- Email: [email protected]
The ontogeny and extrahepatic expression of complement factor C3 in Atlantic salmon (Salmo salar)
Fish & Shellfish Immunology 23 (2007) 542e552 www.elsevier.com/locate/fsi
The ontogeny and extrahepatic expression of complement factor C3 in Atlantic salmon (Salmo salar) Marie Løvoll a,*, Hanne Johnsen a, Hani Boshra b, Jarl Bøgwald a, J. Oriol Sunyer b, Roy A. Dalmo a a
Department of Marine Biotechnology, Norwegian College of Fishery Science, University of Tromsø, Breivika, N-9037 Tromsø, Norway b Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA Received 8 August 2006; revised 3 January 2007; accepted 15 January 2007 Available online 20 January 2007
Abstract Fish embryos and hatchlings are exposed to pathogens long before maturation of their lymphoid organs. Little is known about defence mechanisms during the earliest stages of life, but innate mechanisms may be essential for survival. The complement system in fish is well developed and represents a major part of innate immunity. Complement factor 3 (C3) is central subsequent to activation of all pathways of the complement system, leading to inflammatory reactions, such as chemotaxis, opsonisation and lysis of pathogens. Hepatocytes represent the major source of C3, but modern molecular biological methods have confirmed that C3 is synthesised at multiple sites. Our main objective was to study the ontogeny of C3 in Atlantic salmon by mapping the commencement of synthesis and localisation of proteins. Eggs, embryos, hatchlings and adult fish were analysed for the presence of C3 mRNA and proteins. From immunohistochemical studies, C3 proteins were detected at several extrahepatic sites, such as the skeletal muscle, developing notochord and chondrocytes of the gill arch. Immunoblotting revealed presence of C3 proteins in the unfertilised egg, but C3 mRNA was only detected after fertilisation by real-time RT-PCR. Taken together, the results implicated the maternal transfer of C3 proteins as well as novel non-immunological functions during development. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Complement component C3; Ontogeny; Extrahepatic; Atlantic salmon
1. Introduction The complement system plays a vital role in innate immunity and by augmenting B-cell proliferation, it also affects acquired immunity [1e3]. Activation of the complement cascade occurs through three partially overlapping pathways, where complement factor 3 (C3) is the key protein. Activation leads to the assembly of a pore forming complex capable of lysing pathogens, and also promotes inflammation and clearance of pathogens through generation of anaphylatoxins and opsonins. C3 belongs to the a2-macroglobulin family and consists of two disulfide-linked chains; a and b. In di- and tetraploid fish, such as teleosts, several complement factors are encoded by multiple genes giving * Corresponding author. Tel.: þ47 7764 6345; fax: þ47 7764 6020. E-mail address: [email protected] (M. Løvoll). 1050-4648/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2007.01.002
M. Løvoll et al. / Fish & Shellfish Immunology 23 (2007) 542e552
543
rise to subtypes displaying structural and functional diversity [4,5]. In the common carp (Cyprinus carpio), five C3 variants have been characterised [6], while the rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar) both possess three subtypes. The trout subtypes have been fully characterised [7,8], but only partial sequences have been described for Atlantic salmon (S. salar). From BLAST analysis (Basic Local Alignment Search Tool, http:// www.ncbi.nlm.nih.gov/blast/), only few nucleotide differences between species were revealed. Hepatocytes represent the major source of most plasma complement proteins, but modern molecular biological methods have confirmed that complement proteins are synthesised at multiple sites [9,10]. Ontogenic appearance and mapping of extrahepatic synthesis of complement components in rainbow trout (O. mykiss) revealed widespread production of several complement components [11]. Studies on Atlantic halibut larvae (Hippoglossus hippoglossus) and Atlantic cod (Gadus morhua) revealed presence of C3 mRNA and proteins in several tissues post hatch [12e14]. Following several reports on extrahepatic synthesis of complement, non-immunological functions in e.g. reproduction, development, ossification, signal transduction and metabolism have been suggested [10,15]. Embryos and hatchlings are exposed to pathogens long before they are able to mount a mature immune response [16e18]. In Atlantic salmon, the thymus and kidney are fully lymphoid by the time of hatching, but surface immunoglobulins are not present until around the start of feeding [19]. Absence of immunoglobulin producing cells makes the developing embryo highly vulnerable against pathogen attack [20,21]. The embryo relies solely on physical barriers (mucus and epithelium from the skin, gills and intestine), innate immune cells (macrophages and granulocytes) [22] and other innate mechanisms as defence against invading pathogens. At these early stages, maternally derived immune defence components in the yolk, such as complement, may play a vital role in protection. Maternal transfer of C3, a2M, serum amyloid A and C1r/s mRNAs along with C3 and Ig proteins has been described in carp (C. Carpio) [23], several maternally derived complement proteins have been detected in rainbow trout (O. mykiss) [11] and transfer of C3 proteins has been described in the spotted wolffish (Anarhichas minor) [24]. The main objective of this work was to study the ontogeny and extrahepatic expression of C3 in Atlantic salmon (S. salar) by mapping the commencement of synthesis and localisation of proteins. 2. Materials and methods 2.1. Sample collection and preparation Unfertilised Atlantic salmon (S. salar) eggs and sperm were obtained from AquaGen (Trondheim, Norway), mixed and disinfected with buffodine. Incubation of fertilised eggs and maintenance of hatchlings was performed in upwelling incubators and aquaria supplied with aerated, running water at 6 C for 15 weeks (Ka˚rvika Aquaculture Research Station, Tromsø, Norway). Hatchlings were maintained under a photoperiod of 12 h light/12 h darkness. The embryos hatched at day 77e84 and yolk-sac resorption was completed w119 days post fertilisation. Samples were collected weekly. Hatchlings were overanaesthetized using 0.01% benzocaine. Five adult Atlantic salmon (S. salar) (w5 kg) were kindly provided by a commercial fish farm (Sotra Fiskeindustri, Glesvær, Norway). The adult fish had been vaccinated once (Norvax Fur Vib; Intervet Norbio, Norway) 16 months prior to sacrifice. Tissue samples from the gills, skin, muscle, heart, pylorus, intestine, gonads, spleen, head kidney and liver were dissected. Samples for RNA purification were submerged in RNAlater (Ambion, Austin, Texas, USA), kept at room temperature overnight and stored at 20 C. Samples for hemolytic assays and immunoblotting were directly frozen at 20 C. Samples for immunohistochemistry were fixed in 4% paraformaldehyde in phosphate buffered saline (PBS) for two days, transferred to 70% ethanol, dehydrated and embedded in paraffin wax. 2.2. Purification of total RNA Total RNA was extracted using the TRIzol method [25]. Three eggs, embryos or hatchlings at each time-point were pooled and homogenised in 3 ml TRIzol reagent using a rotor-stator homogeniser (UltraTurrax; IKAÒWerke, Staufen, Germany). For additional removal of DNA and proteins, the water phase of the initial TRIzol/chloroform separation was added to a second volume of TRIzol reagent. To remove any contaminating genomic DNA, samples were treated with DNase (TURBO DNA-freeÔ, Ambion). Purified RNA was confirmed to be intact by gel electrophoresis. RNA concentrations and purity were measured spectrophotometrically (NanoDrop Technologies, Wilmington, USA).
M. Løvoll et al. / Fish & Shellfish Immunology 23 (2007) 542e552
544
2.3. Quantitative real-time reverse transcription-polymerase chain reaction (qPCR) RNA was reversely transcribed using random hexamers (TaqMan RT-reagents, Applied Biosystems, CA, USA). Reaction volumes of 50 ml contained 100 ng of total RNA. PCRs were performed in duplicates with an ABI PRISMÒ 7000 Sequence Detection System (Applied Biosystems) as described earlier [11]. Primers were designed from partial cDNA sequences deposited in the GenBank (accession numbers are listed in Table 1) using the Primer Express software (version 2.0; Applied Biosystems) and synthesised by MedProbe (Oslo, Norway). The amplification efficiency of each primer set was assessed from 2-fold liver cDNA dilutions (standard curves) according to the equation: E ¼ 10(1/slolpe) [26] (Table 1). The relative expression ratio (R) of the C3 gene was calculated based on primer efficiencies (E) and the Ct deviations (DCt) of the unknown samples versus a calibrator, and normalised to the corresponding reference gene according to the equation R ¼ (Etarget)DCt target (calibrator-sample)/(Ereference)DCt reference (calibrator-sample) [27]. cDNA to be used with the 18S primers was diluted 1:10 000. Melting curves were generated for the amplicons, verifying specific amplification and absence of dimers [28]. In addition, the PCR products were analysed by gel electrohoresis. Presence of any genomic DNA contamination of the RNA was tested by subjecting parallel samples to PCR without preceding reverse transcription. All data were captured using Sequence Detection Software (SDS version 1.1; Applied Biosystems). 2.4. Antibodies Antibodies recognizing individual chains of different C3 were generated in rabbits by immunization with SDSPAGE purified a- and b-chains [8]. Briefly, 500 mg of purified C3-1 were run on 7.5% SDS-PAGE under reducing conditions. The a- and b-chains were detected using a Zinc Stain Kit (Bio-Rad, Hercules, CA, USA). The individual a- and b-chains were excised from the gel and extracted protein samples were sent to Cocalico Biologicals, Inc. (Reamstown, PA, USA) for antibody production in rabbits. 2.5. Immunoblotting All reagents were purchased from Invitrogen and used in compliance with the manufacturer’s protocol as previously described [11]. Briefly, proteins were extracted from eight pooled eggs, embryos or five hatchlings at each time-point. Samples were homogenised in 2.5 ml 0.1 M Tris, pH 7.6 and protease inhibitors (Protease Inhibitor Cocktail; Sigma-Aldrich, Steinberg, Germany) using the rotor-stator homogeniser (UltraTurrax). The homogenates were centrifuged at 4500 g for 15 min at 4 C, the supernatants were centrifuged another three times at 14 000 g for 5 min at 4 C. Protein extracts were stored at 20 C. Protein concentrations and purity were measured spectrophotometrically (NanoDrop Technologies). Twenty micrograms of proteins from each sampled time-point were reduced, denatured and fractionated by gel electrophoresis. Proteins were transferred to a polyvinylidene difluoride-membrane and incubated with a mixture of rabbit antisera raised against the rainbow trout C3-1a- and b-chains (1:1000 dilution). Normal rabbit serum (Sigma-Aldrich) served as the control. 2.6. Immunohistochemistry The immunostaining procedure was performed as described earlier [29]. Briefly, serial sections of 5 mm were mounted onto SuperFrostÒ slides, melted at 60 C, dewaxed in xylene, rehydrated via ethanol to water and demasked by boiling in 0.1 M citric acid (pH 6.0) for 5 min. Following antigen retrieval and blocking, the sections were Table 1 Primers, amplicon lengths, GenBank accession numbers and primer efficiencies (E) for qPCR Gene C3 18S
Oligonucleotides Forward Reverse Forward Reverse
0
0
5 -tccctggtggtcaccagtacac-3 50 -atgatggtggactgtgtggatc-30 50 -tgtgccgctagaggtgaaatt-30 50 -cgaacctccgactttcgttct-30
The C3 primers annealed to the sequence encoding the a-chain.
Amplicon (bp)
GenBank acc. no.
E
157
BI468074
1.95
101
AJ427629
1.98
M. Løvoll et al. / Fish & Shellfish Immunology 23 (2007) 542e552
545
incubated with mixed polyclonal rabbit anti-rainbow trout C3-1a and C3-1b sera (1:1000 dilution). Normal rabbit serum (Sigma-Aldrich) served as control. 2.7. Hemolytic assay The ability of homogenates prepared from eggs, embryos and hatchlings to lyse foreign cells was measured. Four samples from each time-point were homogenised in Hank’s Balanced Saline Solution (HBSSþ/þ). 100 ml homogenates (duplicates of two-fold serial dilutions) were incubated with 100 ml of 1% sheep red blood cells in each well of a 96 well microtray (Nunc, Denmark). The tray was incubated for 1.5 h at room temperature followed by centrifugation at 148 g for 10 min at 4 C. The supernatants (100 ml) were collected from each well and transferred to a non-absorbent flat bottom microtray (Nunc). The optical density at 405 nm (OD405) was read. To normalise the results, OD405 values of homogenates without added red blood cells were subtracted from the OD405 values of the test samples. Replacing homogenates with HBSSþ/þ served as negative control (0% lysis) and a replacement of homogenates by H2O served as positive control (100% lysis). To inhibit the hemolytic activity mediated by complement, the homogenates were heated at 45 C for 30 min or HBSSþ/þ was supplemented with 10 mM ethylenediamine tetraacetic acid (EDTA).
3. Results Using real-time RT-PCR to compare mRNA levels can be influenced by fluctuating levels of the reference gene between different developmental stages, tissues and individuals. 18S rRNA has been found to be a well behaved endogenous control in salmon [30,31], and also in this study the 18S rRNA levels were constant. The level of 18S rRNA in homogenates of the unfertilised egg was higher than in homogenates from the later time-points studied. Any genomic contamination was excluded by negative controls (minus reverse transcriptase). Specificity of anti C3-1a and anti C3-1b was previously confirmed in rainbow trout [11] and cross-binding to the Atlantic salmon C3 aand b-chains was confirmed in serum from adult salmon (Fig. 1). Specific cross-binding to the salmon C3 subtypes was not evaluated. Serum from human or fish are known to contain several degradation fragments of C3 in addition to native C3. The bands that are immunoreactive with the antibody are consistent with degradation fragments previously reported for trout C3-1 [8].
Fig. 1. Cross-binding of anti-rainbow trout C3-1 serum to Atlantic salmon serum. Lane 1: size marker, lane 2: Atlantic salmon serum, lane 3: rainbow trout serum.
546
M. Løvoll et al. / Fish & Shellfish Immunology 23 (2007) 542e552
3.1. Ontogenic appearance of C3 mRNA and protein C3 mRNA levels were detected by real-time RT-PCR throughout the study period (Fig. 2). Expression levels are given in fold increase compared to day 14. No mRNA was detected prior to fertilisation (0 d), but by day 14 transcription had commenced. Slight decreases in mRNA levels were detected prior to hatch (70e77 d) and on day 98e105, but in general, transcription increased steadily throughout the study period. Immunoblotting revealed signals of increasing intensities from the C3 b-chain (w70 kDa) in homogenates from all time-points, including in the unfertilised egg (0 d). The C3 a-chain (w112 kDa) was detected as a weak signal from day 98 and onwards. Negative controls using a 1:500 dilution of normal rabbit serum revealed signals from a protein of w80 kDa. These signals were, however, faint and not in conflict with the interpretation of the immunoblot using the rabbit anti-trout C3-1a and C3-1b sera.
3.2. Immunohistochemical detection of C3 proteins during ontogeny Localisation of C3 proteins in embryos, yolk-sac hatchlings and first feeding hatchlings was studied by immunohistochemistry. Complement C3 proteins were detected at several extrahepatic sites including in the neurons of the spinal chord, the epithelial cells of the oesophagus and intestine, skeletal muscle, muscle cells of the heart and chondrocytes and lamellae of the gill arches. Strong colouring was also detected in the yolk-sac, possibly of periblast cells, which are involved in conversion of yolk-proteins into embryo nutrition (Fig. 3). While the levels of C3 proteins in the liver seemed to increase throughout development, the extrahepatic C3 levels decreased.
Fig. 2. Expression of C3 mRNA (real-time RT-PCR) and C3 proteins (immunoblotting) during ontogeny. Eggs, embryos and hatchlings were sampled weekly from prior to fertilisation (0 d), during embryo development and hatch (77e84 d) throughout complete yolk-sac resorption (84e119 d). C3 mRNA was detected by real-time RT-PCR and the expression levels are given in fold increase compared to the C3 mRNA levels detected in homogenates from day 14. C3 proteins were detected in homogenates from the corresponding time-points using immunoblotting. C3 proteins were visualised by chemiluminescence and exposure to an X-ray-sensitive film for three hours.
M. Løvoll et al. / Fish & Shellfish Immunology 23 (2007) 542e552
547
3.3. Hemolytic activity in embryos and hatchlings Compared to serum from adult rainbow trout, homogenates from all time-points showed very low hemolytic activities and no hemolysis was detected in homogenates of unfertilised eggs (results not shown). Slightly higher activities were detected 14e28 days post fertilisation, as well as in homogenates from the first time-point after hatch (day 84). Interestingly, the hemolytic activity of the homogenates was not inhibited by heat and was enhanced by addition of EDTA, suggesting non-complement mediated activity (Fig. 4).
Fig. 3. A selection of micrographs of tissue sections from Atlantic salmon embryos and hatchlings at different stages of development. Sections to the left were stained for C3 proteins with FastRed TR/naphtol AS-MX and counterstained with Harris’ hematoxylin solution (blue). At day 35 post fertilisation: (A) overview, (B) bodies of neurons in the spinal chord surrounded by striated muscle, (C) epithelial cells of the oesophagus and columnar epithelial cells of the intestine. At day 70 post fertilisation: (D) hepatocytes of the liver and periblast cells of the yolk-sac. At day 91 post fertilisation: (E) chondrocytes of the gill arch, (F) skeletal muscle cells. A.1eF.1 represent the corresponding normal rabbit serum controls.
548
M. Løvoll et al. / Fish & Shellfish Immunology 23 (2007) 542e552
3.4. Quantitation of C3 mRNA in adult salmon C3 transcripts were detected in all the tissues studied (Fig. 5). The highest extrahepatic levels were detected in the heart followed by gonadal tissue, skeletal muscle, intestine and skin. The mean Ct values in liver and extrahepatic tissues before normalisation to 18S rRNA were 15.9 0.9 and 28.2 2.4, respectively. The statistical analysis was conducted using SSC-Stat. Expression levels are given in percentage of average liver C3 mRNA levels. 4. Discussion Being a central component of all activation pathways, C3 is crucial for a complete innate immune response to occur. For lysis of pathogens to occur, all components of the membrane attack complex are essential. Whether these
Fig. 3 (continued).
M. Løvoll et al. / Fish & Shellfish Immunology 23 (2007) 542e552
549
0,4 Sample 45°C EDTA EDTA, 45°C
OD405
0,3
0,2
0,1
0 day 14
serum
Fig. 4. Effects of EDTA and heat on the hemolytic activity of homogenates of four embryos (14 d.p.f.) and serum from adult salmon. Values are given as OD405 values after subtraction of OD405 values of homogenates/serum without added sheep red blood cells.
components are present in the developing salmon embryo, is not known. We have earlier shown presence of C5 and C7 mRNA during rainbow trout ontogeny, though at very low levels and not until 28 days post hatch [11]. In the absence of a lytic pathway, C3 degradation products can still function as opsonins and anaphylatoxins, thereby promoting phagocytosis. This does of course require the presence of phagocytic cells. At 4 days post fertilisation, the rainbow trout spleen anlage is just appearing, and the kidney is not yet lymphoid. Pending maturation of the main phagocytic sites, it has been suggested that macrophages in the gills, skin and gut may protect the hatchlings [32]. In zebrafish (Danio rerio), macrophages differentiate from multipotent myeloid-erythroid progenitors 12e20 h post fertilisation [33]. They are able to phagocytose apoptotic cells and bacteria, and also respond to chemotaxis [34]. In carp
% of liver mRNA levels
1,4 1,2 1 0,8 0,6 0,4 0,2
intestine
kidney
gonads
spleen
pylorus
heart
muscle
skin
gills
0
Fig. 5. Relative expression levels (ordinate axis) of C3 mRNA in extrahepatic tissues (abscissa axis) (n ¼ 5). Expression levels are given in percentage of liver C3 mRNA levels (liver ¼ 100%). The data were analysed by SSC-Stat and expressed as box plots where the median is the line within each box. The boxes indicate the interquartile range; the upper and lower boxes represent the upper and lower 25% of the group, respectively. The tails represent the minimum and maximum values of the group and the horizontal lines indicate the mean values.
550
M. Løvoll et al. / Fish & Shellfish Immunology 23 (2007) 542e552
(C. carpio), macrophages have been identified from 2 days post fertilisation in the head kidney [35]. The ontogenic appearance of macrophages in salmon is, however, unclear and whether they may serve protection prior to hatch is uncertain. Besides the traditional functions of C3 in immunology, several functions during development have been suggested, including tagging apoptotic cells for removal [36e39] as well as mediating stem-cell commitment and differentiation [10]. Quantitation of C3 mRNA revealed steadily increasing levels from day 14 post fertilisation. Inconsistencies, such as the slight decrease observed prior to hatch and the increase at day 91, may be valid or result from a few embryos with especially low or high C3 mRNA levels leading to reduced and increased mean values of expression in the pooled samples. We have earlier shown the corresponding expression profiles of the C3 subtypes for rainbow trout during ontogeny [11]. The salmon and trout profiles differed after hatch by increasing and deflating expression levels, respectively. The salmon C3 primers were designed based on the C3-1 a-chain. Whether this region is unique to the C3-1 subtype is not known and the primers may possibly have annealed to C3-3 and C3-4 as well. From rainbow trout ontogeny studies, the C3-3 and C3-4 levels were found to be approximately 4% and 0.2% of the C3-1 levels, respectively [11]. Transcripts in unfertilised eggs must in general be maternally derived, as transcription starts in correspondence to cell division [40]. Maternal mRNAs are highly protected by e.g. methylation, 50 -capping and massive binding of proteins that may complicate RNA purification. 18S rRNA belongs to the many housekeeping genes that are maternally transferred in all organisms. In this study, the level of maternally transferred 18S rRNA was slightly higher than the embryo’s own synthesis post fertilisation. C3 mRNA was not detected in the unfertilised egg, which was in contrast to studies on carp (C. carpio) where maternally derived C3 mRNA was described [23]. The tissue-distribution study of C3 mRNA in adult salmon showed that the liver was indeed the main supplier of C3. C3 transcripts were, however, detected in all extrahepatic tissues studied, particularly in the heart, followed by gonadal tissue and skeletal muscle. Compared to the levels of complement mRNA in the liver, extrahepatic synthesis was low, but considering the Ctvalues separately, the values indeed implied high enough levels for extrahepatic complement to be of biological significance. Similar studies on rainbow trout showed that the highest extrahepatic levels of synthesis occurred in the gills, skin, skeletal muscle and heart (manuscript in preparation). The individual variation was high, which may result from inconsistent tissue dissection or simply reflect the natural variation in outbred salmon populations. Immunoblotting revealed presence of the C3 b-chain in homogenates from all time-points studied, including in the unfertilised eggs. From day 98, the intensity of the signals increased and the a-chain was visualised. Absence of the a-chain at the earliest time-points has been described earlier and was probably due to the chain’s susceptibility to proteolytic attack [11,23,41]. Our results coincide with studies on fertilised cod eggs (G. morhua) where presence of the C3 b-chain was detected from day 7 post fertilisation and onwards [42]. The C3 proteins detected in the unfertilised egg must be of maternal origin and short protein half-lives may be a reason for the absence of the a-chain at the very earliest time-points. By day 98, the hatchling’s own synthesis may have reached a detectable level where the a-chain could be visualised. The normal rabbit serum showed a weak signal at w80 kDa, but could not account for the signals detected using the anti-C3 serum. Immunohistological studies revealed widespread distribution of C3 proteins at extrahepatic sites, such as in the notochord, intestine, muscle and gill arches. These results coincide with studies on halibut (H. hippoglossus) and cod (G. morhua) where C3 mRNA and protein were detected in the neuronal cells of the spinal chord, epithelial cells of the intestine, chondrocytes and surrounding muscle [13,43,44]. In humans, it has been hypothesised that C3 may be involved in the ossification of cartilage [45,46]. Normal rabbit serum showed no staining in control sections evaluated by immunohistochemistry. The negligible background detected by immunoblotting may be due to higher sensitivity of this method compared to immunohistochemistry. In addition, immunoblotting was conducted under reducing conditions that breaks disulfide bridges, thus changing the three-dimensional configurations of the proteins. More epitopes may be exposed, resulting in a higher background level. Fish complement is more heat labile and has lower optimal reaction temperatures (10e27 C) than mammalian complement. Optimal reaction temperatures vary not only between cold- and warm water fish, but also between species. Generally, incubation for 20e30 min at 45 C completely abolishes the hemolytic activity of e.g. rainbow trout (O. mykiss), goldfish (Carassius auratus), catfish (Ictalurus punctatus) and tilapia (Oreochromis mossambicus) sera [47]. Addition of EDTA, which binds divalent cations, effectively inhibits the activity of both the alternative and the classical pathways. In this study, the hemolytic activity was not inhibited by heat, and addition of EDTA seemed to increase, rather than inhibit the activity. This excluded complement as the main mediator of hemolysis, suggesting that other factors may have been involved either in conjunction with or independent of complement. Similar results
M. Løvoll et al. / Fish & Shellfish Immunology 23 (2007) 542e552
551
were reported on cod (G. morhua) where serum was unusually insensitive to heat and EDTA augmented the hemolytic activity [12]. In 2004, Magnado´ttir and Lange extended these results by suggesting an inhibitory effect of Apolipoprotein A-I (which exists in close association with C3) on the hemolytic activity in cod (G. morhua) [48]. According to our results, this may concern Atlantic salmon as well. Halibut serum has been described as highly sensitive to storage, especially at 20 C [49]. Similar degrees of instability have not been detected in trout, goldfish, catfish and tilapia sera [47], but whether this may have concerned the homogenates in this study was not tested. In conclusion, this study revealed widespread extrahepatic distribution of C3 proteins, particularly in the skeletal muscle, chondrocytes of the gill arch and developing notochord of developing embryos and hatchlings. C3 proteins were detected in the unfertilised egg, but C3 mRNA was not detected until 14 days post fertilisation. Taken together, these results strongly suggest maternal transfer of C3 proteins. Acknowledgements The financial support of the European Commission grant IMAQUANIM (contract no. 007103) and the University of Tromsø is acknowledged. References [1] Dempsey PW, Allison MDD, Akkaraju S, Goodnow CC, Fearon DT. C3d of complement as a molecular adjuvant: bridging innate and acquired immunity. Science 1996;271:348e50. [2] Morgan BP, Marchbank KJ, Longhi MP, Harris CL, Gallimore AM. Complement: central to innate immunity and bridging to adaptive responses. Immunology Letters 2005;97:171e9. [3] Carroll MC. The complement system in B cell regulation. Molecular Immunology 2004;41:141e6. [4] Nakao M, Mutsuro J, Nakahara M, Kato Y, Yano T. Expansion of genes encoding complement components in bony fish: biological implications of the complement diversity. Developmental and Comparative Immunology 2003;27:749e62. [5] Sunyer JO, Zarkadis IK, Lambris JD. Complement diversity: a mechanism for generating immune diversity. Immunology Today 1998;19(11):519e23. [6] Nakao M, Mutsuro J, Obo R, Fujiki K, Nonaka M, Yano T. Molecular cloning and protein analysis of divergent forms of the complement component C3 from a bony fish, the common carp (Cyprinus carpio): presence of variants lacking the catalytic histidine. European Journal of Immunology 2000;30:858e66. [7] Zarkadis IK, Sarrias MR, Sfyroera G, Sunyer JO, Lambris JD. Cloning and structure of three rainbow trout C3 molecules: a plausible explanation for their functional diversity. Developmental & Comparative Immunology 2001;25:11e24. [8] Sunyer JO, Zarkadis IK, Sahu A, Lambris JD. Multiple forms of complement C3 in trout that differ in binding to complement activators. Proceedings of the National Academy of Sciences of the USA 1996;93:8546e51. [9] Morgan BP, Gasque P. Extrahepatic complement biosynthesis: where, when and why? Clinical and Experimental Immunology 1997; 107:1e7. [10] Mastellos D, Lambris JD. Complement: more than a ‘guard’ against invading pathogens. TRENDS in Immunology 2002;23:485e91. [11] Løvoll M, Kilvik T, Boshra H, Bøgwald J, Sunyer JO, Dalmo RA. Maternal transfer of complement components C3-1, C3-3, C3-4, C4, C5, C7, Bf and Df to offspring in rainbow trout (Oncorhynchus mykiss). Immunogenetics 2006;58:168e79. [12] Lange S, Bambir SH, Dodds AW, Bowden T, Bricknell I, Espelid S, et al. Complement component C3 transcription in Atlantic halibut (Hippoglossus hippoglossus L.) larvae. Fish & Shellfish Immunology 2006;20:285e94. [13] Lange S, Dodds AW, Gudmundsdo´ttir S, Bambir S, Magnado´ttir B. The ontogenic transcription of complement component C3 and Apolipoprotein A-I tRNA in Atlantic cod (Gadus morhua L.) e a role in development and homeostasis? Developmental and Comparative Immunology 2005;29:1065e77. [14] Magnadottir B, Lange S, Gudmundsdottir S, Bogwald J, Dalmo RA. Ontogeny of humoral immune parameters in fish. Fish & Shellfish Immunology 2005;19:429e39. [15] Mastellos D, Andronis C, Persidis A, Lambris JD. Novel biological networks modulated by complement. Clinical Immunology 2005; 115(3):225e35. [16] Ellis AE. Ontogeny of the immune response in Salmo salar. Histogenesis of the lymphoid organs and appearance of membrane immunoglobulin and mixed leucocyte reactivity. Symposia on Developmental Immunobiology 1977;225e31. [17] Razquin BE, Castillo A, Lo´pez-Fierro P, Alvarez F, Zapata AG, Villena AJ. Ontogeny of IgM-producing cells in the lymphoid organs of rainbow trout, Salmo gairdneri Richardson: an immuno- and enzyme-histochemical study. Journal of Fish Biology 1990;36:159e73. [18] Zapata AG, Torroba M, Varas A, Jime´nez E. Immunity in fish larvae. Developments in Biological Standardizations 1997;90:23e32. [19] Ellis A.E, Ontogeny of the immune system in teleost fish. In: Ellis, editor. Fish Vaccination 1988. p. 20e31. [20] Ogundele MO. Role and significance of the complement system in mucosal immunity: particular reference to the human breast milk complement. Immunology and Cell Biology 2001;79:1e10. [21] Schroder MB, Villena AJ, Jorgensen TO. Ontogeny of lymphoid organs and immunoglobulin producing cells in Atlantic cod (Gadus morhua L.). Developmental & Comparative Immunology 1998;22:507e17.
552
M. Løvoll et al. / Fish & Shellfish Immunology 23 (2007) 542e552
[22] Rombout JHWM, Huttenhuis HB, Picchietti S, Scapigliati G. Phylogeny and ontogeny of fish leucocytes. Fish & Shellfish Immunology 2005;19:441e55. [23] Huttenhuis HBT, Grou CPO, Taverne-Thiele AJ, Taverne N, Rombout JHWM. Carp (Cyprinus carpio L.) innate immune factors are present before hatching. Fish & Shellfish Immunology 2006;20:586e96. [24] Ellingsen T, Strand C, Monsen E, Bogwald J, Dalmo RA. The ontogeny of complement component C3 in the spotted wolffish (Anarhichas minor Olafsen). Fish & Shellfish Immunology 2005;18(5):351e8. [25] Chomczynski P, Sacchi N. Single-step method or RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Analytical Biochemistry 1987;162:156e9. [26] Bogerd J, Blomenrohr M, Andersson E, van der Putten HHAGM, Tensen CP, Vischer HF, et al. Discrepancy between molecular structure and ligand selectivity of a testicular follicle-stimulating hormone receptor of the African catfish (Clarias gariepinus). Biological Reproduction 2001;64:1633e43. [27] Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Research 2001;29:e45. [28] Ririe KM, Rasmussen RP, Wittwer CT. Product differentiation by analysis of DNA melting curves during the polymerase chain reaction. Analytical Biochemistry 1997;245:154e60. [29] Abelseth TK, Stensvag K, Espelid S, Nygaard R, Ellingsen T, Bogwald J, et al. The spotted wolffish (Anarhichas minor Olafsen) complement component C3: isolation, characterisation and tissue distribution. Fish & Shellfish Immunology 2003;15:13e27. [30] Jorgensen SM, Kleveland EJ, Grimholt U, Gjoen T. Validation of reference genes for real-time polymerase chain reaction studies in Atlantic salmon. Marine Biotechnology 2006;0:1e11. [31] Olsvik PA, Lie KK, Jordal AEO, Nilsen TO, Hordvik I. Evaluation of potential reference genes in real-time RT-PCR studies in Atlantic salmon. BMC Molecular Biology 2005;6:21. [32] Grace MF. The functional histogenesis of the immune system in rainbow trout, Salmo gairdneri Richardson 1836. PhD Thesis, Hull University, UK; 1981. [33] Crowhurst MO, Layton JE, Lieschke GJ. Developmental biology of zebrafish myeloid cells. International Journal of Developmental Biology 2002;46:483e92. [34] Herbomel P, Thisse B, Thisse C. Ontogeny and behaviour of early macrophages in the zebrafish embryo. Development 1999;126:3735e45. [35] Romano N, Picchietti S, Taverne-Thiele JJ, Taverne N, Abelli L, Mastrolia L, et al. Distribution of macrophages during fish development: an immunohistochemical study in carp (Cyprinus carpio, L.). Anatomy and Embryology 1998;198:31e41. [36] Kircheis R, Kircheis L, Oshima H, Kohchi C, Soma G, Mizuno D. Selective lysis of early embryonic cells by the alternative pathway of complement-a possible mechanism for programmed cell death in embryogenesis. In Vivo 1996;10:389e403. [37] Cole DS, Morgan BP. Beyond lysis: how complement influences cell fate. Clinical Science 2003;104:455e66. [38] Nauta AJ, Daha MR, Kooten Cv, Roos A. Recognition and clearance of apoptotic cells: a role for complement and pentraxins. Trends in Immunology 2003;24:148e54. [39] Henson PM, Hume DA. Apoptotic cell removal in development and tissue homeostasis. Trends in Immunology 2006;27:244e50. [40] Picton H, Briggs D, Gosden R. The molecular basis of oocyte growth and development. Molecular and Cellular Endocrinology 1998; 145:27e37. [41] Lange S, Dodds AW, Magnado´ttir B. Isolation and characterization of complement component C3 from Atlantic cod (Gadus morhua L.) and Atlantic halibut (Hippoglossus hippoglossus L.). Fish & Shellfish Immunology 2004;16:227e39. [42] Magnadottir B, Lange S, Steinarsson A. Gudmundsdottir S. The ontogenic development of innate immune parameters of cod (Gadus morhua L.). Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology 2004;139:217e24. [43] Lange S, Bambir S, Dodds AW, Bowden T, Bricknell I, Espelid S, et al. Complement component C3 transcription in Atlantic halibut (Hippoglossus hippoglossus L.) larvae. Fish & Shellfish Immunology 2006;20:285e94. [44] Lange S, Bambir S, Dodds AW, Magnado´ttir B. An immunohistochemical study on complement component C3 in juvenile Atlantic halibut (Hippoglossus hippoglossus L.). Developmental & Comparative Immunology 2004;28:593e601. [45] Andrades JA, Nimni ME, Becerra J, Eisenstein R, Davis M, Sorgente N. Complement proteins are present in developing endochondral bone and may mediate cartilage cell death and vascularization. Experimental Cell Research 1996;227:208e13. [46] Sato T, Abe E, Jin C, Hong M, Katagiri T, Kinoshita T, et al. The biological roles of the third component of complement in osteoclast formation 10.1210/en.133.1.397. Endocrinology 1993;133:397e404. [47] Sakai DK. Repertoire of complement in immunological defense mechanisms of fish. Annual Review of Fish Diseases 1992;2:223e47. [48] Magnadottir B, Lange S. Is Apolipoprotein A-I a regulating protein for the complement system of cod (Gadus morhua L.)? Fish & Shellfish Immunology 2004;16:265e9. [49] Lange S, Magnadottir B. Spontaneous haemolytic activity of Atlantic halibut (Hippoglossus hippoglossus L.) and sea bass (Dicentrarchus labrax) serum. Comparative Biochemistry and Physiology Part B 2003;136:99e106.
The ontogeny and extrahepatic expression of complement factor C3 in Atlantic salmon (Salmo salar) Marie Løvoll a,*, Hanne Johnsen a, Hani Boshra b, Jarl Bøgwald a, J. Oriol Sunyer b, Roy A. Dalmo a a
Department of Marine Biotechnology, Norwegian College of Fishery Science, University of Tromsø, Breivika, N-9037 Tromsø, Norway b Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA Received 8 August 2006; revised 3 January 2007; accepted 15 January 2007 Available online 20 January 2007
Abstract Fish embryos and hatchlings are exposed to pathogens long before maturation of their lymphoid organs. Little is known about defence mechanisms during the earliest stages of life, but innate mechanisms may be essential for survival. The complement system in fish is well developed and represents a major part of innate immunity. Complement factor 3 (C3) is central subsequent to activation of all pathways of the complement system, leading to inflammatory reactions, such as chemotaxis, opsonisation and lysis of pathogens. Hepatocytes represent the major source of C3, but modern molecular biological methods have confirmed that C3 is synthesised at multiple sites. Our main objective was to study the ontogeny of C3 in Atlantic salmon by mapping the commencement of synthesis and localisation of proteins. Eggs, embryos, hatchlings and adult fish were analysed for the presence of C3 mRNA and proteins. From immunohistochemical studies, C3 proteins were detected at several extrahepatic sites, such as the skeletal muscle, developing notochord and chondrocytes of the gill arch. Immunoblotting revealed presence of C3 proteins in the unfertilised egg, but C3 mRNA was only detected after fertilisation by real-time RT-PCR. Taken together, the results implicated the maternal transfer of C3 proteins as well as novel non-immunological functions during development. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Complement component C3; Ontogeny; Extrahepatic; Atlantic salmon
1. Introduction The complement system plays a vital role in innate immunity and by augmenting B-cell proliferation, it also affects acquired immunity [1e3]. Activation of the complement cascade occurs through three partially overlapping pathways, where complement factor 3 (C3) is the key protein. Activation leads to the assembly of a pore forming complex capable of lysing pathogens, and also promotes inflammation and clearance of pathogens through generation of anaphylatoxins and opsonins. C3 belongs to the a2-macroglobulin family and consists of two disulfide-linked chains; a and b. In di- and tetraploid fish, such as teleosts, several complement factors are encoded by multiple genes giving * Corresponding author. Tel.: þ47 7764 6345; fax: þ47 7764 6020. E-mail address: [email protected] (M. Løvoll). 1050-4648/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2007.01.002
M. Løvoll et al. / Fish & Shellfish Immunology 23 (2007) 542e552
543
rise to subtypes displaying structural and functional diversity [4,5]. In the common carp (Cyprinus carpio), five C3 variants have been characterised [6], while the rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar) both possess three subtypes. The trout subtypes have been fully characterised [7,8], but only partial sequences have been described for Atlantic salmon (S. salar). From BLAST analysis (Basic Local Alignment Search Tool, http:// www.ncbi.nlm.nih.gov/blast/), only few nucleotide differences between species were revealed. Hepatocytes represent the major source of most plasma complement proteins, but modern molecular biological methods have confirmed that complement proteins are synthesised at multiple sites [9,10]. Ontogenic appearance and mapping of extrahepatic synthesis of complement components in rainbow trout (O. mykiss) revealed widespread production of several complement components [11]. Studies on Atlantic halibut larvae (Hippoglossus hippoglossus) and Atlantic cod (Gadus morhua) revealed presence of C3 mRNA and proteins in several tissues post hatch [12e14]. Following several reports on extrahepatic synthesis of complement, non-immunological functions in e.g. reproduction, development, ossification, signal transduction and metabolism have been suggested [10,15]. Embryos and hatchlings are exposed to pathogens long before they are able to mount a mature immune response [16e18]. In Atlantic salmon, the thymus and kidney are fully lymphoid by the time of hatching, but surface immunoglobulins are not present until around the start of feeding [19]. Absence of immunoglobulin producing cells makes the developing embryo highly vulnerable against pathogen attack [20,21]. The embryo relies solely on physical barriers (mucus and epithelium from the skin, gills and intestine), innate immune cells (macrophages and granulocytes) [22] and other innate mechanisms as defence against invading pathogens. At these early stages, maternally derived immune defence components in the yolk, such as complement, may play a vital role in protection. Maternal transfer of C3, a2M, serum amyloid A and C1r/s mRNAs along with C3 and Ig proteins has been described in carp (C. Carpio) [23], several maternally derived complement proteins have been detected in rainbow trout (O. mykiss) [11] and transfer of C3 proteins has been described in the spotted wolffish (Anarhichas minor) [24]. The main objective of this work was to study the ontogeny and extrahepatic expression of C3 in Atlantic salmon (S. salar) by mapping the commencement of synthesis and localisation of proteins. 2. Materials and methods 2.1. Sample collection and preparation Unfertilised Atlantic salmon (S. salar) eggs and sperm were obtained from AquaGen (Trondheim, Norway), mixed and disinfected with buffodine. Incubation of fertilised eggs and maintenance of hatchlings was performed in upwelling incubators and aquaria supplied with aerated, running water at 6 C for 15 weeks (Ka˚rvika Aquaculture Research Station, Tromsø, Norway). Hatchlings were maintained under a photoperiod of 12 h light/12 h darkness. The embryos hatched at day 77e84 and yolk-sac resorption was completed w119 days post fertilisation. Samples were collected weekly. Hatchlings were overanaesthetized using 0.01% benzocaine. Five adult Atlantic salmon (S. salar) (w5 kg) were kindly provided by a commercial fish farm (Sotra Fiskeindustri, Glesvær, Norway). The adult fish had been vaccinated once (Norvax Fur Vib; Intervet Norbio, Norway) 16 months prior to sacrifice. Tissue samples from the gills, skin, muscle, heart, pylorus, intestine, gonads, spleen, head kidney and liver were dissected. Samples for RNA purification were submerged in RNAlater (Ambion, Austin, Texas, USA), kept at room temperature overnight and stored at 20 C. Samples for hemolytic assays and immunoblotting were directly frozen at 20 C. Samples for immunohistochemistry were fixed in 4% paraformaldehyde in phosphate buffered saline (PBS) for two days, transferred to 70% ethanol, dehydrated and embedded in paraffin wax. 2.2. Purification of total RNA Total RNA was extracted using the TRIzol method [25]. Three eggs, embryos or hatchlings at each time-point were pooled and homogenised in 3 ml TRIzol reagent using a rotor-stator homogeniser (UltraTurrax; IKAÒWerke, Staufen, Germany). For additional removal of DNA and proteins, the water phase of the initial TRIzol/chloroform separation was added to a second volume of TRIzol reagent. To remove any contaminating genomic DNA, samples were treated with DNase (TURBO DNA-freeÔ, Ambion). Purified RNA was confirmed to be intact by gel electrophoresis. RNA concentrations and purity were measured spectrophotometrically (NanoDrop Technologies, Wilmington, USA).
M. Løvoll et al. / Fish & Shellfish Immunology 23 (2007) 542e552
544
2.3. Quantitative real-time reverse transcription-polymerase chain reaction (qPCR) RNA was reversely transcribed using random hexamers (TaqMan RT-reagents, Applied Biosystems, CA, USA). Reaction volumes of 50 ml contained 100 ng of total RNA. PCRs were performed in duplicates with an ABI PRISMÒ 7000 Sequence Detection System (Applied Biosystems) as described earlier [11]. Primers were designed from partial cDNA sequences deposited in the GenBank (accession numbers are listed in Table 1) using the Primer Express software (version 2.0; Applied Biosystems) and synthesised by MedProbe (Oslo, Norway). The amplification efficiency of each primer set was assessed from 2-fold liver cDNA dilutions (standard curves) according to the equation: E ¼ 10(1/slolpe) [26] (Table 1). The relative expression ratio (R) of the C3 gene was calculated based on primer efficiencies (E) and the Ct deviations (DCt) of the unknown samples versus a calibrator, and normalised to the corresponding reference gene according to the equation R ¼ (Etarget)DCt target (calibrator-sample)/(Ereference)DCt reference (calibrator-sample) [27]. cDNA to be used with the 18S primers was diluted 1:10 000. Melting curves were generated for the amplicons, verifying specific amplification and absence of dimers [28]. In addition, the PCR products were analysed by gel electrohoresis. Presence of any genomic DNA contamination of the RNA was tested by subjecting parallel samples to PCR without preceding reverse transcription. All data were captured using Sequence Detection Software (SDS version 1.1; Applied Biosystems). 2.4. Antibodies Antibodies recognizing individual chains of different C3 were generated in rabbits by immunization with SDSPAGE purified a- and b-chains [8]. Briefly, 500 mg of purified C3-1 were run on 7.5% SDS-PAGE under reducing conditions. The a- and b-chains were detected using a Zinc Stain Kit (Bio-Rad, Hercules, CA, USA). The individual a- and b-chains were excised from the gel and extracted protein samples were sent to Cocalico Biologicals, Inc. (Reamstown, PA, USA) for antibody production in rabbits. 2.5. Immunoblotting All reagents were purchased from Invitrogen and used in compliance with the manufacturer’s protocol as previously described [11]. Briefly, proteins were extracted from eight pooled eggs, embryos or five hatchlings at each time-point. Samples were homogenised in 2.5 ml 0.1 M Tris, pH 7.6 and protease inhibitors (Protease Inhibitor Cocktail; Sigma-Aldrich, Steinberg, Germany) using the rotor-stator homogeniser (UltraTurrax). The homogenates were centrifuged at 4500 g for 15 min at 4 C, the supernatants were centrifuged another three times at 14 000 g for 5 min at 4 C. Protein extracts were stored at 20 C. Protein concentrations and purity were measured spectrophotometrically (NanoDrop Technologies). Twenty micrograms of proteins from each sampled time-point were reduced, denatured and fractionated by gel electrophoresis. Proteins were transferred to a polyvinylidene difluoride-membrane and incubated with a mixture of rabbit antisera raised against the rainbow trout C3-1a- and b-chains (1:1000 dilution). Normal rabbit serum (Sigma-Aldrich) served as the control. 2.6. Immunohistochemistry The immunostaining procedure was performed as described earlier [29]. Briefly, serial sections of 5 mm were mounted onto SuperFrostÒ slides, melted at 60 C, dewaxed in xylene, rehydrated via ethanol to water and demasked by boiling in 0.1 M citric acid (pH 6.0) for 5 min. Following antigen retrieval and blocking, the sections were Table 1 Primers, amplicon lengths, GenBank accession numbers and primer efficiencies (E) for qPCR Gene C3 18S
Oligonucleotides Forward Reverse Forward Reverse
0
0
5 -tccctggtggtcaccagtacac-3 50 -atgatggtggactgtgtggatc-30 50 -tgtgccgctagaggtgaaatt-30 50 -cgaacctccgactttcgttct-30
The C3 primers annealed to the sequence encoding the a-chain.
Amplicon (bp)
GenBank acc. no.
E
157
BI468074
1.95
101
AJ427629
1.98
M. Løvoll et al. / Fish & Shellfish Immunology 23 (2007) 542e552
545
incubated with mixed polyclonal rabbit anti-rainbow trout C3-1a and C3-1b sera (1:1000 dilution). Normal rabbit serum (Sigma-Aldrich) served as control. 2.7. Hemolytic assay The ability of homogenates prepared from eggs, embryos and hatchlings to lyse foreign cells was measured. Four samples from each time-point were homogenised in Hank’s Balanced Saline Solution (HBSSþ/þ). 100 ml homogenates (duplicates of two-fold serial dilutions) were incubated with 100 ml of 1% sheep red blood cells in each well of a 96 well microtray (Nunc, Denmark). The tray was incubated for 1.5 h at room temperature followed by centrifugation at 148 g for 10 min at 4 C. The supernatants (100 ml) were collected from each well and transferred to a non-absorbent flat bottom microtray (Nunc). The optical density at 405 nm (OD405) was read. To normalise the results, OD405 values of homogenates without added red blood cells were subtracted from the OD405 values of the test samples. Replacing homogenates with HBSSþ/þ served as negative control (0% lysis) and a replacement of homogenates by H2O served as positive control (100% lysis). To inhibit the hemolytic activity mediated by complement, the homogenates were heated at 45 C for 30 min or HBSSþ/þ was supplemented with 10 mM ethylenediamine tetraacetic acid (EDTA).
3. Results Using real-time RT-PCR to compare mRNA levels can be influenced by fluctuating levels of the reference gene between different developmental stages, tissues and individuals. 18S rRNA has been found to be a well behaved endogenous control in salmon [30,31], and also in this study the 18S rRNA levels were constant. The level of 18S rRNA in homogenates of the unfertilised egg was higher than in homogenates from the later time-points studied. Any genomic contamination was excluded by negative controls (minus reverse transcriptase). Specificity of anti C3-1a and anti C3-1b was previously confirmed in rainbow trout [11] and cross-binding to the Atlantic salmon C3 aand b-chains was confirmed in serum from adult salmon (Fig. 1). Specific cross-binding to the salmon C3 subtypes was not evaluated. Serum from human or fish are known to contain several degradation fragments of C3 in addition to native C3. The bands that are immunoreactive with the antibody are consistent with degradation fragments previously reported for trout C3-1 [8].
Fig. 1. Cross-binding of anti-rainbow trout C3-1 serum to Atlantic salmon serum. Lane 1: size marker, lane 2: Atlantic salmon serum, lane 3: rainbow trout serum.
546
M. Løvoll et al. / Fish & Shellfish Immunology 23 (2007) 542e552
3.1. Ontogenic appearance of C3 mRNA and protein C3 mRNA levels were detected by real-time RT-PCR throughout the study period (Fig. 2). Expression levels are given in fold increase compared to day 14. No mRNA was detected prior to fertilisation (0 d), but by day 14 transcription had commenced. Slight decreases in mRNA levels were detected prior to hatch (70e77 d) and on day 98e105, but in general, transcription increased steadily throughout the study period. Immunoblotting revealed signals of increasing intensities from the C3 b-chain (w70 kDa) in homogenates from all time-points, including in the unfertilised egg (0 d). The C3 a-chain (w112 kDa) was detected as a weak signal from day 98 and onwards. Negative controls using a 1:500 dilution of normal rabbit serum revealed signals from a protein of w80 kDa. These signals were, however, faint and not in conflict with the interpretation of the immunoblot using the rabbit anti-trout C3-1a and C3-1b sera.
3.2. Immunohistochemical detection of C3 proteins during ontogeny Localisation of C3 proteins in embryos, yolk-sac hatchlings and first feeding hatchlings was studied by immunohistochemistry. Complement C3 proteins were detected at several extrahepatic sites including in the neurons of the spinal chord, the epithelial cells of the oesophagus and intestine, skeletal muscle, muscle cells of the heart and chondrocytes and lamellae of the gill arches. Strong colouring was also detected in the yolk-sac, possibly of periblast cells, which are involved in conversion of yolk-proteins into embryo nutrition (Fig. 3). While the levels of C3 proteins in the liver seemed to increase throughout development, the extrahepatic C3 levels decreased.
Fig. 2. Expression of C3 mRNA (real-time RT-PCR) and C3 proteins (immunoblotting) during ontogeny. Eggs, embryos and hatchlings were sampled weekly from prior to fertilisation (0 d), during embryo development and hatch (77e84 d) throughout complete yolk-sac resorption (84e119 d). C3 mRNA was detected by real-time RT-PCR and the expression levels are given in fold increase compared to the C3 mRNA levels detected in homogenates from day 14. C3 proteins were detected in homogenates from the corresponding time-points using immunoblotting. C3 proteins were visualised by chemiluminescence and exposure to an X-ray-sensitive film for three hours.
M. Løvoll et al. / Fish & Shellfish Immunology 23 (2007) 542e552
547
3.3. Hemolytic activity in embryos and hatchlings Compared to serum from adult rainbow trout, homogenates from all time-points showed very low hemolytic activities and no hemolysis was detected in homogenates of unfertilised eggs (results not shown). Slightly higher activities were detected 14e28 days post fertilisation, as well as in homogenates from the first time-point after hatch (day 84). Interestingly, the hemolytic activity of the homogenates was not inhibited by heat and was enhanced by addition of EDTA, suggesting non-complement mediated activity (Fig. 4).
Fig. 3. A selection of micrographs of tissue sections from Atlantic salmon embryos and hatchlings at different stages of development. Sections to the left were stained for C3 proteins with FastRed TR/naphtol AS-MX and counterstained with Harris’ hematoxylin solution (blue). At day 35 post fertilisation: (A) overview, (B) bodies of neurons in the spinal chord surrounded by striated muscle, (C) epithelial cells of the oesophagus and columnar epithelial cells of the intestine. At day 70 post fertilisation: (D) hepatocytes of the liver and periblast cells of the yolk-sac. At day 91 post fertilisation: (E) chondrocytes of the gill arch, (F) skeletal muscle cells. A.1eF.1 represent the corresponding normal rabbit serum controls.
548
M. Løvoll et al. / Fish & Shellfish Immunology 23 (2007) 542e552
3.4. Quantitation of C3 mRNA in adult salmon C3 transcripts were detected in all the tissues studied (Fig. 5). The highest extrahepatic levels were detected in the heart followed by gonadal tissue, skeletal muscle, intestine and skin. The mean Ct values in liver and extrahepatic tissues before normalisation to 18S rRNA were 15.9 0.9 and 28.2 2.4, respectively. The statistical analysis was conducted using SSC-Stat. Expression levels are given in percentage of average liver C3 mRNA levels. 4. Discussion Being a central component of all activation pathways, C3 is crucial for a complete innate immune response to occur. For lysis of pathogens to occur, all components of the membrane attack complex are essential. Whether these
Fig. 3 (continued).
M. Løvoll et al. / Fish & Shellfish Immunology 23 (2007) 542e552
549
0,4 Sample 45°C EDTA EDTA, 45°C
OD405
0,3
0,2
0,1
0 day 14
serum
Fig. 4. Effects of EDTA and heat on the hemolytic activity of homogenates of four embryos (14 d.p.f.) and serum from adult salmon. Values are given as OD405 values after subtraction of OD405 values of homogenates/serum without added sheep red blood cells.
components are present in the developing salmon embryo, is not known. We have earlier shown presence of C5 and C7 mRNA during rainbow trout ontogeny, though at very low levels and not until 28 days post hatch [11]. In the absence of a lytic pathway, C3 degradation products can still function as opsonins and anaphylatoxins, thereby promoting phagocytosis. This does of course require the presence of phagocytic cells. At 4 days post fertilisation, the rainbow trout spleen anlage is just appearing, and the kidney is not yet lymphoid. Pending maturation of the main phagocytic sites, it has been suggested that macrophages in the gills, skin and gut may protect the hatchlings [32]. In zebrafish (Danio rerio), macrophages differentiate from multipotent myeloid-erythroid progenitors 12e20 h post fertilisation [33]. They are able to phagocytose apoptotic cells and bacteria, and also respond to chemotaxis [34]. In carp
% of liver mRNA levels
1,4 1,2 1 0,8 0,6 0,4 0,2
intestine
kidney
gonads
spleen
pylorus
heart
muscle
skin
gills
0
Fig. 5. Relative expression levels (ordinate axis) of C3 mRNA in extrahepatic tissues (abscissa axis) (n ¼ 5). Expression levels are given in percentage of liver C3 mRNA levels (liver ¼ 100%). The data were analysed by SSC-Stat and expressed as box plots where the median is the line within each box. The boxes indicate the interquartile range; the upper and lower boxes represent the upper and lower 25% of the group, respectively. The tails represent the minimum and maximum values of the group and the horizontal lines indicate the mean values.
550
M. Løvoll et al. / Fish & Shellfish Immunology 23 (2007) 542e552
(C. carpio), macrophages have been identified from 2 days post fertilisation in the head kidney [35]. The ontogenic appearance of macrophages in salmon is, however, unclear and whether they may serve protection prior to hatch is uncertain. Besides the traditional functions of C3 in immunology, several functions during development have been suggested, including tagging apoptotic cells for removal [36e39] as well as mediating stem-cell commitment and differentiation [10]. Quantitation of C3 mRNA revealed steadily increasing levels from day 14 post fertilisation. Inconsistencies, such as the slight decrease observed prior to hatch and the increase at day 91, may be valid or result from a few embryos with especially low or high C3 mRNA levels leading to reduced and increased mean values of expression in the pooled samples. We have earlier shown the corresponding expression profiles of the C3 subtypes for rainbow trout during ontogeny [11]. The salmon and trout profiles differed after hatch by increasing and deflating expression levels, respectively. The salmon C3 primers were designed based on the C3-1 a-chain. Whether this region is unique to the C3-1 subtype is not known and the primers may possibly have annealed to C3-3 and C3-4 as well. From rainbow trout ontogeny studies, the C3-3 and C3-4 levels were found to be approximately 4% and 0.2% of the C3-1 levels, respectively [11]. Transcripts in unfertilised eggs must in general be maternally derived, as transcription starts in correspondence to cell division [40]. Maternal mRNAs are highly protected by e.g. methylation, 50 -capping and massive binding of proteins that may complicate RNA purification. 18S rRNA belongs to the many housekeeping genes that are maternally transferred in all organisms. In this study, the level of maternally transferred 18S rRNA was slightly higher than the embryo’s own synthesis post fertilisation. C3 mRNA was not detected in the unfertilised egg, which was in contrast to studies on carp (C. carpio) where maternally derived C3 mRNA was described [23]. The tissue-distribution study of C3 mRNA in adult salmon showed that the liver was indeed the main supplier of C3. C3 transcripts were, however, detected in all extrahepatic tissues studied, particularly in the heart, followed by gonadal tissue and skeletal muscle. Compared to the levels of complement mRNA in the liver, extrahepatic synthesis was low, but considering the Ctvalues separately, the values indeed implied high enough levels for extrahepatic complement to be of biological significance. Similar studies on rainbow trout showed that the highest extrahepatic levels of synthesis occurred in the gills, skin, skeletal muscle and heart (manuscript in preparation). The individual variation was high, which may result from inconsistent tissue dissection or simply reflect the natural variation in outbred salmon populations. Immunoblotting revealed presence of the C3 b-chain in homogenates from all time-points studied, including in the unfertilised eggs. From day 98, the intensity of the signals increased and the a-chain was visualised. Absence of the a-chain at the earliest time-points has been described earlier and was probably due to the chain’s susceptibility to proteolytic attack [11,23,41]. Our results coincide with studies on fertilised cod eggs (G. morhua) where presence of the C3 b-chain was detected from day 7 post fertilisation and onwards [42]. The C3 proteins detected in the unfertilised egg must be of maternal origin and short protein half-lives may be a reason for the absence of the a-chain at the very earliest time-points. By day 98, the hatchling’s own synthesis may have reached a detectable level where the a-chain could be visualised. The normal rabbit serum showed a weak signal at w80 kDa, but could not account for the signals detected using the anti-C3 serum. Immunohistological studies revealed widespread distribution of C3 proteins at extrahepatic sites, such as in the notochord, intestine, muscle and gill arches. These results coincide with studies on halibut (H. hippoglossus) and cod (G. morhua) where C3 mRNA and protein were detected in the neuronal cells of the spinal chord, epithelial cells of the intestine, chondrocytes and surrounding muscle [13,43,44]. In humans, it has been hypothesised that C3 may be involved in the ossification of cartilage [45,46]. Normal rabbit serum showed no staining in control sections evaluated by immunohistochemistry. The negligible background detected by immunoblotting may be due to higher sensitivity of this method compared to immunohistochemistry. In addition, immunoblotting was conducted under reducing conditions that breaks disulfide bridges, thus changing the three-dimensional configurations of the proteins. More epitopes may be exposed, resulting in a higher background level. Fish complement is more heat labile and has lower optimal reaction temperatures (10e27 C) than mammalian complement. Optimal reaction temperatures vary not only between cold- and warm water fish, but also between species. Generally, incubation for 20e30 min at 45 C completely abolishes the hemolytic activity of e.g. rainbow trout (O. mykiss), goldfish (Carassius auratus), catfish (Ictalurus punctatus) and tilapia (Oreochromis mossambicus) sera [47]. Addition of EDTA, which binds divalent cations, effectively inhibits the activity of both the alternative and the classical pathways. In this study, the hemolytic activity was not inhibited by heat, and addition of EDTA seemed to increase, rather than inhibit the activity. This excluded complement as the main mediator of hemolysis, suggesting that other factors may have been involved either in conjunction with or independent of complement. Similar results
M. Løvoll et al. / Fish & Shellfish Immunology 23 (2007) 542e552
551
were reported on cod (G. morhua) where serum was unusually insensitive to heat and EDTA augmented the hemolytic activity [12]. In 2004, Magnado´ttir and Lange extended these results by suggesting an inhibitory effect of Apolipoprotein A-I (which exists in close association with C3) on the hemolytic activity in cod (G. morhua) [48]. According to our results, this may concern Atlantic salmon as well. Halibut serum has been described as highly sensitive to storage, especially at 20 C [49]. Similar degrees of instability have not been detected in trout, goldfish, catfish and tilapia sera [47], but whether this may have concerned the homogenates in this study was not tested. In conclusion, this study revealed widespread extrahepatic distribution of C3 proteins, particularly in the skeletal muscle, chondrocytes of the gill arch and developing notochord of developing embryos and hatchlings. C3 proteins were detected in the unfertilised egg, but C3 mRNA was not detected until 14 days post fertilisation. Taken together, these results strongly suggest maternal transfer of C3 proteins. Acknowledgements The financial support of the European Commission grant IMAQUANIM (contract no. 007103) and the University of Tromsø is acknowledged. References [1] Dempsey PW, Allison MDD, Akkaraju S, Goodnow CC, Fearon DT. C3d of complement as a molecular adjuvant: bridging innate and acquired immunity. Science 1996;271:348e50. [2] Morgan BP, Marchbank KJ, Longhi MP, Harris CL, Gallimore AM. Complement: central to innate immunity and bridging to adaptive responses. Immunology Letters 2005;97:171e9. [3] Carroll MC. The complement system in B cell regulation. Molecular Immunology 2004;41:141e6. [4] Nakao M, Mutsuro J, Nakahara M, Kato Y, Yano T. Expansion of genes encoding complement components in bony fish: biological implications of the complement diversity. Developmental and Comparative Immunology 2003;27:749e62. [5] Sunyer JO, Zarkadis IK, Lambris JD. Complement diversity: a mechanism for generating immune diversity. Immunology Today 1998;19(11):519e23. [6] Nakao M, Mutsuro J, Obo R, Fujiki K, Nonaka M, Yano T. Molecular cloning and protein analysis of divergent forms of the complement component C3 from a bony fish, the common carp (Cyprinus carpio): presence of variants lacking the catalytic histidine. European Journal of Immunology 2000;30:858e66. [7] Zarkadis IK, Sarrias MR, Sfyroera G, Sunyer JO, Lambris JD. Cloning and structure of three rainbow trout C3 molecules: a plausible explanation for their functional diversity. Developmental & Comparative Immunology 2001;25:11e24. [8] Sunyer JO, Zarkadis IK, Sahu A, Lambris JD. Multiple forms of complement C3 in trout that differ in binding to complement activators. Proceedings of the National Academy of Sciences of the USA 1996;93:8546e51. [9] Morgan BP, Gasque P. Extrahepatic complement biosynthesis: where, when and why? Clinical and Experimental Immunology 1997; 107:1e7. [10] Mastellos D, Lambris JD. Complement: more than a ‘guard’ against invading pathogens. TRENDS in Immunology 2002;23:485e91. [11] Løvoll M, Kilvik T, Boshra H, Bøgwald J, Sunyer JO, Dalmo RA. Maternal transfer of complement components C3-1, C3-3, C3-4, C4, C5, C7, Bf and Df to offspring in rainbow trout (Oncorhynchus mykiss). Immunogenetics 2006;58:168e79. [12] Lange S, Bambir SH, Dodds AW, Bowden T, Bricknell I, Espelid S, et al. Complement component C3 transcription in Atlantic halibut (Hippoglossus hippoglossus L.) larvae. Fish & Shellfish Immunology 2006;20:285e94. [13] Lange S, Dodds AW, Gudmundsdo´ttir S, Bambir S, Magnado´ttir B. The ontogenic transcription of complement component C3 and Apolipoprotein A-I tRNA in Atlantic cod (Gadus morhua L.) e a role in development and homeostasis? Developmental and Comparative Immunology 2005;29:1065e77. [14] Magnadottir B, Lange S, Gudmundsdottir S, Bogwald J, Dalmo RA. Ontogeny of humoral immune parameters in fish. Fish & Shellfish Immunology 2005;19:429e39. [15] Mastellos D, Andronis C, Persidis A, Lambris JD. Novel biological networks modulated by complement. Clinical Immunology 2005; 115(3):225e35. [16] Ellis AE. Ontogeny of the immune response in Salmo salar. Histogenesis of the lymphoid organs and appearance of membrane immunoglobulin and mixed leucocyte reactivity. Symposia on Developmental Immunobiology 1977;225e31. [17] Razquin BE, Castillo A, Lo´pez-Fierro P, Alvarez F, Zapata AG, Villena AJ. Ontogeny of IgM-producing cells in the lymphoid organs of rainbow trout, Salmo gairdneri Richardson: an immuno- and enzyme-histochemical study. Journal of Fish Biology 1990;36:159e73. [18] Zapata AG, Torroba M, Varas A, Jime´nez E. Immunity in fish larvae. Developments in Biological Standardizations 1997;90:23e32. [19] Ellis A.E, Ontogeny of the immune system in teleost fish. In: Ellis, editor. Fish Vaccination 1988. p. 20e31. [20] Ogundele MO. Role and significance of the complement system in mucosal immunity: particular reference to the human breast milk complement. Immunology and Cell Biology 2001;79:1e10. [21] Schroder MB, Villena AJ, Jorgensen TO. Ontogeny of lymphoid organs and immunoglobulin producing cells in Atlantic cod (Gadus morhua L.). Developmental & Comparative Immunology 1998;22:507e17.
552
M. Løvoll et al. / Fish & Shellfish Immunology 23 (2007) 542e552
[22] Rombout JHWM, Huttenhuis HB, Picchietti S, Scapigliati G. Phylogeny and ontogeny of fish leucocytes. Fish & Shellfish Immunology 2005;19:441e55. [23] Huttenhuis HBT, Grou CPO, Taverne-Thiele AJ, Taverne N, Rombout JHWM. Carp (Cyprinus carpio L.) innate immune factors are present before hatching. Fish & Shellfish Immunology 2006;20:586e96. [24] Ellingsen T, Strand C, Monsen E, Bogwald J, Dalmo RA. The ontogeny of complement component C3 in the spotted wolffish (Anarhichas minor Olafsen). Fish & Shellfish Immunology 2005;18(5):351e8. [25] Chomczynski P, Sacchi N. Single-step method or RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Analytical Biochemistry 1987;162:156e9. [26] Bogerd J, Blomenrohr M, Andersson E, van der Putten HHAGM, Tensen CP, Vischer HF, et al. Discrepancy between molecular structure and ligand selectivity of a testicular follicle-stimulating hormone receptor of the African catfish (Clarias gariepinus). Biological Reproduction 2001;64:1633e43. [27] Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Research 2001;29:e45. [28] Ririe KM, Rasmussen RP, Wittwer CT. Product differentiation by analysis of DNA melting curves during the polymerase chain reaction. Analytical Biochemistry 1997;245:154e60. [29] Abelseth TK, Stensvag K, Espelid S, Nygaard R, Ellingsen T, Bogwald J, et al. The spotted wolffish (Anarhichas minor Olafsen) complement component C3: isolation, characterisation and tissue distribution. Fish & Shellfish Immunology 2003;15:13e27. [30] Jorgensen SM, Kleveland EJ, Grimholt U, Gjoen T. Validation of reference genes for real-time polymerase chain reaction studies in Atlantic salmon. Marine Biotechnology 2006;0:1e11. [31] Olsvik PA, Lie KK, Jordal AEO, Nilsen TO, Hordvik I. Evaluation of potential reference genes in real-time RT-PCR studies in Atlantic salmon. BMC Molecular Biology 2005;6:21. [32] Grace MF. The functional histogenesis of the immune system in rainbow trout, Salmo gairdneri Richardson 1836. PhD Thesis, Hull University, UK; 1981. [33] Crowhurst MO, Layton JE, Lieschke GJ. Developmental biology of zebrafish myeloid cells. International Journal of Developmental Biology 2002;46:483e92. [34] Herbomel P, Thisse B, Thisse C. Ontogeny and behaviour of early macrophages in the zebrafish embryo. Development 1999;126:3735e45. [35] Romano N, Picchietti S, Taverne-Thiele JJ, Taverne N, Abelli L, Mastrolia L, et al. Distribution of macrophages during fish development: an immunohistochemical study in carp (Cyprinus carpio, L.). Anatomy and Embryology 1998;198:31e41. [36] Kircheis R, Kircheis L, Oshima H, Kohchi C, Soma G, Mizuno D. Selective lysis of early embryonic cells by the alternative pathway of complement-a possible mechanism for programmed cell death in embryogenesis. In Vivo 1996;10:389e403. [37] Cole DS, Morgan BP. Beyond lysis: how complement influences cell fate. Clinical Science 2003;104:455e66. [38] Nauta AJ, Daha MR, Kooten Cv, Roos A. Recognition and clearance of apoptotic cells: a role for complement and pentraxins. Trends in Immunology 2003;24:148e54. [39] Henson PM, Hume DA. Apoptotic cell removal in development and tissue homeostasis. Trends in Immunology 2006;27:244e50. [40] Picton H, Briggs D, Gosden R. The molecular basis of oocyte growth and development. Molecular and Cellular Endocrinology 1998; 145:27e37. [41] Lange S, Dodds AW, Magnado´ttir B. Isolation and characterization of complement component C3 from Atlantic cod (Gadus morhua L.) and Atlantic halibut (Hippoglossus hippoglossus L.). Fish & Shellfish Immunology 2004;16:227e39. [42] Magnadottir B, Lange S, Steinarsson A. Gudmundsdottir S. The ontogenic development of innate immune parameters of cod (Gadus morhua L.). Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology 2004;139:217e24. [43] Lange S, Bambir S, Dodds AW, Bowden T, Bricknell I, Espelid S, et al. Complement component C3 transcription in Atlantic halibut (Hippoglossus hippoglossus L.) larvae. Fish & Shellfish Immunology 2006;20:285e94. [44] Lange S, Bambir S, Dodds AW, Magnado´ttir B. An immunohistochemical study on complement component C3 in juvenile Atlantic halibut (Hippoglossus hippoglossus L.). Developmental & Comparative Immunology 2004;28:593e601. [45] Andrades JA, Nimni ME, Becerra J, Eisenstein R, Davis M, Sorgente N. Complement proteins are present in developing endochondral bone and may mediate cartilage cell death and vascularization. Experimental Cell Research 1996;227:208e13. [46] Sato T, Abe E, Jin C, Hong M, Katagiri T, Kinoshita T, et al. The biological roles of the third component of complement in osteoclast formation 10.1210/en.133.1.397. Endocrinology 1993;133:397e404. [47] Sakai DK. Repertoire of complement in immunological defense mechanisms of fish. Annual Review of Fish Diseases 1992;2:223e47. [48] Magnadottir B, Lange S. Is Apolipoprotein A-I a regulating protein for the complement system of cod (Gadus morhua L.)? Fish & Shellfish Immunology 2004;16:265e9. [49] Lange S, Magnadottir B. Spontaneous haemolytic activity of Atlantic halibut (Hippoglossus hippoglossus L.) and sea bass (Dicentrarchus labrax) serum. Comparative Biochemistry and Physiology Part B 2003;136:99e106.