolymph. This is why the TRAP of hemolymph we tested. In fact, the TRAP of B. mori hemolymph was very high. It could be caused by the mulberry (Morus alba) leaves (the only food of the B. mori larvae). It was reported that mulberry leaves contain antioxidant components such as β-caroten and α-tocopherol (Yen et al. 1996). An extremely high TRAP can destroy ROS at the moment they arise. The production of ROS was therefore measured in isolated hemocytes freed of endogenous antioxidants but also in this type of experiments the ROS production was not observed. None of the several experimental approaches used in our study demonstrated any production of reactive oxygen metabolites by B. mori hemocytes as part of their immune response. This study was conducted as part of the research project Z 500 4920 and was supported by grant no. S 500 4009 of the Grant Agency of the Academy of Sciences of the Czech Republic. The authors thank Dr. K. Souek for his expert help with fluorimetric determinations. REFERENCES AKHURST R.J., DUNPHY G.B.: Tripartite interaction between symbiotically associated entomopathogenic bacteria, nematodes, and their insect hosts, pp. 1–23 in N E. Beckage, S.N. Thompson, B. Federici (Eds): Parasites and Pathogens of Insects, Vol. 2. Academic Press, New York 1993. ANDERSON R.S., HOLMES B., GOOD R.A.: Comparative biochemistry of phagocytosing insect hemocytes. Comp.Biochem.Physiol. 46B, 595–602 (1973). ARAKAWA T.: Superoxide generation in vitro in lepidopteran larval hemolymph. J.Insect Physiol. 40, 165–171 (1994). ARAKAWA T.: Superoxide generative reaction in insect hemolymph and its mimic model system with surfactants in vitro. Insect Biochem.Mol.Biol. 25, 247–253 (1995a). ARAKAWA T.: Possible involment of an enzymatic system for superoxide generation in lepidopteran larval hemolymph. Arch.Insect Biochem.Physiol. 29, 281–291 (1995b). AZUMI K., KURIBAYASHI F., KANEGASAKI S., YOKOSAWA H.: Zymosan induces production of superoxide anions by hemocytes of the solitary ascidian Halocynthia roretzi. Comp.Biochem.Physiol. 133, 567–574 (2002).
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BELL K.L., SMITH V.J.: In vitro superoxide production by hyaline cells of the shore crab Carcinus maenas (L.). Dev.Comp.Immunol. 17, 211–219 (1993). BENOV L., SZTEJNBERG L., FRIDOVICH I.: Critical evaluation of the use of hydroethidine as a measure of superoxide anion radical. Free Rad.Biol.Med. 25, 826–831 (1998). ÍŽ M., LOJEK A.: Improved dextran preparation of human leukocytes for chemiluminescence analysis of the oxidative burst of polymorphonuclear cells. Clin.Lab.Haematol. 19, 49–51 (1997). DRÁBIKOVÁ K., JANINOVÁ V., NOSÁL R., DANIHELOVÁ E.: Human blood platelets, PMN leukocytes and their interactions in vitro. Responses to selective and non-selective stimuli. Gen.Physiol.Biophys. 19, 393–404 (2000). GARDINER E.M.M., STRAND M.R.: Monoclonal antibodies bind distinct classes of hemocytes in the moth Pseudoplusia includens. J.Insect Physiol. 45, 113–126 (1999). GLUPOV V.V., KHVOSHEVSKAYA M.F., LOZINSKAYA Y.L., DUBOVSKI I.M., MARTEMYANOV V.V., SOKOLOVA J.Y.: Application of the nitroblue tetrazolium-reduction method for studies on the production of reactive oxygen species in insect hemocytes. Cytobios 106, 165–178 (2001). ITO T., MATSUTANI T., MORI K., NOMURA T.: Phagocytosis and hydrogen peroxide production by phagocytes of the sea urchin Strongylocentrotus nudus. Dev.Comp.Immunol. 16, 287–294 (1992). KUBALA L., LOJEK A., ÍŽ M., VONDRÁEK J., DUŠKOVÁ M., SLAVÍKOVÁ H.: Determination of phagocyte activity in whole blood of carp (Cyprinus carpio) by luminol-enhanced chemiluminescence. Vet.Med.Czech 41, 323–327 (1996). LAMBERT C., NICOLAS J.L.: Specific inhibition of chemiluminescent activity by pathogenic vibrios in hemocytes of two marine bivalves: Pecten maximus and Crassostrea gigas. J.Invertebr.Pathol. 71, 53–63 (1998). LAVINE M.D., STRAND M.R.: Insect hemocytes and their role in immunity. Insect Biochem.Mol.Biol. 32, 1295–1309 (2002). LOJEK A., KUBALA L., ÍŽOVÁ H., ÍŽ M.: A comparison of neutrophil chemiluminescence in cuvettes and microtitre plates. Luminescence 17, 1–4 (2002). MARNILA P., TIISKA A., LAGERSPETZ K., LILIUS E.M.: Phagocyte activity in the frog Rana temporaria: whole blood chemiluminescence method and the effects of temperature and thermal acclimation. Comp.Biochem.Physiol. 111, 609–614 (1995). MAZET I., PENDLAND J., BOUCIAS D.: Comparative analysis of phagocytosis of fungal cells by insect hemocytes versus horse neutrophils. Dev.Comp.Immunol. 18, 455–466 (1994). NAKAMURA M., MORI K., INOOKA S., NOMURA T.: In vitro production of hydrogen peroxide by the amebocytes of the scallop, Patinopecten yessoensis (JAY). Dev.Comp.Immunol. 9, 407–417 (1985). ORDAS M.C., NOVOA B., FIGUERAS A.: Modulation of the chemiluminescence response of Mediterranean mussel (Mytilus galloprovincialis) hemocytes. Fish Shellfish Immunol. 10, 611–622 (2000). PEREIRA L.S., OLIVEIRA P.L., BARJA-FIGALDO C., DAFFRE S.: Production of reactive oxygen species by hemocytes from the cattle tick Boophilus microplus. Exp.Parasitol. 99, 66–72 (2001). ROTHE G., VALET G.: Flow cytometric analysis of respiratory burst activity in phagocytes with hydroethidine and 2´,7´-dichlorofluorescein. J.Leukoc.Biol. 47, 440–448 (1990). SLAVÍKOVÁ H., LOJEK A., HAMAR J., DUŠKOVÁ M., KUBALA L., VONDRÁEK J., ÍŽ M.: Total antioxidant capacity of serum increased in early but not late period after intestinal ischemia in rats. Free Rad.Biol.Med. 25, 9–18 (1998). VALEMBOIS P., LASSEGUES M.: In vitro generation of reactive oxygen species by free celomic cells of the annelid Eisenia foetida andrei: an analysis by chemiluminescence and nitro-blue tetrazolium reduction. Dev.Comp.Immunol. 19, 195–204 (1995). VOWELLS S.J., SEKHSARIA S., MALECH H.L., SHALIT M., FLEISHER T.A.: Flow cytometric analysis of the granulocyte respiratory burst: a comparison study of fluorescent probes. J.Immunol.Meth. 178, 89–97 (1995). WHITTEN M.M.A., RATCLIFFE N.A.: In vitro superoxide activity in the hemolymph of the West Indian leaf cockroach, Blaberus discoidalis. J.Insect Physiol. 45, 667–675 (1999). YAMASHITA M., IWABUCHI K.: Bombyx mori prohemocyte division and differentiation in individual microcultures. J.Insect Physiol. 47, 325–331 (2001). YEN G.H., WU S.C., DUH P.D.: Extraction and identification of antioxidant components from the leaves of mulberry (Morus alba L.). J.Agric.Food Chem. 44, 1687–1690 (1996).
