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Calreticulin is Transiently Induced after Immunogen Treatment in the Fat Body of the Silkworm Bombyx mori. INTRODUCTION. Calreticulin (CRT) is one of the ...
Journal of Insect Biotechnology and Sericology 75, 79-84 (2006)

Calreticulin is Transiently Induced after Immunogen Treatment in the Fat Body of the Silkworm Bombyx mori Tadashi Takahashi1, Hiroki Murakami1, Shigeo Imanishi2, Masao Miyazaki3, Katsuyoshi Kamiie4, Koichi Suzuki1, Hideharu Taira1 and Tetsuro Yamashita1* 1

Faculty of Agriculture, Iwate University, 3-18-8 Ueda, Morioka, Iwate 020-8550, Japan, 2 National Institute of Agrobiological Sciences, 1-2 Owashi, Tsukuba, Ibaraki 305-8634, Japan, 3 Sphingolipid Expression Laboratory, Supra-biomolecular System Group, Frontier Research System, Institute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan, and 4 Faculty of Pharmaceutical Sciences, Aomori University, 2-3-1 Koubata, Aomori-shi 030-0943, Japan (Received September 29, 2005; Accepted December 15, 2005)

 Calreticulin (CRT) is a multifunctional endoplasmic reticulum (ER) lumen protein, which is also involved in innate immunity. To study the involvement of CRT in the insect immune response, silkworm larvae of Bombyx mori were treated with lipopolysaccharide (LPS). LPS treatment induced an increase in CRT mRNA in the fat body only, and not in hemocytes, the midgut, silk gland, or Malpighian tubule. Time course analysis indicated that significant CRT mRNA expression by the fat body was induced 6 h after LPS injection, and returned to the normal level within 24 h. The NIAS-Bm-aff3 cell line, which was established from the fat body of B. mori, showed a similar expression pattern on LPS exposure. Since the fat body is capable of a humoral response in insect innate immunity, our results suggests that CRT is induced by the activation of the humoral immune system in the fat body of Bombyx mori.  Key words: Bombyx mori, calreticulin, fat body, innate immunity, lipopolysaccharide

INTRODUCTION  Calreticulin (CRT) is one of the best-characterized molecular chaperones, and it participates in the maturation of glycoproteins in the endoplasmic reticulum (ER) (Michalak et al., 1999; Ellgaard and Helenius, 2001). However, it is also believed to be involved in many cellular functions, including both acquired and innate immunity (Johnson et al., 2001). As an example of the former, CRT participates in the functional MHC class I loading complex in the ER and is thought to act as a structural scaffold for other components of the loading complex (Bouvier, 2003; Paulsson and Wang, 2003). CRT is also an auto-antigen often found in patients with autoimmune diseases, such as systemic lupus erythematosus (SLE) (Eggleton et al., 1997). In innate immunity, CRT functions to extend the phagocytotic cap of Dictyostelium cells (Muller-Taubenberger et al., 2001), as a co-receptor of complement C1q (Ghebrehiwet and Peerschke, 2004), and in the uptake of apoptotic cells (Ogden et al., 2001; Vandivier et al., 2002).  Insects possess innate immunity, but not acquired immunity, as a defense system, and the insect immune response can be further subdivided into humoral and cellular defense responses (Hultmark, 2003). Humoral defenses include the production of antimicrobial peptides, reactive intermediates of oxygen or nitrogen, and the *To whom correspondence should be addressed. Tel & Fax: +81-19-621-6157. Email: [email protected]

complex enzymatic cascades that regulate coagulation or melanization of the hemolymph (Tzou et al., 2002). In contrast, cellular defense refers to hemocyte-mediated immune responses, like phagocytosis, nodulation, and encapsulation (Lavine and Strand, 2002). Some papers have reported the involvement of CRT in insect immunity. In Galleria mellonella, CRT bound to DEAE-Sepharose beads that were injected into larvae as a foreign substance, and anti-CRT antibodies inhibited the gathering of hemocytes around the beads. Therefore, CRT was suggested to be an early-stage encapsulation protein (Choi et al., 2002). The cell surface CRT of Pieris rapae was reported to be a phagocytosis-related protein because the uptake of yeast cells by hemocytes was inhibited by treatment with anti-CRT antibodies (Asgari and Schmidt, 2003). These reports indicate that CRT participates in the cellular response of insect immunity, although little is known of the molecular mechanism involved.  In this study, we examined if CRT was involved in the humoral response in the fat body of Bombyx mori. We found that CRT was induced specifically and transiently in the fat body with LPS treatment, as well as in the NIAS-Bm-aff3 cell line established from the B. mori fat body.

MATERIALS AND METHODS Insect  Silkworm (Bombyx mori, DAIZO) larvae were reared on an artificial diet, Silkmate 1(M) (Nihon Nosankogyo Co., Japan) under a 12L:12D photoperiod at 25°C. Fifth-

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instar larvae were used for the experiments. Immunization  The LPS solution, from Escherichia coli O111:B4 (Wako), was prepared at a concentration of 4 μg/μl in isotonic solution for B. mori (1.67 mM Na2HPO4, 1.66 mM KH2PO4, 147.5 mM NaCl, 1.34 mM KCl, 3 mM CaCl2), and injected into 3-day/5th-instar larvae. The E. coli suspension was prepared by culturing E. coli (JM109) with LB medium until OD600 = 0.6. One milliliter of the culture medium was centrifuged at 4,000 g for 5 min, and the pellet was washed with the isotonic solution, resuspended in 1 ml of the same solution, and boiled for 3 min. Then 3-day/5th-instar larvae were immunized with 5 μl of the E. coli suspension per larva.  After breeding for the indicated time, the larvae were paralyzed on ice for 30 min and hemolymph was collected by cutting off their feet. The hemolymph was centrifuged at 1,000 g for 5 min at 4°C; and the supernatant (plasma) was removed; and the hemocyte pellet was resuspended in the isotonic solution. The larvae used to harvest hemolymph and the major organs; the fat body, midgut, silk gland, and Malpighian tubule were dissected. The hemocytes and major organs were immediately frozen and preserved at −80°C until use. cDNA cloning and nucleotide sequencing of B. mori calreticulin  Total RNA was isolated from fat bodies at the prepupal stage. The reverse transcription polymerase chain reaction (RT-PCR) was performed using a GeneAmp RNA PCR Core Kit (Applied Biosystems, Foster City, CA, USA). First strand cDNA was synthesized from 1 μg of total RNA and oligo d(T)16 primer, and the product was used as the template for PCR. The PCR primers CRT-F (5’-GCTGTTGTACTGGTCGTTGTC-3’) and CRT-RV (5’-AGGTACTTAATGTCTTGG-3’) were designed based on homology to CRT in SilkBase, the B. mori EST database (http://papilio.ab.a.u-tokyo.ac.jp/silkbase/). The amplified PCR fragment was cloned into pCR2.1 vector (Invitrogen, Carlsbad, CA, USA); 5’-RACE was performed with a SMARTII cDNA synthesis kit (Clontech, Palo Alto, CA, USA). The cDNA sequence was determined using an automated DNA sequencer (ABI3100) and registered in the NCBI database as Acc#AB090887 (nucleotide) and Acc#BAC57964 (protein). Preparation of antiserum  Polypeptide was synthesized using an automated peptide synthesis system (PSSM-8 system; Shimadzu, Kyoto, Japan) based on the sequence of calreticulin residues 380-398, and purified using an HPLC system (Shimadzu). The peptides were coupled with keyhole limpet hemocya-

nin (Sigma, St. Louis, MO, USA) via m-maleimidobenzoyl-N-hydroxysuccinimide ester (Sigma) and mixed with Freund’s complete (first administration) or incomplete (second and later) adjuvant (Gibco; Invitrogen) and used to immunize a rabbit. Western blotting  The silkworm organs were homogenized and lysed in RIPA buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% NP-40, 1% DOC, 1% SDS, 1 mM EDTA, 10 μg/ml leupeptin, 10 μg/ml pepstatin, and 1 mM PMSF). The tissue lysates were centrifuged at 15,000 g, and the concentration of protein in the supernatant was determined using a BCA assay kit (Pierce, Rockford, IL, USA). The protein extracts were prepared at a concentration of 2 μg/μl with 2 × SDS-PAGE sample buffer (135 mM Tris-HCl (pH 6.8), 10% 2-mercaptoethanol, 10% glycerol, 4% SDS, and 0.002% bromophenol blue) and separated in a 10% polyacrylamide gel. The gel was electroblotted onto PVDF membrane (Millipore, Bedford, MA, USA). The membranes were blocked with TBS-T (10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.05% Tween 20) buffer containing 1% polyvinyl-pyrrolidone (Sigma) at 4°C, for 16 h. The membranes were probed with anti-calreticulin antiserum diluted 4,000-fold for 1 h at room temperature. The membrane was washed with TBS-T three times and then incubated with Protein A-HRP (Amersham Pharmacia Biotech, Hercules, CA, USA) diluted 8,000-fold for 1 h at room temperature. After washing with TBS-T, the membrane was incubated with ECL reagents (Amersham) and exposed to X-ray film (Fuji film, Tokyo, Japan). Northern blotting  Total RNA was isolated from the organs or NIAS-Bmaff3 cells using TRIzol (Invitrogen), separated on a 1% agarose-MOPS gel, and blotted onto Hybond-N+ membrane (Amersham). The membrane was hybridized with CRT or glyceraldehyde-3-phosphate dehydrogenase (GAPDH, as an internal control) probes labeled using an AlkPhos DIRECT kit (Amersham), at 55°C, for 8 h. Probe fragments were obtained by RT-PCR and cloned into TOPO pCRII vector (Invitrogen). The primers used were CRT-F (5’-GCTGTTGTACTGGTCGTTGTC-3’), CRTRV161 (5’-ATCCTCTGGGTCGCTGAAGA-3’), GAPDH-F (5’-TGAGGGCTTGATGACTACTG-3’), and GAPDH-RV (5’-ATTCCAGTCAGCTTGCCATT-3’). The membrane-hybridizing probe was washed twice with primary wash buffer (2 M urea, 0.1% SDS, 50 mM Na phosphate, 150 mM NaCl, 1 mM MgCl 2, 0.2% blocking reagent, pH 7.0) at 55°C, and then washed twice with 2 × secondary wash buffer (100 mM Tris, 200 mM NaCl, 2 mM MgCl2, pH 10.0) at room temperature. The membranes were incubated with CDP-Star detection reagent

Induction of Calreticulin by LPS Treatment

(Amersham) and exposed to X-ray film (Fuji film). Cell line and culture  NIAS-Bm-aff3 cells derived from B. mori, (N509·N510) × (C509·C510), fat bodies were cultured in IPL-41 insect cell culture medium (Gibco; Invitrogen) containing 10% FBS (Gibco; Invitrogen) at 25°C. LPS treatment was performed in 0.1% FBS-IPL41 medium.

RESULTS AND DISCUSSION Cloning and sequencing calreticulin cDNA  The SilkBase database was searched for EST clones as

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candidates for B. mori CRT (Mita et al., 2003). Forward and CRT reverse primers were designed based on wdV40077 (forward primer) and fbVm1076 (reverse primer); PCR was performed and an approximately 1.2-kbp fragment was obtained, as expected. This fragment was subcloned into pCR2.1 vector and its sequence was confirmed. In addition, 5’-RACE was performed and the cDNA sequence is shown in Fig. 1. The 5’-UTR was 47 bp and the following 1147 bp encoded 398 amino acids. The deduced amino acid sequence contained two calreticulin family signatures, three calreticulin repeats, and an ER retention signal (HDEL) at the C-terminal (Fig. 1). The deduced amino acid sequence of the cDNA shared 76

 Fig. 1. Nucleotide and amino acid sequences of calreticulin in Bombyx mori. Bold letters indicate the calreticulin family signature and underlined letters the calreticulin repeat. The HDEL sequence located at the C-terminal is an ER retention signal.

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and 89% identity with Drosophila melanogaster CRT and Galleria mellonella CRT, respectively. LPS injection induces fat body CRT expression  The hemocytes, fat body, midgut, silk gland, Malpighian tubule, and plasma were removed from 4-day/5th-instar silkworm larvae and the protein extracts were subjected to Western blotting. Twenty micrograms of the proteins were electrophoresed on a 10% polyacrylamide gel, and probed with anti-CRT antibody. CRT was detected in all organs, and was especially abundant in the fat body and hemocytes, but not in plasma (Fig. 2). Since CRT is the ER luminal protein that contains ER retention signal at the C-terminal (Fig. 1), CRT should not be secreted into the hemolymph. The abundant existence of CRT in the fat body and hemocyte might be accounted for the physiological function of these tissues.  Therefore, to investigate the involvement of CRT in the immune response of B. mori, the effect of immunogen treatment on CRT expression was analyzed. B. mori larvae were injected with LPS (20 μg/larvae), and total RNA was isolated from hemocytes, the fat body, midgut, silk gland, and Malpighian tubule 0 and 12 h after injection. Total RNA was isolated from each organ, and Northern blotting was performed using a CRT probe, with the GAPDH probe as an internal control. Figure 3 shows that following LPS treatment, CRT mRNA increased only in the fat body, and not the other tissues in which CRT mRNA was also detected by Northern analysis. This suggests that CRT was specifically upregulated in the fat body, but not the other organ. Recent research on the association between CRT and insect immunity has focused on hemocytes and cellular immunity. Our result suggests that CRT functions in the fat body and in humoral immunity, because the fat body is thought to act in the humoral response, but not in the cellular response.  The time course of the accumulation of CRT mRNA after immunization was assayed. Northern blotting was performed with total RNA extracted from fat bodies 0, 6, 12, 24, 36, and 48 h after LPS or E. coli treatment. Figure 4A shows that the CRT mRNA level increased markedly at

 Fig. 2. Western blot analysis of CRT protein in the major organs of Bombyx mori. Hemocytes (H), fat body (F), midgut (MG), silk gland (S), Malpighian tubule (MT), and plasma (P) were collected from 4-day/5th-instar larvae of B. mori. Twenty micrograms of lysates were separated in a 10% polyacrylamide gel and probed with anti-CRT antibody.

 Fig. 3. Effect of LPS treatment on the induction of CRT mRNA in the major organs. Three-day/5th-instar larvae were immunized with 20 µg of LPS, and 0 or 12 h after immunization, total RNA was extracted from the hemocytes, fat body, midgut, silk gland, and Malpighian tubule. 20 µg of total RNA were used for Northern blotting; GAPDH probe was used as a control.

 Fig. 4. The time course analysis for CRT mRNA expression after immunogen injection. Three-day/5th-instar larvae were immunized with LPS (A) or E. coli (B), and the fat bodies were removed 0, 6, 12, 24, 36, and 48 h after immunization. Twenty micrograms of total RNA were used for Northern blotting. GAPDH was used as a control.

Induction of Calreticulin by LPS Treatment

6 h, reached a maximum at 12 h, and then decreased until 24 h. In contrast, the upregulation of CRT mRNA remained in effect until 48 h after E. coli immunization (Fig. 4B). LPS is a cell wall component of gram-negative bacteria that can stimulate the innate immune system. It has been reported that E. coli entering B. mori larvae are phagocytosed and digested by hemocytes, which is followed by LPS release into the hemolymph (Taniai et al., 1997). Therefore, the prolonged stimulation on injection of E. coli may explain the time lag between phagocytosis and the release of LPS. These results indicate that the LPS responsiveness of CRT is specific to the fat body and is transient in vivo. The effect of LPS treatment on NIAS-Bm-aff3 cells established from the B. mori fat body  To investigate whether NIAS-Bm-aff3 cells (Fig. 5A), which were established from the B. mori fat body, re-

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spond to LPS, Northern blotting was performed with NIAS-Bm-aff3 cells treated with LPS in IPL-41 medium containing 0.1% FBS. Figure 5B indicates that the CRT mRNA level increased in a dose-dependent manner for LPS concentrations between 1 and 103 ng/ml, but was lower at 104 ng/ml than at 103 ng/ml. Time course Northern and Western blotting analyses were performed with NIAS-Bm-aff3 cell extracts treated with 103 ng/ml LPS. CRT mRNA increased gradually until 9 h, remained constant until 12 h, and then returned to baseline by 24 h after LPS treatment. Western blotting showed that the pattern of CRT protein expression was about 3 h later than that of CRT mRNA (Fig. 5C). These results suggest that LPS interacts with fat body cells directly and activates CRT gene expression.  The fat body is one of the most important organs in insects, corresponding to the mammalian liver, and it also functions in the insect humoral immune response (Tzou et

 Fig. 5. CRT induction in NIAS-Bm-aff3 cells with LPS treatment. A, Microphotograph of a NIAS-Bm-aff3 cell (Bar, 100 µm). NIAS-Bm-aff3 cells were cultured in IPL41 insect medium containing 10% FBS at 27°C. B, Dose-dependency of CRT induction in response to LPS in NIAS-Bm-aff3 cells. NIAS-Bm-aff3 cells were incubated with 0.1% FBS-IPL41 medium containing stepwise diluted LPS. After 12 h, total RNA was extracted from the cells and 6 µg of total RNA was used for Northern blotting with a CRT or GAPDH probe (control). C, NIAS-Bm-aff3 cells were incubated in 0.1% FBS-IPL41 insect medium containing 1 µg/ml LPS. After 0, 3, 6, 9, 12, 18, and 24 h, CRT protein or mRNA was detected using Western blotting with 2.5 µg of protein or Northern blotting with 20 µg of total RNA, respectively.

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al., 2002). If the microbes invade the insect body cavity, the fat body recognizes the immunogen and produces antimicrobial peptides (AMPs), each of which has a specific spectrum for killing microbes (Hultmark, 2003; Leclerc and Reichhart, 2004). Although antibacterial peptides such as cecropin B are synthesized in the fat body (Kato et al., 1993), expression analysis is usually performed with nonfat body-derived cell lines, such as embryos (Taniai and Tomita, 2000; Johnson et al., 2001). Our result indicated that NIAS-Bm-aff3 cells should be useful for elucidating the LPS signaling pathway, which probably includes CRT, in fat body cells. To clarify the function of CRT in the network of the humoral immune system of insects, further studies are needed to identify the cellular components associated with CRT.  Recently, we cloned the 5’-upstream region of the B. mori CRT gene and performed dual luciferase assay with stepwise truncated CRT promoter sequence. It was shown that about 400-800 bp upstream region contained cis-acting elements for the expression of CRT gene by LPS treatment (Takahashi et al., unpublished data). We found seven CATT(A/T) sequences in this region, which are reported to be LPS response elements in B. mori (Taniai and Tomita, 2000) . Research examining the mechanisms regulating the CRT gene expression by LPS stimulation using NIAS-Bm-aff3 cells is now in progress.

ACKNOWLEDGMENTS  This work was supported in part by a Grant-in-Aid for the 21st Century COE Program from the Ministry of Education, Culture, Sports, Science and Technology of Japan to Iwate University. M. Miyazaki is a special postdoctoral fellow of RIKEN.

REFERENCES Asgari, S., and Schmidt, O. (2003) Is cell surface calreticulin involved in phagocytosis by insect hemocytes? J. Insect Physiol. 49, 545-550. Bouvier, M. (2003) Accessory proteins and the assembly of human class I MHC molecules: a molecular and structural perspective. Mol. Immunol. 39, 697-706. Choi, J.Y., Whitten, M.M., Cho, M.Y., Lee, K.Y., Kim, M.S., Ratcliffe, N.A., and Lee, B.L. (2002) Calreticulin enriched as an early-stage encapsulation protein in wax moth Galleria mellonella larvae. Dev. Comp. Immunol. 26, 335-343. Eggleton, P., Reid, K.B., Kishore, U., and Sontheimer, R.D. (1997) Clinical relevance of calreticulin in systemic lupus erythematosus. Lupus 6, 564-571. Ellgaard, L., and Helenius, A. (2001) ER quality control: to-

wards an understanding at the molecular level. Curr. Opin. Cell Biol. 13, 431-437. Ghebrehiwet, B., and Peerschke, E.I. (2004) cC1q-R (calreticulin) and gC1q-R/p33: ubiquitously expressed multi-ligand binding cellular proteins involved in inflammation and infection. Mol. Immunol. 41, 173-183. Hultmark, D. (2003) Drosophila immunity: paths and patterns. Curr. Opin. Immunol. 15, 12-19. Johnson, S., Michalak, M., Opas, M., and Eggleton, P. (2001) The ins and outs of calreticulin: from the ER lumen to the extracellular space. Trends Cell Biol. 11, 122-129. Kato, Y., Taniai, K., Hirochika, H., and Yamakawa, M. (1993) Expression and characterization of cDNAs for cecropin B, an antibacterial protein of the silkworm, Bombyx mori. Insect Biochem. Mol. Biol. 23, 285-290. Lavine, M.D., and Strand, M.R. (2002) Insect hemocytes and their role in immunity. Insect Biochem. Mol. Biol. 32, 1295-1309. Leclerc, V., and Reichhart, J.M. (2004) The immune response of Drosophila melanogaster. Immunol. Rev. 198, 59-71. Michalak, M., Corbett, E.F., Mesaeli, N., Nakamura, K., and Opas, M. (1999) Calreticulin: one protein, one gene, many functions. Biochem. J. 344 Pt 2, 281-292. Mita, K., Morimyo, M., Okano, K., Koike, Y., Nohata, J., Kawasaki, H., Kadono-Okuda, K., Yamamoto, K., Suzuki, M. G., Shimada, T., Goldsmith, M. R., and Maeda, S. (2003) The construction of an EST database for Bombyx mori and its application. Proc. Natl. Acad. Sci. USA 100, 1412114126. Muller-Taubenberger, A., Lupas, A.N., Li, H., Ecke, M., Simmeth, E., and Gerisch, G. (2001) Calreticulin and calnexin in the endoplasmic reticulum are important for phagocytosis. EMBO J. 20, 6772-6782. Ogden, C.A., deCathelineau, A., Hoffmann, P.R., Bratton, D., Ghebrehiwet, B., Fadok, V.A., and Henson, P.M. (2001) C1q and mannose binding lectin engagement of cell surface calreticulin and CD91 initiates macropinocytosis and uptake of apoptotic cells. J. Exp. Med. 194, 781-795. Paulsson, K., and Wang, P. (2003) Chaperones and folding of MHC class I molecules in the endoplasmic reticulum. Biochim. Biophys. Acta 1641, 1-12. Taniai, K., and Tomita, S. (2000) A novel lipopolysaccharide response element in the Bombyx mori cecropin B promoter. J. Biol. Chem. 275, 13179-13182. Taniai, K., Wago, H., and Yamakawa, M. (1997) In vitro phagocytosis of Escherichia coli and release of lipopolysaccharide by adhering hemocytes of the silkworm, Bombyx mori. Biochem. Biophys. Res. Commun. 231, 623-627. Tzou, P., De Gregorio, E., and Lemaitre, B. (2002) How Drosophila combats microbial infection: a model to study innate immunity and host-pathogen interactions. Curr. Opin. Microbiol. 5, 102-110. Vandivier, R.W., Ogden, C.A., Fadok, V.A., Hoffmann, P.R., Brown, K.K., Botto, M., Walport, M.J., Fisher, J.H., Henson, P.M., and Greene, K.E. (2002) Role of surfactant proteins A, D, and C1q in the clearance of apoptotic cells in vivo and in vitro: calreticulin and CD91 as a common collectin receptor complex. J. Immunol. 169, 3978-3986.